Unveiling population diversity, biosurfactant and antibacterial agents production of Bacillus pumilus group species

Fabiana Raquel Gouveia Pinto Nevado Branquinho UNVEILING POPULATION DIVERSITY, BIOSURFACTANTS AND ANTIBACTERIAL AGENTS PRODUCTION OF BACILLUS PUMILUS GROUP SPECIES Tese do 3º Ciclo de Estudos conducente ao Grau de Doutoramento em Ciências Farmacêuticas na especialidade de Microbiologia Trabalho efetuado sob a orientação e co-orientação de: Professora Doutora Luísa Vieira Peixe, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal Professora Doutora Maria Manuela Estevez Pintado, Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Porto, Portugal Fevereiro 2014 UNVEILING POPULATION DIVERSITY, BIOSURFACTANTS AND ANTIBACTERIAL AGENTS PRODUCTION OF BACILLUS PUMILUS GROUP SPECIES Projeto financiado pela Fundação para a Ciência e Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior em Portugal (SFRH / BD / 61410 / 2009, QREN - POPH - Tipologia 4.1 - Formação Avançada e PEst-C/EQB/LA0006/2011) DE ACORDO COM A LEGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTA TESE. Faculdade de Farmácia da Universidade do Porto, 17 de Fevereiro de 2014. Aos meus pais e madrinha Agradecimentos/Acknowledgements No final de mais uma etapa da minha vida, gostaria de manifestar algumas palavras de sincero agradecimento e de apreço a todos os que me acompanharam e apoiaram no decorrer destes últimos anos, contribuindo para a concretização deste meu desafio. Em primeiro lugar gostaria de agradecer às minhas orientadoras científicas, Professora Doutora Luísa Peixe e Professora Doutora Manuela Pintado, por todo o apoio, entrega e dedicação concedidos ao longo destes anos. Indiscutivelmente, as suas qualidades científicas e humanas, constante incentivo, encorajamento, ensinamento e amizade, foram determinantes preponderantes e fontes inesgotáveis de estímulo e inspiração durante este percurso. Ao Professor Hugo Osório, que de uma forma exímia me possibilitou a aprendizagem dos primeiros passos no mundo da espectrometria de massa, o meu sincero agradecimento pela oportunidade que me concedeu e pelos constantes desafios que me lançou. A sua paciência, disponibilidade e amizade foram também elementos decisivos para o meu crescimento científico e pessoal. Aos Professores Pedro Pereira e Sandra Ribeiro, pelo vosso precioso suporte e pela oportunidade de poder trabalhar com a vossa equipa no laboratório de Cristalografia do IBMC e explorar os meus conhecimentos na área da proteómica. Vorrei esprimere un sentito ringraziamento alle professoresse Giovanna Martinotti, Letizia Fracchia e Gianna Allegrone, per la avermi dato la possibilità di integrare il loro gruppo di ricerca. Il piacevolissimo periodo che ho potuto trascorrere a Novara è stato possibile solo grazie alle loro straordinarie qualità professionali e umane. La disponibilitá dimostrata nel superamento delle sfide quotidiane é stata determinante per il progresso del lavoro sui biosurfattanti. Un ringraziamento speciale anche vá al mio collega Massimo Cavallo, per tutto l'appoggio che mi hai dato in DSF. À Professora Doutora São José Nascimento pela oportunidade concedida na realização deste trabalho no serviço de Microbiologia da Faculdade de Farmácia da Universidade do Porto. A todos os restantes Professores do Serviço de Microbiologia e do CBQF, quero vii também manifestar o meu agradecimento pela simpatia, carinho e disponibilidade demonstrados desde o meu primeiro dia de trabalho. Ao REQUIMTE e à Faculdade de Farmácia da Universidade do Porto, agradeço a oportunidade de desenvolvimento científico, facultando-me todas as condições necessárias à execução deste projeto, e pela aceitação como aluna de Doutoramento da instituição. À Fundação para a Ciência e Tecnologia, agradeço o apoio financeiro deferido para a realização deste projeto de investigação, quer nacional quer internacionalmente. Aos colegas e amigos do laboratório de Microbiologia da FFUP, do CBQF da ESB e do laboratório de Cristalografia do IBMC, manifesto também o meu profundo agradecimento pela vossa paciência e por conseguirem pintar sempre um Sol, mesmo nos dias que se suspeitavam mais cinzentos. Todavia, não poderia deixar de destacar o meu reconhecimento e apreço: à Eduarda Silveira, amiga pilar e modelo de coragem a seguir, o meu bem-haja por todos os teus ensinamentos de vida que me possibilitaram um desenvolvimento pessoal imensurável, por teres amparado as minhas quedas com a tua sensibilidade tão característica e por teres estado sempre na primeira linha de batalha com o teu sorriso rejuvenescedor; ao João Pires, que com sua jovialidade me conseguiu manter sempre crente de que todos os sonhos são possíveis e (podem) ter uma explicação científica, o meu obrigada por teres sido o “biosurfactante” ideal nos momentos em que a tensão necessitava ser reduzida; à Ângela Novais, à Filipa Grosso e à Clara Sousa por todo o carinho, amizade e disponibilidade e ainda pela vossa incansável ajuda nesta reta final do trabalho; à Teresa Gonçalves, à Carla Rodrigues, à Joana Mourão e à Liliana Silva pelo vosso companheirismo e amizade; à Vicenza Ferraro, à Manuela Amorim, à Maria Gião e à Ana Amaro pela vossa ajuda demonstrada em todos os instantes, mas acima de tudo pela vossa amizade incondicional. Aos técnicos do laboratório de Microbiologia, Cristina Pinto da Costa e Nuno Oliveira, pela plena ajuda prestada e pelas palavras de afago. Aos meus familiares e amigos (os diamantes de sempre, com quem tenho uma ligação sem limites, com força iónica suficiente para perpetuar todos os momentos) agradeço por todo o apoio, pela boa disposição constante, pela paciência e compreensão, pela vossa sinceridade e encorajamento, e por terem sido com os vossos sorrisos os sinais de pontuação genuínos na escrita deste capítulo da minha jornada. viii Aos meus maiores mestres: pai, mãe e madrinha, a quem dedico esta tese, por me terem passado os vossos valores simples mas grandiosos, por ter sempre encontrado em vocês o pilar, o conforto, o incentivo e a compreensão nos momentos bons, menos bons e em todas as minhas mudanças e desafios; o meu bem-haja pelo vosso carinho inesgotável e por acreditarem em mim, mesmo nos momentos em que eu própria duvidei. Raquel ix xvi A diferenciação destas espécies foi também suportada pelos perfis proteicos e de composição celular obtidos por MALDI-TOF/MS e FTIR, respetivamente, demonstrando a adequabilidade destas metodologias, rápidas e de baixo custo, na sua diferenciação. Todos os isolados pertencentes ao grupo de B. pumilus revelaram ausência de genes de resistência a antibióticos e genes de virulência, características necessárias para assegurar a sua inocuidade aquando de uma possível aplicação na indústria alimentar e/ou farmacêutica. A maioria dos isolados de B. pumilus eB. safensis estudados revelaram propriedades compatíveis com a produção de compostos biosurfactantes. A caracterização obtida por LC/ESI-MS/MS revelou a presença de uma mistura de péptidos antimicrobianos contendo variantes de pumilacidina e surfactina detetadas apenas em isolados de B. safensis, enquanto que em B. pumilus apenas uma variante especifica de pumilacidina foi verificada. Decorrente deste estudo, a maioria dos isolados previamente identificados como B. pumilus são agora classificados como B. safensis, ou como pertencentes a uma possível nova espécie, a que foi proposta a designação de Bacillus invictus sp. nov. Verificou-se ainda que a reclassificação taxonómica é congruente com a os tipos de péptidos antibacterianos produzidos pelas diferentes espécies. Para além do contributo para a correta e rápida discriminação das espécies do grupo B. pumilus, o presente trabalho demonstra ainda, pela primeira vez, a plasticidade genómica destas bactérias, o que pode justificar a sua dispersão em diferentes ambientes e, consequentemente, a sua capacidade de produção de um conjunto diversificado de biocompostos, com uma ampla gama de atividades. Table of Contents Research Aims ............................................................................................................... xxxix Outline of the Thesis .......................................................................................................... xli Chapter 1: Introduction .................................................................................................. 47 I. Bacillus spp.: phylogeny, antimicrobial resistance and virulence .......................... 49 1. Bacillus spp. .................................................................................................................. 49 1.1. Bacillus RNA group 1 .................................................................................................. 53 2. Bacillus pumilus group .................................................................................................. 53 2.1. General features ........................................................................................................ 53 2.2. Evolution and phylogenetic boundaries ..................................................................... 55 2.2.1 Phenotypic based phylogeny .................................................................................... 55 2.2.2 Genotypic based phylogeny ...................................................................................... 56 2.2.3. Chemotaxonomic based phylogeny ........................................................................ 60 2.2.3.1 Application of MALDI-TOF MS in Bacillus systematics ......................................... 61 2.2.3.2 Application of FTIR in Bacillus Systematics ........................................................... 62 3. Clinical Relevance, antimicrobial Resistance and Virulence within Bacillus spp. .......... 63 3.1 Clinical Relevance ....................................................................................................... 63 3.2. Antimicrobial Susceptibility of Bacillus spp. ................................................................ 64 3.3. Virulence potential of Bacillus spp. ............................................................................. 68 3.3.1. B. cereus group ........................................................................................................ 68 3.3.2. B. pumilus group. .................................................................................................... 70 4. Biotechnological and industrial applications of Bacillus pumilus group members .......... 70 II. Antimicrobial peptides of Bacillus spp. .................................................................... 73 5. Antimicrobial peptides (AMPs) types from Bacillus spp ................................................. 73 5.1. Bacteriocins ................................................................................................................ 75 5.2. Bacteriocin-like inhibitory substances (BLIS) .............................................................. 84 5.3. Unusual peptides ........................................................................................................ 84 5.4. Other antibiotic compounds (miscellaneous) ............................................................. 85 xvii 5.5. Polyketides and non-ribosomal peptides ................................................................... 86 5.6. Non-ribosomal peptides (NRPs) ................................................................................. 88 III. Biosurfactants of Bacillus spp. ................................................................................ 91 6. General features of Bacillus spp. biosurfactants ............................................................ 91 6.1. Biosurfactants basic structure and classification ....................................................... 92 6.2. Biosurfactants diversity .............................................................................................. 94 6.3. Lipopeptides Biosurfactants (LPBSs): a high diversity of structures .......................... 95 6.3.1. Surfactin family ....................................................................................................... 98 6.3.2. Iturin family ............................................................................................................ 100 6.3.3. Fengycin family ..................................................................................................... 100 6.3.4. Other Lipopeptide Compounds: Kurstakins .......................................................... 101 6.4. Biosynthesis of LPBSs: from genes to biomolecules ............................................... 101 6.4.1. The non-ribosomal peptide synthesis machinery .................................................. 101 6.4.2. Diversity of NRPSs in Bacillus spp. ....................................................................... 104 6.4.2.1. Surfactin and Lichenysin Synthetases ............................................................... 106 6.4.2.2. Fengycin Synthetase ......................................................................................... 106 6.4.2.3. Iturin, Bacillomycin and Mycosubtilin Synthetases ............................................ 106 6.4.3. Gene Regulation in Bacillus spp. ........................................................................... 107 6.5. Natural functions of LPBSs ...................................................................................... 110 6.5.1. Antagonism ........................................................................................................... 110 6.5.2. Motility ................................................................................................................... 110 6.5.3. Plant pathogenesis and induction of systemic resistance ..................................... 111 6.5.4. Biofilm formation and development ....................................................................... 111 6.5.5. Chelation of metal ions and degradation of xenobiotics ....................................... 112 6.6. Applications of LPBSs .............................................................................................. 113 6.6.1. Lipopeptides in pharmaceutical industry ............................................................... 113 6.6.2. Lipopeptides in cosmetic industry .......................................................................... 120 6.6.3. Lipopeptides in food industry ................................................................................ 120 6.6.4. Lipopeptides in biotech and nanotech industry ...................................................... 121 6.6.5. Environmental application ..................................................................................... 122 7. References. ................................................................................................................. 123 xviii Chapter 2: Diversity, differentiation by high-throughput methods and biosafety of B. pumilus group species ............................................................................................. 165 2.1. Phylogenetic and clonality analysis of Bacillus pumilus isolates uncovered a highly heterogeneous population of different closely related species and clones ...................... 167 2.2. Bacillus invictus sp. nov., isolated from medicinal products in Portugal ................... 207 2.3. Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF/MS ..... 237 2.4. Differentiation of Bacillus pumilus and Bacillus safensis by Fourier transform infrared spectroscopy and chemometry ........................................................................................ 267 2.5. Safety profile of Bacillus subtilis complex: antibiotic and virulence features ............. 285 Chapter 3. Characterization of the antibacterial compound produced by Bacillus safensis Bs1, the medicine-slug isolate ...................................................................... 305 3.1. Characterization of a new antimicrobial peptide with anti-MRSA activity produced by Bacillus safensis Bs1................................................................................................... 307 Chapter 4. Biosurfactants in B. pumilus group species: diversity and anti-biofilm activity of a surfactin-like biosurfactant ...................................................................... 351 4.1. Species-specific surfactin-like biosurfactants combination within Bacillus pumilus group ................................................................................................................................ 353 4.2. Linezolid resistant ST2/CC5 Staphylococcus epidermidis – biofilm producer .......... 389 4.3. Anti-biofilm activity in Staphylococcus epidermidis and structural characterization of Bacillus safensis biosurfactant ......................................................................................... 399 Chapter 5. Conclusions and Future Perspectives ..................................................... 433 Annexes ......................................................................................................................... 437 xix List of Figures Chapter 1: Introduction ................................................................................................................. 47 I. Bacillus spp.: phylogeny, antimicrobial resistance and virulence ............................. 49 Figure 1. Schematic outline of the phylogenetic relationships between aerobic representatives of Gram-positive bacteria based on 16S rRNA gene sequences. Areas of the triangles represent closeness’s of the number of species included in the taxa covered by the triangle. The circle indicates the uncertainty of the order at which the lineages diverge from each other. ..................... 50 Figure 2. Detailed neighbour-joining tree of Bacillus species of RNA groups 1, 2 and 5. The dotted area indicates the uncertainty of the order at which the lineages diverge from each other. The area was chosen somewhat arbitrarily and cover more recent branching points. The bar indicates 10% nucleotide substitutions. B, Bacillus; T, type strain.. ......................................................................... 51 Figure 3. Detailed neighbor-joining tree of Bacillus species members of RNA groups 3, 4 and 6, other Bacillus species, and lineages containing reclassified Bacillus species. The dotted areas indicate the uncertainty of the order at which the lineages diverge from each other. The areas are chosen somewhat arbitrarily and may more recent branching points. The bar indicates 10% nucleotide substitutions. Gra, Gracilibacillus; Amb, Amphibacillus; B, Bacillus; Bv, Brevibacillus; Ex, Exiguobacterium; Hb, Halobacillus; Pb, Paenibacillus; Sb, Salibacillus; T, Type strain; Vg, Virgibacillus. ...................................................................................................................................... 52 Figure 4. Phylogenetic affiliation based on 16S rRNA genes of B. pumilus group members recovered from marine sources. The tree was constructed using the neighbor-joining method. Bootstrap values over 50% (1000 replications) were shown at each node. Bar, % estimated substitution. B. cereus ATCC 14579T was used as outgroup ........................................................... 57 Figure 5. Phylogenetic tree based on seven concatenated housekeeping genes of marine B. pumilus group isolates. Groups represent: (A) B. altitudinis, (B) B. xiamenensis, (D) B. pumilus, (F) B. safensis and (C and E) could not be assigned. Tree constructed using the neighbor-joining method. Bootstrap values over 50% (1000 replications) were shown at each node. Bar, % estimated substitution. B. cereus ATCC 14579T was used as the outgroup .................................... 59 II. Antimicrobial peptides of Bacillus spp.... .......................................................................... 73 Figure 6. Dendrogram showing the relatedness of Bacillus spp. bacteriocins according to primary amino acid sequence homologies. Shaded characters indicate identical amino acid residues. ...... 80 xxi Figure 7. (a) Comparison of subtilin and ericin structures with that of the lactococcal lantibiotic nisin A. Conserved residues at identical positions to all four bacteriocins are highlighted in green, while those conserved only in subtilin and ericins are depicted in yellow; other conserved residues are in light red. (b) Structures of the single-peptide lantibiotics sublancin, mersacidin and subtilosin A. The structure of the A1 subunit of haloduracin is also included for comparison with mersacidin. The conserved residues are highlighted in orange color. Cysteines involved in disulfide bridge formation are highlighted in blue. For subtilosin A, residues involved in sulfur to a-carbon linkages are shown in green, while residues involved in head-to-tail amide bond formation are in light blue. (c) Comparison of the two-peptide lantibiotics haloduracin and lichenicidin from Bacillus, and lacticin 3147 from Lactococcus lactis. Conserved residues of the A1 peptides are highlighted in orange and light yellow. A conserved Pro residue between lichenicidin A1 subunit and mersacidin is shown in light blue. Conserved residues of the A2 peptides are highlighted in green, deep yellow, light orange and violet. The C-terminal parts of the A1 subunits also share two conserved loops of 11 and eight amino acid residues. The C-terminal parts of A2 peptides share the same pattern of lanthionine (Ala–S–Ala) and methyllanthionine (Abu–S–Ala) bridges. Non-identical residues are in white ................................................................................................................................................. 82 Figure 8. Chemical structures of miscellaneous antibiotics bacilysocin 2, 3,3´-neotrehalosadiamine (NTD) 3 and amicoumacin 4. ............................................................................................................ 85 Figure 9. Chemical structures of polyketides antibiotics a) difficidin, b) bacillaene and c) macrolactin .......................................................................................................................................................... 87 Figure 10. Chemical structures of polypeptide antibiotics a) gramicidin, b) bacitracin, c) polymyxin B and d) daptomycin. ........................................................................................................................... 89 III. Biosurfactants of Bacillus spp.. ......................................................................................... 91 Figure 11. Representative structures of different biosurfactants ..................................................... 93 Figure 12. Relationship of surface tension, interfacial tension and CMC related to surfactant concentration ................................................................................................................................... 93 Figure 13. Detailed structure of some representative biosurfactants produced by Bacillus spp. ..... 98 Figure 14. Overview of NRPS relationship between genes, modules and domains. Each gene is related with a single protein, which can then be prepared through one of more modules. These modules are organized into domains, represented here by colors (A, adenylation domain; C, condensation domain; PCP, peptidyl carrier protein and E, epimerisation domain) ..................... 102 xxii Figure 15. General representation of biosynthesis and structure of surfactin (srf) operon. Assembly line of surfactin biosynthesis with condensation domains are colored in gray, adenylation domains in red and peptidyl carrier proteins in green. Epimerization domains are shown in blue, the thioesterase domain in orange ....................................................................................................... 103 Figure 16. Surfactin biosynthesis by the Modular Peptide Synthetase. (A) The srf operon (top) presente three genes srfA-A, srfA-B, and srfA-C, which coded for surfactin synthetase subunits, shown below the genes. Bars indicate the module position within the protein, whereas the individual domains are shown as colored balls: A, adenylation domain; PCP, peptidyl carrier protein domain; C, condensation domain; E, epimerization domain; TE, thioesterase domain. 4′- phosphopantetheinyl cofactors with active thiol groups were shown with the corresponding peptides attached at their current synthesis states. The growing peptide chain is passed from left to right, until the linear product at the last PCP domain is cyclized to the lipopeptide by the TE domain. (B) The SNAC (S-N-acetyl cysteamine) acyl peptide can be cyclized by the genetically excised SrfTE domain. The native peptide (R = DLeu) and the soluble substrate (R = DOrn) are illustrated. ....................................................................................................................................... 103 Figure 17. Multidomain organization of the representative gene clusters encoding NRPSs in Bacillus spp. genes encompassing each peptide synthetase operon, including their sizes and organization within the modules. Surfactin, liquenysin, fengycin/plipastatin, mycosubtilin, Iturin A and bacillomycin D are represented ............................................................................................... 105 Figure 18. Overall model of gene regulation involved in surfactin biosynthesis. Closed-head arrows indicate positive regulation whereas closed circles indicate negative regulation. .......................... 109 Figure 19. Application sector and exploited properties of surfactin ............................................... 113 Chapter 2: Diversity, differentiation by high-throughput methods and biosafety of B. pumilus group species ................................................................................................................ 165 2.1. Phylogenetic and clonality analysis of Bacillus pumilus isolates uncovered a highly heterogeneous population of different closely related species and clones .......................... 167 Figure 1. Neighbour-joining (NJ) tree based on the 16S rRNA gene sequences comparison, showing the relationship of B. pumilus group members, namely B. safensis, B. pumilus and B. altitudinis. Genetic distances were constructed using Kimura´s 2-parameter model. Numbers at branch points indicated bootstrap percentages (1000 replications) from NJ analysis and only values greater than 70 % were shown. Bacillus subtilis subsp. subtilis str. 168, B. amyloliquefaciens DSM7T, B. licheniformis ATCC 14580T and B. cereus ATCC 14579T were used as outgroups. Accession numbers were given in parentheses. Symbols represent: B. pumilus from terrestrial () xxiii and marine () settings; B. safensis from terrestrial () and marine () settings and B. altitudinis from terrestrial () and marine () settings. Bar: genetic distance of 0.005. ................................ 188 Figure 2. Neighbour-joining (NJ) tree based on the gyrB gene sequences comparison, showing the relationship of B. pumilus group members, namely B. safensis, B. pumilus and B. altitudinis. Genetic distances were constructed using Kimura´s 2-parameter model. Numbers at branch points indicated bootstrap percentages (1000 replications) from NJ analysis and only values greater than 70 % were shown. Bacillus subtilis subsp. subtilis str. 168, B. amyloliquefaciens DSM7T, B. licheniformis ATCC 14580T and B. cereus ATCC 14579T were used as outgroups. DSM7T, B. licheniformis ATCC 14580T and B. cereus ATCC 14579T were used as outgroups. Accession numbers were given in parentheses. Symbols represent: B. pumilus from terrestrial () and marine () settings; B. safensis from terrestrial () and marine () settings and B. altitudinis from terrestrial () and marine () settings. Bar: genetic distance of 0.05 ............................................ 190 Figure 3. Neighbour-joining (NJ) tree based on the hypervariable region of rpoB gene sequences comparison, showing the relationship of B. pumilus group members, namely B. safensis, B. pumilus and B. altitudinis. Genetic distances were constructed using Kimura´s 2-parameter model. Numbers at branch points indicated bootstrap percentages (1000 replications) from NJ analysis and only values greater than 70 % were shown. Bacillus subtilis subsp. subtilis str. 168, B. amyloliquefaciens DSM7T, B. licheniformis ATCC 14580T and B. cereus ATCC 14579T were used as outgroups. DSM7T, B. licheniformis ATCC 14580T and B. cereus ATCC 14579T were used as outgroups. Accession numbers were given in parentheses. Symbols represent: B. pumilus from terrestrial () and marine () settings; B. safensis from terrestrial () and marine () settings and B. altitudinis from terrestrial () and marine () settings. Bar: genetic distance of 0.02 ............... 192 Figure 4. a) Interand b) intra-species similarity ranges of 16S rRNA, gyrB and rpoB genes in Bacillus pumilus group members. ................................................................................................... 194 Figure 5. Dendrogram resulting from cluster analysis of PFGE fingerprints of ApaI-digested DNA from typeable B. pumilus (a), B. safensis (b) and B. altitudinis (c) isolates. First and second columns (right) represented the isolate name and PFGE-types, respectively. Similarity values were achieved using UPGMA method. Band lines were automatically generated by InfoQuest software to better elucidate its position in the gel ............................................................................................. 195 Figure S1. Phylogenetic tree inferred from GyrB protein sequences in B. pumilus group members. Genetic distances were constructed using Jones-Taylor-Thornton (JTT) model. Numbers at branch points indicate bootstrap percentages from Neighbor-Joining method analysis and only values greater than 70 % were shown. B. cereus was used as the outgroup taxon. Symbols represent: B. pumilus from terrestrial () and marine () settings; B. safensis from terrestrial () and marine xxiv () settings and B. altitudinis from terrestrial () and marine () settings. Bar: genetic distance of 0.05. ................................................................................................................................................ 203 Figure S2. Phylogenetic tree inferred from RpoB protein sequences (based on hypervariable region of the rpoB gene) in B. pumilus group members. Genetic distances were constructed using JonesTaylor-Thornton (JTT) model. Numbers at branch points indicate bootstrap percentages from Neighbor-Joining method analysis and only values greater than 70 % were shown. B. cereus was used as the outgroup taxon. Symbols represent: B. pumilus from terrestrial () and marine () settings; B. safensis from terrestrial () and marine () settings and B. altitudinis from terrestrial () and marine () settings. Bar: genetic distance of 0.05. ........................................................... 205 2.2. Bacillus invictus sp. nov., isolated from healthy products in Portugal .......................... 207 Figure 1. a) Mass spectra comparison of Bi.FFUP1T and closed related B. safensis FO-36bT and B. pumilus ATCC 7061T generated by MALDI-TOF/MS. Species-specific peaks are indicated by (*). A. u. means arbitrary intensity. b) Score plot corresponding to the first three components of the PCA regression model of isolates Bi.FFUP1T, Bi.FFUP2, Bi.FFUP3, B. safensis FO-36bT, B. pumilus ATCC 7061T and B. pumilus ATCC 14884 analyzed ................................................................................. 223 Figure 2. a) FTIR-ATR spectra of Bacillus invictus sp. nov. Bi.FFUP1T and closely related B. safensis FO-36bT and B. pumilus ATCC 7061T processed with SNV and Savitzky-Golay (9 points filter size, 2nd degree polynomial, 2nd derivative) in the region 1200-900 cm-1. b) Score plot corresponding to the first three components of the PCA regression model of isolates Bi.FFUP1T, Bi.FFUP2, Bi.FFUP3, B. safensis FO-36bT, B. pumilus ATCC 7061T and B. pumilus ATCC 14884 analyzed. ..................... 224 Figure 3. Neighbour-joining (NJ) tree based on the rpoB gene sequences comparison, showing the relationship of strains Bi.FFUP1, Bi.FFUP2 and Bi.FFUP3 with related species. Genetic distances were constructed using Jukes-Cantor model. Numbers at branch points indicated bootstrap percentages from both NJ (before the slash ‘/’) and ML analyses (after the slash ‘/’). As NJ tree was very similar to the ML tree, only the first is shown. B. subtilis subsp. subtilis str. 168, B. amyloliquefaciens DSM7T, B. licheniformis ATCC 14580T and B. cereus ATCC 14579T were used as outgroups. Accession numbers were given in parentheses. Bar: genetic distance of 0.02. ............................. 225 Figure 4. Neighbour-joining (NJ) tree based on the gyrB gene sequences comparison, showing the relationship of strains Bi.FFUP1, Bi.FFUP2 and Bi.FFUP3 with related species. Genetic distances were constructed using Jukes-Cantor model. Numbers at branch points indicated bootstrap percentages from both NJ (before the slash ‘/’) and ML analyses (after the slash ‘/’). As NJ tree was very similar to the ML tree, only the first is shown. B. subtilis subsp. subtilis str. 168, B. amyloliquefaciens DSM7T, B. licheniformis ATCC 14580T and B. cereus ATCC 14579T were used as outgroups. Accession numbers were given in parentheses. Bar: genetic distance of 0.05. ............................. 226 xxv Table1. Phylogenetic assignment and pulsotypes of Bacillus pumilus group isolates from diverse terrestrial sources. ........................................................................................................ 181 Table2. PCR primers sequences and amplification conditions for detection of 16S rRNA, gyrB and rpoB genes ........................................................................................................................ 183 Table3. Gene sequences of 16S rRNA, gyrB and rpoB for Bacillus pumilus group isolates deposited on GenBank and included in the phylogenetic analysis. .......................................... 184 2.2. Bacillus invictus sp. nov., isolated from healthy products in Portugal .................... 207 Table 1. Differential metabolic profiles of strain Bi.FFUP1T and closely related B. pumilus ATCC 7061T, B. safensis FO-036bT, B. xiamenensis HYC-10T and B. altitudinis 41KF2bT. ............... 219 Table 2. Bacillus spp. used in this study, their sources and correspondent GenBank accession numbers. .................................................................................................................................... 220 Table 3. Major fatty acids profiles of novel isolates from Bacillus sp. and their closely related species.. .................................................................................................................................... 221 Table 4. DNA-DNA hybridization relatedness (%±SD) of the Bacillus invictus sp. nov. with reference strains B. pumilus ATCC 14884, B. pumilus ATCC 7061T, B. safensis FO-036bT and B. altitudinis 41KF2bT.. .............................................................................................................. 222 Table S1. Physiological and phenotypic characteristics of B. invictus strains Bi.FFUP1T, Bi.FFUP2 and Bi.FFUP3 (data from this study). . .......................................................................................... 233 Table S2. PCR primers sequences and amplification conditions for detection of 16S rDNA, rpoB and gyrB genes. ........................................................................................................................ 235 2.3. Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF/MS .... 237 Table 1. Origins of Bacillus spp. isolates (n=27) included in this study. ................................... 257 Table 2. Species-specific ion peaks values (average) of B. pumilus and B. safensis isolates. 259 Table 3. Overview of biomarkers tentative assignment of MALDI-TOF/MS mass signals of B. pumilus group species. Protein identity was determined by the TagIdent software and compared with ribosomal subunit proteins developed by Hotta et al [23] described in Materials and Methods section ........................................................................................................................ 260 xxxii Table 4. Candidate species-specific biomarkers assignments of B. pumilus and B.safensis. 262 2.4. Differentiation of Bacillus pumilus and Bacillus safensis by Fourier transform infrared spectroscopy and chemometry ............................................................................... 267 Table 1. Features of Bacillus pumilus and B. safensis included in this study.. ......................... 282 2.5. Safety profile of Bacillus subtilis complex: antibiotic and virulence features ......... 285 Table 1. Susceptibility profile of Bacillus spp. tested.. ............................................................. 290 Table S1. Bacillus subtilis complex isolates included in this study. ........................................ 299 Table S2. Primers and amplification conditions used for detection of enterotoxin and resistance genes ........................................................................................................................................ 301 Chapter 3. Characterization of the antibacterial compound produced by Bacillus safensis Bs1, the medicine-slug isolate.. ............................................................................................. 305 3.1. Characterization of the antibacterial compound produced by Bacillus safensis Bs1, the medicine-slug isolate ...................................................................................................... 307 Table 1. Strains used in antimicrobial activity assays... ............................................................ 326 Table 2. Prediction of antimicrobial regions accordingly CAMP database... ............................ 327 Table S1. Comparison resulting sequences producing significant alignment and blast results from homologous sequences using BLAST tool inside the CAMP database... ....................... 345. Chapter 4. Biosurfactants in B. pumilus group species: diversity and anti-biofilm activity of a surfactin-like biosurfactant ............................................................................................. 351 4.1. Species-specific surfactin-like biosurfactants combination within Bacillus pumilus group ....................................................................................................................................... 351 Table 1. Primers sequences and amplification conditions for screening the main families of lipopeptides biosynthetic genes in Bacillus spp.. ...................................................................... 373 Table2. Surface tension and emulsification properties of Bacillus spp. studied... .................... 374 xxxiii Table 3. PCR amplification results for main families of lipopeptides in B. pumilus, B. safensis, B. altitudinis and B. subtilis isolates.... ........................................................................................... 376 Table 4. LC/ESI-MS/MS analysis of pumilacidin, surfactin and fengycin homologues produced by B. safensis, B. pumilus and B. subtilis isolates.... ................................................................ 378 4.2 Linezolid resistant ST2/CC5 Staphylococcus epidermidis – biofilm producer ......... 389 Table 1. Epidemiological features and antibiotic resistance characterization of linezolid resistant Staphylococcus epidermidis isolates recovered in a Portuguese hospital... ............................ 397 4.3. Anti-biofilm activity in Staphylococcus epidermidis and structural characterization of Bacillus safensis biosurfactant ............................................................................................ 399 Table 1. Biofilm producing Staphylococccus epidermidis. ....................................................... 421 Table 2. LC/ESI-MS/MS analysis of pumilacidin and surfactin homologues produced by Bacillus safensis Bs1 strain .................................................................................................................... 422 Annexes .................................................................................................................................... 437 Table A1. Bacillus pumilus group members isolates (n=41) (B. pumilus, B. safensis and B. altitudinis) characterized in this thesis and correspondent assigned GenBank accession numbers.... ................................................................................................................................. 439 Table A2. Control, reference and type strains used in this thesis.... ........................................ 441 Table A3. Oligonucleotides used for the characterization of Bacillus isolates. .... ................... 443 xxxiv List of Abbreviations ACN Acetonitrile AMP Antimicrobial peptide AMPA Antimicrobial Sequence Scanning System ANN Artificial Neural Network APPA Amino acid (Z)-l-2-Amino-5-Phosphono-3-Pentenoic Acid ARDB Antibiotic resistance genes database aroE Shikimate dehydrogenase gene ATCC American Type Culture Collection ATP Adenosine triphosphate BLIS Bacteriocin-like inhibitory substance CAMP Collection of Anti-Microbial Peptides database CBD Calgary biofilm device CC Complex Type CCUG Culture Collection of University of Göteborg CD Circular Dichroism CFS Cell-Free Supernatant CFU Colony Forming Unit CHCA α-cyano-4-hydroxycinnamic acid CLSI Clinical and Laboratory Standards Institute CMC Critical Micelle Concentration CMV Cytomegalovirus CNS Coagulase-negative Staphylococci CytK Cytotoxin K Da Dalton DDH DNA-DNA Hybridization DHB 2,5-dihydroxybenzoic acid DNA Deoxyribonucleic Acid DPG DiphosphatIdylglycerol DSM DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH E24 Emulsification Index EFSA European Food Safety Authority EPS Extracellular Polymeric Substances ESI Electrospray Ionization xxxv ESI-MS EUCAST FA FAME FEEDAP FPLC FTIR-ATR GCg GISA gyrB Hbl HCA HCl HIV IL JTT Kb kDa LAB LB LC/ESI-MS/MS LinR LinS LPBS LZD-R MALDI-TOF/MS MBEC MDR MEL MH MIC MIS MK ML Electrospray Ionization with Tandem Mass Spectrometry European Committee on Antimicrobial Susceptibility Testing Ferulic acid Fatty Acid Methyl Ester Panel on Additives and Products or Substances used in Animal Feed Fast Protein Liquid Chromatography Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance Genomic G+C content Glycopeptide-intermediate-susceptible Staphylococcus aureus β-subunit of DNA gyrase gene Hemolysin BL Hierarchical Clustering Analysis Chloridric Acid Human immunodeficiency virus Interleukin Jones-Taylor-Thornton Kilobase KiloDalton Lactic acid bacteria Luria Bertani-Miller medium Liquid Chromatography – Electrospray Tandem Mass Spectrometry Linezolid Resistant Linezolid-Susceptible Lipopeptides biosurfactants Linezolid-Resistant Staphylococcus epidermidis Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry Minimal Biofilm Eradication Concentration Multidrug Resistant Mannosylerythritol lipid Mueller Hinton agar Minimal Inhibitory Concentration Sherlock Microbial Identification System Menaquinone Maximum-Likelihood xxxvi MLST Multilocus Sequence Typing mN/m millinewton/meter MRSA Methicillin-Resistant Staphylococcus aureus MRSE Methicillin-Resistant Staphylococcus epidermidis MS Mass Spectrometry MS/MS Tandem Mass Spectrometry mutL DNA mismatch repair protein gene MW Molecular Weight NaCl Sodium chloride NASA National Aeronautics and Space Administration Nhe Non-hemolytic enterotoxin NJ Neighbour-Joining NRP Nonribosomal peptide NRPS Non-ribosomal peptide synthetase NTD 3,3´-neotrehalosadiamine OD Optical Density ORF Open reading frame PAS Periodic Acid-Schiff PBP Penicilin-Binding-Proteins PBS Phosphate buffer saline PC Principal Component PCA Principal Component Analysis PCP Peptidyl carrier protein PCR Polymerase chain reaction PFGE Pulsed-Field Gel Electrophoresis PFGE-types Pulsed-Field Gel Electrophoresis-types PG Phosphatidylglycerol PGPR Plant Growth-Promoters PK Polyketides PKS Polyketide synthetase PLSDA Partial Least Square Discriminant Analysis PMF Peptide Mass Fingerprinting PRSP Penicillin-resistant Streptococcus pneumoniae PVDF Polyvinylidene Fluoride Membrane pycA Pyruvate carboxylase A gene pyre Orotate phosphoribosyltransferase gene QPS Qualified Presumption of Safety xxxvii RF Random Forests RNA Ribonucleic acid RP-HPLC Reverse-phase high-performance liquid chromatography rpoB β-subunit of RNA polymerase gene Rt Retention Time rRNA Ribosomal RNA SA Sinapinic acid SAR Systemic Acquired Resistance SASP Small Acid-Soluble Spore Protein SCAN Scientific Committee on Animal Nutrition SCCmec Staphylococcal Cassette Chromosome mec SD Standard Deviation SDS-PAGE Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis SNAC S-N-acetyl cysteamine ST Surface Tension Ste1 Methicillinand Linezolid-Resistant Staphylococcus epidermidis SVM Support Vector Machine TEII Type II thioesterases TFA Trifluoroacetic Acid trpB Tryptophan synthase gene TSA Trypticase Soy Agar TSB Trypticase Soy Broth UPGMA Unweighted-Pair Group Method using Average linkages US FDA United States Food and Drug Administration VRE Vancomycin-Resistant Enterococci γ-PGA Poly-γ-Glutamic Acid xxxviii Research Aims The intensive use and misuse of antibiotics have generated a strong selective pressure for the emergence of multi-drug resistant bacteria, which stimulates efforts to identify and develop new antibacterial compounds from natural sources or alternative anti-infectious strategies During several decades, in some regions of Portugal, a terrestrial black slug (Arion ater) was used as a traditional medicine in the treatment of cutaneous infections, including wounds and abscesses. Previous work demonstrated a strong in vitro anti-staphylococci activity of this slug throughout the contribution of bioproducts produced by an isolate identified as Bacillus pumilus. Despite previous reports on the production of a molecule with a putative anti-staphylococci activity by B. pumilus, no known application has been recognized. Moreover, the recently disclosed diversity within this species suggests a neglected potential of bioproducts produced by species from the B. pumilus group. In the present work, and due to the difficulty to accurately distinguish B. pumilus and its closely related counterparts, we deeply characterized an extended collection of isolates previously identified as B. pumilus. Our hypothesis consists on that the medicine-slug isolate corresponds to an Arion ater adapted clonal lineage that produces antimicrobial compound(s) with anti-staphylococci activity, which might be unusual among B. pumilus group strains. These antimicrobial properties confer to the medicine-slug isolate or to its anti-stapphylococci compound(s) a pharmaceutical or biotechnological interest. The global objective of this proposal is to assess the singularity of the medicine-slug isolate and to characterize its antimicrobial compound(s). xxxix The specific objectives are: •To position phylogenetically the medicine-slug isolate, evaluating its niche-clonal specificity. •To investigate the potential of high-throughput approaches such as MatrixAssisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDITOF) and Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance (FTIR-ATR), coupled with chemometric tools, to discriminate within B. pumilus group species. •To investigate, in the slug-medicine isolate and closely related species, the presence of safety features (antibiotic resistance and virulence) required for their application in humans and agriculture. •To characterize the antibacterial compound(s) produced by the medicine-slug isolate. •To evaluate the diversity and anti-adhesive or anti-biofilm properties of biosurfactant compounds produced in slug-medicine isolate and its closely related species, and to provide biochemical characterization of these surface-active compounds. xl Outline I NTRODUCTION C HAPTER 1 I. Bacillus spp.: phylogeny, antimicrobial resistance and virulence II. Antimicrobial peptides of Bacillus spp. G ENERAL C ONCLUSIONS C HAPTER 5 C HAPTER 2 C HAPTER 3 R ESEARCH W ORKS 2.1. Phylogenetic and clonality analysis of Bacillus pumilus isolates uncovered a highly heterogeneous population of different closely related species and clones 2.2. Bacillus invictus sp. nov., isolated from medicinal products in Portugal 2.3. Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF/MS 2.4. Differentiation of Bacillus pumilus and Bacillus safensis by Fourier transform infrared spectroscopy and chemometry 2.5. Safety profile of Bacillus subtilis complex: antibiotic and virulence features 4.1. Species-specific surfactin-like biosurfactants combination within Bacillus pumilus group 4.2. Linezolid resistant ST2/CC5 Staphylococcus epidermidis –biofilm producer 4.3. Anti-biofilm activity in Staphylococcus epidermidis and structural characterization of Bacillus safensis biosurfactant 3.1. Characterization of a new antimicrobial peptide with anti-MRSA activity produced by Bacillus safensis Bs1 Diversity, differentiation by high-throughput methods and biosafety of B. pumilus group species Biosurfactants in B. pumilus group species: diversity and anti-biofilm activity of a surfactin-like biosurfactant Characterization of the antibacterial compound produced by Bacillus safensis Bs1, the medicine-slug isolate III. Biosurfactants of Bacillus spp. C HAPTER 4 xli Chapter 1 I. Bacillus spp.: phylogeny, antimicrobial resistance and virulence 1. Bacillus spp. Members of the genus Bacillus (Kingdom Bacteria; Phylum Firmicutes; Class Bacilli; Order Bacillales; Family Bacillaceae) include Gram-positive, spore-forming, rod-shaped, aerobic or facultative anaerobic bacteria, which are phenotypically and genotypically heterogeneous (Earl et al., 2008; Satomi et al., 2006). This polyphyletic genus was discovered by Cohn in 1872 (Cohn, 1872), includes currently near two hundred species (Parte, 2013), and has been subjected to frequent restructuration’s with the continuous recognition of new species (Lai et al., 2014; Liu et al., 2013; You et al., 2013; Shivaji et al., 2006). Phylogenetically, Ash et al. (1991) clustered Bacillus species into five RNA groups (RNA group 1, RNA group 2, RNA group 3, RNA group 4 and RNA group 5), and more recently, Nielsen et al (1995) reported the presence of an additional RNA group (RNA group 6) for alkaliphilic and alkalitolerant species (Nielsen et al., 1995). For the purpose of this thesis, only RNA group 1, where Bacillus pumilus group species are included, will be detailed. The phylogenetic affiliation of Bacillus spp. and other related This chapter contains the state of the art on the phylogeny, virulence and antibiotic resistance aspects of interest to Bacillus pumilus group, the subject of this thesis. Its general properties and relevance are reviewed, with special emphasis on their diversity, peptides with antimicrobial activity and biosurfactants. Furthermore, important human and environmental safety features will be discussed. 49 Chapter 1 Gram positive representative species, based on 16S rRNA gene is shown in Figure 1 (Felsenstein, 1993). Figure 1. Schematic outline of the phylogenetic relationships between aerobic representatives of Gram-positive bacteria based on 16S rRNA gene sequences. Areas of the triangles represent closeness’s of the number of species included in the taxa covered by the triangle. The circle indicates the uncertainty of the order at which the lineages diverge from each other. Adapted from (Berkeley et al., 2008). A more detailed phylogenetic analysis within Bacillus members, based upon the neighborjoining method is illustrated in Figures 2 and 3. 50 Chapter 1 Figure 2. Detailed neighbour-joining tree of Bacillus species of RNA groups 1, 2 and 5. The dotted area indicates the uncertainty of the order at which the lineages diverge from each other. The area was chosen somewhat arbitrarily and cover more recent branching points. The bar indicates 10% nucleotide substitutions. B, Bacillus; T, type strain. (Adapted from: Berkeley et al., 2008). 51 Chapter 1 Figure 3. Detailed neighbor-joining tree of Bacillus species members of RNA groups 3, 4 and 6, other Bacillus species, and lineages containing reclassified Bacillus species. The dotted areas indicate the uncertainty of the order at which the lineages diverge from each other. The areas are chosen somewhat arbitrarily and may more recent branching points. The bar indicates 10% nucleotide substitutions. Gra, Gracilibacillus; Amb, Amphibacillus; B, Bacillus; Bv, Brevibacillus; Ex, Exiguobacterium; Hb, Halobacillus; Pb, Paenibacillus; Sb, Salibacillus; T, Type strain; Vg, Virgibacillus. (Adapted from: Berkeley et al., 2008). 52 Chapter 1 1.1. Bacillus RNA group 1 This group constitutes the core of Bacillus spp. and displays the name of one of the first organisms of which the complete genome was published (Kunst et al., 1997) - Bacillus subtilis (Figure 2). This group is highly heterogeneous, encompassing several consistent clusters and single-species lineages, including those of the Bacillus pumilus group. One of the subgroups that embraces Bacillus RNA group I encloses B. subtilis relatives, which actually encompasses 19 species including, Bacillus subtilis subs. subtilis, B. vallismortis, B. mojavensis, B. amyloliquefaciens, B. atrophaeus, B. licheniformis, B. pumilus, presented in Figure 2, whereas the species most recently discovered B. subtilis subsp. inaquosorum, B. subtilis subs. spizizenni, B. tequilensis, B. sonorensis, B.axarquiensis, B. malacitensis, B. velezensis, B. safensis, B. aerophilus, B. stratosphericus, B. altitudinis and B. xiamenensis are not (Jeyaram et al., 2011; Earl et al., 2008) A second subgroup encompasses B. cohnii, B. horikoshii and B. halmapalus. A third one harbors B. cereus, B. pseudomycoides, B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides and a novel thermotolerant species, B. cytotoxicus (Guinebretière et al., 2013), which can be compiled under the term B. cereus sensu lato or B. cereus group (Ehling-Schulz & Messelhaeusser, 2013; Kolsto et al., 2009). A fourth subgroup embraces B. simplex and B. psychrosaccharolyticus. Based on the reported characteristics, RNA group 1 can also group other bacterial genera as Virgibacillus, Salibacillus, Halobacillus and Gracilibacillus (Figure 3). Phenotypically, all these Bacillus species produce acids from a wide range of sugars and also present oval endospores, which are generally located centrally/subterminally (Leuschner, 2003). Moreover, some species such as B. cereus and B. liqueniformis are facultative anaerobes. 2. Bacillus pumilus group 2.1. General features Bacillus pumilus group, encompasses B. pumilus, the first species described, and the closely related species B. safensis, B. aerophilus, B. stratosphericus, B. altitudinis and B. xiamenensis (Lai et al., 2014; Liu et al., 2013; Satomi et al., 2006; Shivaji et al., 2006). These bacteria are readily isolated from diverse natural envirnments such as soil and 53 Chapter 1 marine settings, plants and air of high altitudes (Lai et al., 2014; Liu et al., 2013; Freitas et al., 2008; Satomi et al., 2006; Shivaji et al., 2006). General morphologic characteristics of these species include Gram-positive, rod-shaped, aerobic growth and the presence of spore located terminally or sub-terminally (Berkeley et al., 2008). This group has a high economic relevance, derived from the wide range of applications of these microorganisms or their products into biotechnological, environmental and biopharmaceutical purposes (Pérez-García et al., 2011; Aunpad & Na-Bangchang, 2007; Hong et al., 2005; Sanders et al., 2003; Lehman et al., 2001), which will be further detailed in sub-section 4. Although rarely, B. pumilus group species have also been associated with food poisonings and human infections, mainly in immunocompromised patients, including anthrax-like cutaneous lesions (Johnson et al., 2008; Bentur et al., 2007; From et al., 2007a; Tena et al., 2007; Haymore et al., 2006; Ozkocaman et al., 2006; Castagnola et al., 2001) (see sub-section 3, for more detailed information) The B. pumilus species, firstly designated in 1901 (Gottheil, 1901) has been the most extensively described member of this group (Boone et al., 2005), being described from soils, plants, foods, health products, cosmetics, clean room environments, water and even animals (Liu et al., 2013; Branquinho et al., 2012; Ouoba et al., 2004; Wei et al., 1996). Nevertheless, only one genome is completely sequenced (B. pumilus SAFR-032 strain, isolated from the Spacecraft Assembly Facility at National Aeronautics and Space Administration (NASA)), comprising 3.7 Mb and coding for 3679 proteins. A large range of applications has been attributed to B. pumilus, mainly related with the production of peptides with antimicrobial activity or its use as animal and human probiotics (EFSA, 2011; Hong et al., 2005; Sanders et al., 2003), phytosanitary products (Pérez-García et al., 2011) or plant growth promoters (Joo et al., 2005). In addition, although infrequently, it has also been reported their involvement in human and animal diseases (Kimouli M et al., 2012; Johnson et al., 2008; Bentur et al., 2007; From et al., 2007a; Callegan et al., 2006; Haymore et al., 2006; Ozkocaman et al., 2006; Galanos et al., 2003; Castagnola et al., 2001; Peltola et al., 2001; Turnbull, 1997). B. safensis was initially reported by NASA as one of the major contaminants in spacecraft, associated with contamination of clean room assembly-facility surfaces due to its highly resistance to gamma and UV radiation (Satomi et al., 2006). More recently 54 Chapter 1 recently it was identified in African oil and locust bean seeds for the production of fermented food condiments (Agbobatinkpo et al., 2013; Ahaotu et al., 2013), plant rhizosphere and from marine environments (Liu et al., 2013). Nevertheless, only one partially sequenced genome (3.68 Mb) of B. safensis VK strain, isolated from the rhizosphere of a cumin plant growing in the saline desert of India is available (Kothari et al., 2013). B. altitudinis, primarily isolated from cryogenic tubes used for collecting air samples from high altitudes (Shivaji et al., 2006), seems to be the most common species from this group in marine environments (Liu et al 2013). No information of complete genome sequences is available. B. stratosphericus and B. aerophilus were originally isolated from air samples (Shivaji et al., 2006). Soils and estuarine sediments were also reported as habitats for B. stratosphericus (Zhang et al., 2012; Yadav et al., 2011). Nevertheless, the type strai of these species are no longer available in public collections. Finally, B. xiamensis was recently identified (Lai et al., 2014) in the intestinal tract contents of a flathead mullet, Mugil cephalus, captured from the sea off Xiamen Island, China. One draft genome of strain HYC-10, containing 3.6 Mb and comprising 3687 protein-coding genes is available. 2.2. Evolution and phylogenetic boundaries Many methods have been applied to identify and discriminate among closed related species, as B. pumilus group members. Beyond the classical morphological, physiological and biochemical characteristics, phylogenetic analysis based on single or multilocus sequence typing (MLST) of housekeeping genes, have been frequently used to depict phylogenetic affiliations among members of Bacillus genus (Helgason et al., 2000). 2.2.1 Phenotypic based phylogeny Members of the B. pumilus group present very similar phenotypic characteristics, which hinders its identification merely based on biochemical and/or morphological properties. Nevertheless, some distinct aspects related with carbohydrates and amino acid metabolization profiles can be assigned, as summarized in Table 1. 55 Chapter 1 Table 1. Biochemical and physiological characteristics of B. pumilus group species. B. pumilus ATCC 7061T B. safensis FO036bT B. xiamenensis HYC-10T B. altitudinis 41KF2bT Temperature range (optimum) (ºC) 30 30 30–37 30–37 NaCl range (optimum) (%, w/v) 1–3 1–3 1–3 1–3 pH range (optimum) 6–8 6–8 6–8 6–8 Carbohydrates acid production profile Cellobiose - + + + D-Arabinose + + - + Erythritol + + - - Glucose - + - - Inositol + - - - Maltose + - - + Mannitol + + + + Mannose + - + + Raffinose + - + - Rhamnose - - + + Sorbitol - - + + Starch - - + + Amino acid utilization L-Arginine - - - + 2.2.2 Genotypic based phylogeny B. pumilus group species present a remarkable high level of 16S rRNA gene similarity (>99%) (Liu et al., 2013; Satomi et al., 2006). In fact, limitations of 16S rDNA sequences to decipher relationships at species level have been recognized as a result of its conserved nature for different species (Adékambi et al., 2008; Wang et al., 2007; Konstantinidis & Tiedje, 2005; ). In fact, strains belonging to B. subtilis group showing ≥99% 16S rDNA sequences similarity, may not belong to the same species. A phylogenetic tree based on the 16S rRNA genes of marine bacteria belonging to the B. pumilus group is presented in Figure 4. 56 Chapter 1 Despite the high similarity observed for 16S rDNA sequences between the isolates analized, B. pumilus members from marine settings are divided into two groups: i) the B. altitudinis and B. xiamenensis and ii) B. pumilus and B. safensis (Liu et al., 2013). Figure 4. Phylogenetic affiliation based on 16S rRNA genes of B. pumilus group members recovered from marine sources. The tree was constructed using the neighbor-joining method. Bootstrap values over 50% (1000 replications) were shown at each node. Bar, % estimated substitution. B. cereus ATCC 14579T was used as outgroup. (Adapted from: Liu et al., 2013). 57 Chapter 1 Some Bacillus spp. are associated with opportunistic infections, e.g. in traumatic or postsurgical wounds, cancer patients or immunocompromised individuals (Wiedmann & Zhang, 2011). Infections of eyes, respiratory tract, central nervous system, septicemia, cases of fulminant necrotizing soft tissue and even endocarditis, have also been reported (Økstad & Kolstø, 2011; Bottone, 2010). In particular, species belonging to B. pumilus group have also occasionally been involved in human diseases (Kimouli et al., 2012; Johnson et al., 2008; Bentur et al., 2007; From et al., 2007a; Callegan et al., 2006; Haymore et al., 2006; Ozkocaman et al., 2006; Castagnola et al., 2001; Galanos et al., 2003; Peltola et al., 2001; Turnbull, 1997). In the hospital setting, B. pumilus group have been reported as causal agents of opportunistic infections mainly in chronically ill and immunosuppressed patients (Bentur et al., 2007; Haymore et al., 2006; Ozkocaman et al., 2006; Castagnola et al., 2001), in most cases associated with contaminated medicines, antiseptics and medical equipment, such as intravenous catheters or other medical devices (Bentur et al., 2007). 3.2. Antimicrobial Susceptibility of Bacillus spp. The development of antibiotic resistance constitutes a serious concern, affecting a wide range of bacteria. Resistance to antimicrobial agents can be due to either (i) intrinsic properties (sometimes called ‘natural resistance’), characteristic of all the strains of a certain species or by (ii) mutation of indigenous genes or the acquisition of resistance genes through mobile genetic elements, such as plasmids or transposons (Commission, 2002). Such intrinsic or acquired properties could make the bacteria capable of rapid inactivation of specific antibiotics through degradation, exportation of the antibiotics out of the cell, through the efflux system, or by alteration of the antibiotic target site (Butaye et al., 2003; Commission, 2002; Roberts et al., 1999). The presence of genes coding for antimicrobial resistance carried by mobile genetic elements, presents a greatest risk for horizontal dissemination of antimicrobial resistance between isolates from the same or even from different species (Devirgiliis et al., 2011; Reenen & Dicks, 2011). According to the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), all bacterial products intended for use as feed additives must be examined to establish its susceptibility profile to a relevant range of antimicrobials of human and veterinary importance, e.g. ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol (EFSA, 2012). Specifically in 64 Chapter 1 the case of Bacillus spp., EFSA determined as a basic requirement the determination of minimum inhibitory concentrations (MIC) and its interpretation according to microbiological cut-off values defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, http://www.eucast.org/) and the European monitoring program, as shown in Table 2. Table 2. Bacillus spp microbiological cut-off values (μg/mL) for different antimicrobials according to FEEDAP panel (EFSA, 2012). Antimicrobials Microbiological cut-off value (μg/mL) Vancomycin 4 Gentamicin 4 Kanamicin 8 Streptomycin 8 Erytromycin 4 Clindamycin 4 Tetracycline 8 Chloramphenicol 8 According to these guidelines, a bacteria presenting a MIC value higher than the cut-off value for a given antibiotic has an acquired resistance mechanism (EFSA, 2012), and according to the FEEDAP Panel when observed for one or more antimicrobials, it is recommended to explore the nature of resistance. The growing health concern about antimicrobial susceptibility profiles in Bacillus spp. is mainly due to the possible source of transfer of antibiotic resistance genes between bacteria (Gevers et al., 2003; Witte, 2000). Antibiotic resistance genes database (ARDB) (http://ardb.cbcb.umd.edu/) provides a centralized compendium of information on antibiotic resistance, providing a list of antibiotic resistance gene sequences identified in Bacillus spp. (Table 3). 65 Chapter 1 Table 3. Antibiotic resistance genes and antibiotic resistance profiles identified in Bacillus spp. members enclosed in ARDB database. Species Antibiotic resistance gene Antibiotic Resistance Profile Acession number B. anthracis str. sterne bacA Bacitracina YP_027565 bl2A_1 Penicillinb YP_029502 fosB Fosfomycinc YP_030068 B. cereus str. E33L bacA Bacitracin YP_082869 AAU18978 Q63DZ3 fosB Fosfomycin Q63CC5 YP_083442 AAU18406 B. cereus str. Q1 bacA Bacitracin YP_002529178 bl2A Penicillin YP_002530963 fosB Fosfomycin YP_002529744 B. cereus str. B4264 bacA Bacitracin YP_002366165 bl2A Penicillin YP_002368151 YP_002367214 fosB Fosfomycin YP_002366749 B. thuringiensis bl2A Penicillin Q45726 B. thuringiensis serovar konkukian str. 97-27 bacA Bacitracin AT61949 YP_035603 B. subtilis subsp. subtilis str. 168 bl2A Penicillin AAT60435 YP_037541 fosB Fosfomycin YP_036189 AAT59669 bacA Bacitracin ZP_03592903 NP_390993 Blt Chloramphenicol, doxor ubicin,fluoroquinolone, puromycind NP_390536 AAC36944 ZP_03592438 Bmr Chloramphenicol, fluoroquinolone AAB81539 ZP_03592168 NP_390281 fosB Fosfomycin ZP_03591517 NP_389667 lmrB Lincomycine NP_388149 ZP_03589930 tetL Tetracyclinef ZP_03593907 NP_391957 BAA05208 66 Chapter 1 tmrB Tunicamycin NP_388196 ZP_03589973 B. subtilis subsp. subtilis str. NCIB 3610 bacA Bacitracin ZP_03597187 Blt Chloramphenicol, doxor ubicin, fluoroquinolone, puromycin ZP_03596719 Bmr Chloramphenicol, fluoroquinolone ZP_03596450 fosB Fosfomycin ZP_03595797 lmrB Lincomycin ZP_03594212 tetL Tetracycline ZP_03598190 tmrB Tunicamycin ZP_03594255 B. pumilus str. SAFR-032 bacA Bacitracin YP_001487997 B. pumilus catA Chloramphenicol P00487 B. licheniformis bcrA Bacitracin P42332 P42334 bl2A Penicillin P00808 ermD Lincosamide, macrolide, streptogramin Bg Q03986 P45438 B. amyloliquefaciens str. FZB42 bacA Bacitracin YP_001422387 ABS75156 fosB Fosfomycin YP_001420711 ABS73480 lmrB Lincomycin ABS72697 YP_001419928 B. clausii str. KSM-K16 bacA Bacitracin BAD65785 YP_176746 Q5WCX5 B. halodurans str. C-125 fosB Fosfomycin NP_242644 BAB05497 B. circulans aph3iva Butirosin, kanamycin, neomycin, paromomycin, ribostamycin CAA27061 P00553 0910185A vanA teicoplanin, vancomycinh CAB61227 CAB61228 CAB61229 CAB61230 CAB61231 CAB61224 vanZ Teicoplanini CAB61225 aUndecaprenyl pyrophosphate phosphatase, which consists in the sequestration of Undecaprenyl pyrophosphate. 67 Chapter 1 bClass A beta-lactamase. This enzyme breaks the beta-lactam antibiotic ring open and deactivates the molecule's antibacterial properties. cGlutathione transferase, metalloglutathione transferase which confers resistance to fosfomycin by catalyzing the addition of glutathione to fosfomycin. dMajor facilitator superfamily transporter. Multidrug resistance efflux pump. eABC transporter system, Macrolide-Lincosamide-Streptogramin B efflux pump. fMajor facilitator superfamily transporter, tetracycline efflux pump. grRNA adenine N-6-methyltransferase, which can methylate adenine at position 2058 of 23S rRNA, conferring resistance to erythromycin. hVanA type vancomycin resistance operon genes, which can synthesize peptidoglycan with modified Cterminal D-Ala-D-Ala to D-alanine--D-lactate. iVanZ confers low-level VanZ confers low-level resistance to the glycopeptide antibiotic teicoplanin. Moreover, other multidrug resistance genes such as cfr (conferring resistance to five chemically unrelated antimicrobial classes, including phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A) have been identified in Bacillus species isolates, located in some of them in a plasmid carrying other resistance genes (Wang et al., 2012; Zhang et al., 2011; Dai et al., 2010). Therefore, the potential dissemination of these resistance genes among different bacterial species is worrisome and should be under surveillance. 3.3. Virulence potential of Bacillus spp. 3.3.1. B. cereus group The first guidance to access virulence (toxigenic) potential of species of the Bacillus genus was developed by the Scientific Committee on Animal Nutrition (SCAN) and published in 2000 (Commission, 2000). In that document, it is assumed that toxins (enterotoxins) found in Bacillus species other than those of Bacillus cereus group would have sufficiently similar properties to be detected by the same detection methods. After 2000, few incidents of food poisoning were reported in non-pathogenic Bacillus-group, which were mostly related with the presence of heat-stable surfactins and similar cyclic lipopeptides. Examples of toxic peptides produced by Bacillus spp. comprise amylosin (B. amyloliquefaciens) (Mikkola et al., 2007), fengycin and surfactin from (B. subtilis and B. mojavensis) (Hwang et al., 2009; From et al., 2007a) or lichenysin (B. licheniformis) (Nieminen et al., 2007). Toxic effects have been detected only when they are produced in amounts able to cause demonstrable cell disruption, which is far from the typical concentration of its production and use (EFSA, 2013). 68 Chapter 1 Pathogenicity potential is much more characterized for B. cereus group species than in other Bacillus species. The main enterotoxins associated with B. cereus virulence and that should be sought by diagnostic tools are the: i) non-hemolytic enterotoxin (Nhe, threecomponent toxin), ii) hemolysin BL (Hbl, three-component toxin), iii) and cytotoxin K (CytK, single-component toxin of the β-barrel pore-forming toxin family) (Stenfors Arnesen et al., 2008; Lund et al., 2000; Lund & Granum, 1996; Beecher & Macmillan, 1991) and iv) emetic toxin cereulide synthetase (Cereulide, cyclic heat stable depsipeptide) (EhlingSchulz et al., 2006b; Ehling-Schulz et al., 2005b) (Table 4). Table 4. Bacillus spp. toxins responsible for gastro-intestinal disorders. (Adapted from: Stenfors Arnesen et al., 2008). Toxin Gene Nature Gatrointestinal disorders infection/intoxication Nhe nhe Protein, 3 components Diarrheal Hbl hbl Protein, 3 components Diarrheal CytK cytK Protein Diarrheal Cereulide ces Cyclic peptide, 1.2 kDa Emetic The protein nhe is the most commonly found enterotoxin gene complex, is probably ubiquitous in B. cereus group members and exhibits haemolytic activity towards erythrocytes from several mammalian species (Fagerlund et al., 2008) (Stenfors Arnesen et al., 2008). The hbl complex constitutes another pore-forming enterotoxin in the B. cereus group exhibiting also haemolytic activity towards erythrocytes from several animal species. (Wiedmann & Zhang, 2011). The cytK is a 34 kDa single-component protein toxin of the β-barrel pore-forming toxin family with necrotic, haemolytic, and enterotoxic effects (Lund et al., 2000). The emetic toxin (cereulide) is a small (1.2. kDa) non-ribosomally synthesized dodecadepsipeptide, which is heat stable, acid and protease resistant (Stenfors Arnesen et al., 2008). Its expression is affected by factors like oxygen, pH, and temperature and is regulated by the transitional state regulator AbrB (Lücking et al., 2009). Whereas the genes encoding the enterotoxins Nhe, Hbl and CytK are chromosomally encoded, the emetic toxin cereulide is encoded by a 208 kb plasmid, pCER270, with similarity to B. anthracis pXO1 and other pXO1-like plasmids (Rasko et al., 2007; Ehling-Schulz et al., 2006a; Hoton et al., 2005), highlighting the risk for lateral gene transfer between strains. 69 Chapter 1 Several other candidate proteins have been suggested as potential contributors to the enterotoxigenic activity of B. cereus, for which more detailed information can be found in a review from Arnesen et al., (Stenfors Arnesen et al., 2008). In the very few reports evaluating the cytotoxicity and production of putative emetic toxins in species other than those of the B. cereus group recovered from foods, water and food plants, the authors found only 8 out of 333 Bacillus strains tested to be putative toxin producers, particularly among B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis isolates, but none of them produced B. cereus-like toxins (From et al., 2005). Although rare, the identification of toxins in non-B. cereus species from water, food, and food environments highlights the risk for food poisining and the need for further characterization of toxins and genes involved to improve detection methods (From et al., 2005). It is also important to refer that the sole presence or absence of an individual toxin gene does not fully explain the pathogenicity or virulence potential of a certain strain, and consequently molecular methods used should always be accompanied by sensitive and accurate toxin quantification systems (Bauer et al., 2009). 3.3.2. B. pumilus group Although rarely, some evidences of toxic properties of Bacillus pumilus have been reported, namely its cytopathic effects in Vero cells, haemolytic activity, lecithinase production, and proteolytic action on casein (From et al., 2005; Hoult & Tuxford, 1991). In addition, pumilacidin from B. pumilus was also reported as presenting toxic properties (From et al., 2007b) . Moreover, it also produces a toxin that has been detected in guinea pigs with experimentally induced enterocolitis associated with clindamycin (Knoop, 1979). 4.Biotechnological and industrial applications of Bacillus pumilus group members Besides the high diversity characterizing Bacillus species at the taxonomic level, the diversity of their metabolic features is also noticeable (Novak et al., 2012; Gaggìa et al., 2010; Schallmey et al., 2004). Beyond their ability to produce a wide range of metabolites with very different natures and structures, they also display broad spectrum of activities, 70 Chapter 1 including enzymes with great interest in detergent industry and food sectors; production of primary metabolites as vitamins (Schallmey et al., 2004) and ribonucleosides (Srivastava et al., 2012) and secondary metabolites comprising antibiotics, insecticides, biosurfactants agents and also growth promoting formulations (Lehman et al., 2013; Martinotti et al., 2013; Mulligan et al., 2014; Fracchia et al., 2012; Lehman et al., 2001; Schallmey et al., 2004), originally designed to enable the bacterium to survive in its natural environment (Stein, 2005). The world market for industrial enzymes is estimated to be 1.6 billion $US, divided between food enzymes (29%), feed enzymes (15%), and general technical enzymes (56%), for which is estimated that enzymes from Bacillus spp. constitute about 50% of the total of enzyme market (Berkeley et al., 2008). B. pumilus is involved in the alkaline serine proteases (subtilisins) production, with a reported primary application in household detergents, as Alcalase (Novo Nordisk) (Schallmey et al., 2004; Chen et al., 1995). Among ribonucleosides, D-Ribose is frequently used as a flavor enhancer in food, pharmaceuticals, cosmetics, health food, and animal feed, as well as for the treatment of myocardiac ischemia and muscular pain, for which some B. pumilus are involved in its production (Srivastava et al., 2012). In addition, poly-γ-glutamic acid (γ-PGA) is a water-soluble, eatable, and biodegradable compound with industrial applications in the food, cosmetics, and in medical fields and also in wastewater treatment as thickener, hemectant, cryoprotectant, drug carrier, highly water adsorbant hydrogel, biopolymer flocculent, heavy metal absorber, and animal feed additive (Shih & Van, 2001). Several Bacillus species, including B. pumilus, have been reported to produce γ-PGA, however, B. licheniformis and B. subtilis are the most widely studied species (Bhat et al., 2013). The biosynthesis of several antibiotics and biosurfactant compounds is also noticeable from species belonging to Bacillus genus and B. pumilus group members in particular. Detailed information regarding theses aspects are given in Section III from this chapter. Probiotic-containing products with Bacillus species are available for human nutrition, as animal feed supplements, and also for aquaculture (Hong et al., 2005; Rolfe, 2000; Verschuere et al., 2000), with potential attributes as colonization, immunostimulation and antimicrobial activity. Originally, many commercial products were sold as products that carry B. subtilis spores, but recent studies have shown that most products are mislabeled and carry other Bacillus species, including B. pumilus (Green et al., 1999; Hoa et al., 71 Chapter 1 2000). In addition, other B. pumilus species were reported as antidiarrhoeal prophylactic agents by its probiotic activity (Sanders et al., 2003; Mazza, 1994). Moreover, it is generally known that several microorganisms exhibit biological activity useful to control plant diseases. Indeed, B. pumilus is not considered an anti-insect pathogen like other members of the genus Bacillus, such as B. thuringiensis or B. sphaericus (Phelps & McKillip, 2002; Schirmer et al., 2002). Nevertheless, some reports identified this species as a biopesticide, with previous studies showing its activity against a broad range of phytopathogenic fungi (Lehman et al., 2013) and as an inducer of systemic acquired resistance (SAR) in plants (EFSA, 2013). Regarding other species belonging to B. pumilus group, no evidences were found concerning its application for industrial or biotechnological purposes. Nevertheless, since appropriated identification methods of members of this group is a critical and still controversial step, which is being suffering considerable changes, it is unknown to which extent species form B. pumilus group are clearly discriminated in many of the available studies. Appropriated delineation of species and/or of clonal lineages adapted to particular niches will thus be of relevance to open new perspectives for biotechnological applications of B. pumilus group species. 72 Chapter 1 II.Antimicrobial peptides of Bacillus spp. Almost 90 years elapsed since Fleming discovered lysozyme, the first natural antimicrobial. Since then, more than 1200 types of antimicrobial peptides (AMPs) have been isolated from bacteria, animals, insects, and plants, and their use had a great impact in human health (Nakatsuji & Gallo, 2012) The intensive and inappropriate use of antibiotic compounds has generated a strong selective pressure for the emergence of multi-drug resistant pathogens leading to a high percentage of morbidity and mortality rates (WHO, 2012), e.g. multidrug-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. (VRE) (CDC, 2013). Moreover, nearly all of the antibiotics used today belong to classes discovered between 1941 and 1968 and it is particularly problematic that only two novel structural classes have become available on the market in the last 40 years: oxozolidiones and cyclic lipopetides (Calza et al., 2004; Jacqueline et al., 2004; Wagenlehner & Naber, 2004). More than ever, efforts are urgently needed to identify and develop new antibacterial compounds, more effective and with novel modes of action (Mills et al., 2011; Barsby et al., 2001). In this sense, the screening of microbial natural products represents an important route to the discovery of novel chemicals, for development of new therapeutic agents and for evaluation of the potential of lesser-known and/or new bacterial taxa (Lazzarini et al., 2000). Therefore, exploration for new compounds from well-known and proficient microorganisms, such as Bacillus, due to the fact that only a small range of their biosynthetic capacity is currently exploited, has been conducted. In fact, recent advances in genome sequencing have highlighted the genus Bacillus as a source of antibiotic-like compounds resulting from their secondary pathways (Fickers, 2012). 5. Antimicrobial peptides (AMPs) types from Bacillus spp. AMPs (AMPs, 2014) are natural products of metabolism that are not essential for normal growth, development or reproduction of an organism, generally comprise 10–50 aminoacid residues and are characterized by cationic amphipathic properties (Seo et al., 2012). In opposition to primary metabolites, these compounds are not ubiquitous in the living 73 Chapter 1 others. Subclass I.1 includes type A lantibiotics with an elongated structure, such as subtilin. Subclass I.2 includes the type B globular lantibiotic mersacidin, and other lantibiotics such as sublancin 168. Subclass I.3 includes the two-component lantibiotics. Subclass I.4 includes the unique cyclic peptide subtilosin A that contains a head-to-tail peptide bond as well as particular sulfide bridges formed between cysteine groups and dehydrated amino acid residues (Abriouel et al., 2011). Moreover, the proposed classification seems to be coherent with clustering obtained from the comparative analysis of published amino acid sequences, as detailed in Figure 6. The subtilin and ericins form a coherent cluster, which is characterized by highly conserved regions in the mature peptide as well as in the leader peptide sequences. Interestingly, the A1 and A2 subunits of haloduracin and lichenicidin also form coherent clusters, suggesting that both two-peptide lantibiotics share a common origin. Mersacidin also shows homology with the A1 subunit of the two-peptide lantibiotics, and especially with the lichenicidin A1 subunit, with 25 conserved residues at identical positions, indicating its relatedness to the two-peptide lantibiotics (Abriouel et al., 2011). Figure 6. Dendrogram showing the relatedness of Bacillus spp. bacteriocins according to primary amino acid sequence homologies. Shaded characters indicate identical amino acid residues. (Adapted from: Abriouel et al., 2011). Bacteriocins of class II include small (0.77–10 kDa), non-modified and linear peptides, which are heat and pH stable (Table 6). This class can be subdivided into four 80 Chapter 1 subclasses. Subclass II.1 includes pediocin-like peptides with a conserved YGNGVXC motif near the N-terminus (Figure 6), and the coagulin produced by B. coagulans I4. Subclass II.2 includes thuricin-like peptides with a conserved DWTXWSXL motif neighboring the N-terminus produced by B. thuringiensis, and cerein MRX1 produced by B. cereus. Comparative amino acid sequence analysis of thuricin-like peptides shows a coherent cluster (Figure 6). Subclass II.3 includes other linear peptides, such as lichenin produced by B. licheniformis, or cereins 7A and 7B. Class III bacteriocins includes large proteins (430 kDa) with phospholipase activity such as megacins A-216 and A-19213 produced by B. megaterium (Table 6). Many other antimicrobial polypeptides of intermediate size (10–30 kDa) and other large antimicrobial proteins produced by bacilli are not included in this classification scheme due to the lack of data on their protein or gene sequences. Moreover, bacteriocin production and secretion seems to be correlated with bacterial stationary phase (Khalil et al., 2009a). Nevertheless, depending on the transcription regulation system, its expression may also be influenced by factors such as carbon sources (Drosinos et al., 2005), cell-density (Riley & Wertz, 2002b), temperature (Diep et al., 1994), or even the presence of a bacteriocin-sensitive strain (Barefoot et al., 1994). Bacteriocins encoding genes are typically located in mobile genetic elements such as plasmids and transposons (Riley & Gillor, 2007) i. In fact, the production of a particular bacteriocin involves the co-expression of other genes that encode proteins associated with immunity and when required, secretion, regulation and biosynthesis (Jack et al., 1995; Nes et al., 1996). Thus, bacteriocins and the associated genes are usually expressed in an operon, but may involve expression of up to four separate operons (Heng et al., 2006). Some of specific aspects of most common bacteriocins produced from Bacillus spp., namely subtilin, ericin, sublancin, mersacidin, subtilosin A, haloduracin and lichenicidin are detailed and compared in Figure 7 a), b) and c). 81 Chapter 1 Figure 7. (a) Comparison of subtilin and ericin structures with that of the lactococcal lantibiotic nisin A. Conserved residues at identical positions to all four bacteriocins are highlighted in green, while those conserved only in subtilin and ericins are depicted in yellow; other conserved residues are in light red. (b) Structures of the single-peptide lantibiotics sublancin, mersacidin and subtilosin A. The structure of the A1 subunit of haloduracin is also included for comparison with mersacidin. The conserved residues are highlighted in orange color. Cysteines involved in disulfide bridge formation are highlighted in blue. For subtilosin A, residues involved in sulfur to a-carbon linkages are shown in green, while residues involved in head-to-tail amide bond formation are in light blue. (c) Comparison of the two-peptide lantibiotics haloduracin and lichenicidin from Bacillus, and lacticin 3147 from Lactococcus lactis. Conserved residues of the A1 peptides are highlighted in orange and light yellow. A conserved Pro residue between lichenicidin A1 subunit and mersacidin is shown in light blue. Conserved residues of the A2 peptides are highlighted in green, deep yellow, light orange and 82 Chapter 1 violet. The C-terminal parts of the A1 subunits also share two conserved loops of 11 and eight amino acid residues. The C-terminal parts of A2 peptides share the same pattern of lanthionine (Ala–S–Ala) and methyllanthionine (Abu–S–Ala) bridges. Non-identical residues are in white. (Adapted from: Abriouel et al., 2011). 83 Chapter 1 Concerning bacteriocins commercially available, unfortunately, only Nisin is currently approved by the US Food and Drug Administration for application as a natural preservative in food. Mersacidin is a compound with remarkably interest. It inhibits the synthesis of S. aureus cell wall, also demonstrating activity against MRSA, with a similar efficiency of vancomycin (Jenssen et al., 2006). Therefore, since its mode of action differs from vancomycin, a possible combination of mersacidin with this antibiotic could be a promising alternative. 5.2. Bacteriocin-like inhibitory substances (BLIS) The term BLIS is often used when the peptide nature of the antimicrobial compound has not been confirmed, whereby ribosomal synthesis is presumed, but may be unknown (Abriouel et al., 2011). Main features of the described BLIS from different Bacillus species are presented in Table 5. Different BLIS with distinct spectra of activity against both Gram-positive and Gramnegative bacteria were described in B. subtilis (Stein et al., 2004; Stein et al., 2002; Stein, 2005; Paik et al., 1998; Bierbaum et al., 1995). Pumilicin was reported in B. pumilus and is a plasmid-encoded peptide (Aunpad & Na-Bangchang, 2007; Lovett et al., 1976). This compound presents a molecular weight of 1994.62 Da, heat stability up to 121 ºC, is active in a pH range of 3–9 and demonstrates remarkable antibacterial activity against MRSA, vancomycin-resistant E. faecalis (VRE) and several Grampositive test bacteria (Aunpad & Na-Bangchang, 2007). 5.3. Unusual peptides Bacillus spp. are able to synthetize other unusual antibiotic peptides, such as rhizocticins and phosphonate oligopeptide which contain C-terminal nonproteinogenic amino acid (Z)- l-2-Amino-5-Phosphono-3-Pentenoic Acid (APPA) (Borisova et al., 2010; Kino, 2010; Kino et al., 2009). These molecules are synthesized by the called L-amino acid ligase, which is able to catalyze the formation of an alpha-peptide bond from L-amino acids in an ATPdependent manner (Tabata et al., 2005). 84 Chapter 1 Rhizocticin A is able to prevent the growth of yeast and filamentous fungi by protein synthesis inhibition but is not active against bacteria (Borisova et al., 2010; Kino et al., 2009; Diddens et al., 1979; Laber et al., 1994). Furthermore, some strains of B. subtilis produce the dipeptide bacilysin composed of L-alanine and the unusual amino acid L-anticapsin (Walker & Abraham, 1970). 5.4. Other antibiotic compounds (miscellaneous) Miscellaneous antibiotics comprise a class of compounds that are structurally different from the remaining AMPs families and from each other and for this reason cannot be classified into the other classes. Examples of representatives agents produced by B. subtilis include bacilysocin 2 (Tamehiro et al., 2002), 3,3´-neotrehalosadiamine (NTD) 3 (Inaoka & Ochi, 2007) and amicoumacin 4 (Pinchuk et al., 2002) (Figure 8). Figure 8. Chemical structures of miscellaneous antibiotics bacilysocin 2, 3,3 ´-neotrehalosadiamine (NTD) 3 and amicoumacin 4. (Adapted from: Sansinenea & Ortiz, 2011). b) 3,3´-Neotrehalosadiamine (NTD) 3 a) Bacilysocin 2 c)Amicoumacin 4 85 Chapter 1 Bacilysocin 2 (Figure 8a) is an antimicrobial phospholipid derived from the major B. subtilis phospholipid phosphatidylglycerol through YtpA-catalysed acyl ester hydrolysis (Tamehiro et al., 2002). NTD 3 (Figure 8b), structurally 3,3´- diamino-3,3´-dideoxy-a,b-trehalose (Tsuno et al., 1986), inhibits S. aureus and Klebsiella pneumoniae and functions as an auto inducer and also as a glucose uptake modulator in B. subtilis by activating its own biosynthetic operon (Inaoka & Ochi, 2007). Amicoumacins 4 (Figure 8c) present antibacterial and anti-inflammatory activities, namely in Helicobacter pylori, which make these compounds attractive for the treatment of chronic gastritis and peptic ulcer in humans (Pinchuk et al., 2002). 5.5. Polyketides and non-ribosomal peptides Polyketides (PKs) are a highly active class of secondary metabolites produced by almost all living organisms, currently used in human and animal health and in agriculture as antibiotic (erythromycin, rifamycin), antifungal, immunosuppressant and antitumor agents (Li et al., 2011; Nakano et al., 2009; Resmi & Soniya, 2012). Structurally, they encompass organic compounds exhibiting remarkable diversity in terms of their structure, which can be divided into three classes, type I, II and III (Chen et al., 2006). Some PKs from B. subtilis and also fungi and plants, are grouped in the third class (Resmi & Soniya, 2012; Li et al., 2011; Nakano et al., 2009). Concerning PKs produced by Bacillus spp., they are synthesized on modularly organized assembly lines by elongation of activated monomers of amino and hydroxyl acid building blocks following the same logic as NRPs (Hertweck et al., 2007). Difficidin, macrolactin and bacillaene are three examples of PKs isolated from B. amyloliquefaciens (Arguelles-Arias et al., 2009; Chen et al., 2007) (Figure 9). 86 Chapter 1 Figure 9. Chemical structures of polyketides antibiotics a) difficidin, b) bacillaene and c) macrolactin (Adapted from: Sansinenea & Ortiz, 2011). Difficidin (Figure 9a) is an unsaturated 22-membered macrocylic polyene lactone phosphate ester with a broad spectrum of antibacterial activity. It inhibits protein biosynthesis and was recently used as a grow suppressive agent against Erwinia amylovara, a devasting plant pathogen causing necrotrophic fire blight disease of apple, pear and other rosaceous plants (Chen et al., 2009b). Bacillaene (Figure 9b) and macrolactin (Figure 9c) are antimicrobial agents that could be potentially useful in human medicine. Both are prokaryotic protein synthesis inhibitors which display antimicrobial activity toward human pathogens such as Serratia marcescens, K. pneumonia and S. aureus (Chen et al., 2009a; Patel et al., 1995). Macrolactin consists of a 24-membered ring lactone which has the ability to inhibit murine melanoma cancer cells as well as mammalian herpes simplex viruses, and also showed effective activity in protecting lymphoblast cells from HIV (Gustafson et al., 1989). Moreover, 17 macrolactins have been described and one of them, 7-O-malonyl macrolactin A, was effective against Gram-positive bacterial pathogens (Romero-Tabarez et al., 2006). c) Macrolactin 12 b) Bacillaene 11 a) Difficidin 10 (Oxydifficidin) 87 Chapter 1 Currently, NRPS comprehend hundreds of molecules, which can be found in Norine database (Caboche et al., 2008). Since the discovery of the linear polypeptide produced by B. brevis named gramicidin (Kapoerchan et al., 2012), in the early 1970’s, (Figure 10a), used primarily as a topical antibiotic against Gram-positive bacteria, many other bioactive NRPs have been isolated and their biosynthetic clusters characterized. A NRPS widely used is Bacitracin (Figure 10b) which is composed by a mixture of related cyclic polypeptides produced by B. licheniformis and related counterparts and is used in human medicine, inhibiting the biosynthesis of the bacterial cell wall by interacting with undecaprenyl pyrophosphate, involved in peptidoglycan synthesis (Stone & Strominger, 1971). This antibiotic was primarily used in topical formulations against Gram positive bacteria namely Streptococcus spp and S. aureus (Awais et al 2008). Recently, 2010, injectable bacitracin was approved by US FDA for the treatment of infants with staphylococcal pneumonia and empyema (Sagent, 2010). Polymyxin B (Figure 10c), a cyclic cationic lipopeptide produced by B. polymyxa, was introduced in 1960 due to the rise of MDR Gram negative bacteria, and is now frequently the last resort for several infections (Kwa et al., 2008). Finally, daptomycin (Figure 10d), a lipopeptide antibiotic, active against Gram-positive bacteria involved in skin and soft tissue infections, is currently the most frequent bactericidal drug on the market and is used as a reserve antibiotic against MDR Gram positive cocci like methicillin-resistant S. aureus (MRSA and vancomycin-resistant enterococci (VRE) (Steenbergen et al., 2005). 5.6. Non-ribosomal peptides (NRPs) 88 Chapter 1 Figure 10. Chemical structures of polypeptide antibiotics a) gramicidin, b) bacitracin, c) polymyxin B and d) daptomycin. Moreover, other interestingly molecules enclosed in this class, namely lipopeptide compounds, will be detailed in section III of this chapter. 89 Chapter 1 Table 7: Primary structure and fatty acid chains of representative LPBSs families produced by Bacillus spp. Name Primary structure of the peptide moiety Main fatty acid chains References Surfactin family Heptapeptide closed by a lactone ring with the β -OH group of the fatty acid chain β-OH fatty acids Surfactin L-Glu-L-XS 2 -D-Leu-L-XS4-L-Asp-D-Leu-L-XS 7 iC 14 , nC 14 iC 15 , aC 15 Peypoux et al., 1999 Lichenysin L-XL1-L-XL2-D-Leu-L-XL4-L-Asp-D-Leu-L-XL7 iC13, aC13, nC13 iC15, aC15 Grangemard et al., 1999; Lin et al., 1994; Bamylocin A Glu-Leu-Met-Leu-Pro-Leu-Leu-Leu C13 Lee et al., 2007 Esperin L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-XE 7 -COOH C 13 , C 14 , C 15 Thomas & Ito, 1969 Iturin family Heptapeptide closed by a lactam ring with the β -NH 2 group of the acid chain β-NH 2 fatty acids Bacillomycin D L-Asn-D-Tyr-D-Asn-L-Pro-L-Glu-D-Ser-L-Thr nC 14 , iC 15 , aC 15 Peypoux et al., 1981 Bacillomycin F L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Thr nC 16 , iC 17 , aC 17 Peypoux et al., 1985 Bacillomycin L or Lc or Bacillopeptin L-Asn-D-Tyr-D-Asn-L-Ser-L-Glu-D-Ser-L-Thr nC14, iC15, aC15 Volpon et al., 2007 Iturin A L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser nC14, iC15, aC15 Peypoux et al., 1978; Isogai et al., 1982 Iturin AL L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser nC16, iC16 Winkelmann et al., 1983 Iturin C L-Asp-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser nC14, iC15, aC15 Peypoux et al., 1986; Volpon et al., 2007 Mycosubtilin L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Ser-L-Asn nC16, iC16, aC17 Peypoux et al., 1986; Duitman et al., 1999 Fengycin family Decapeptide with a lactone ring between carboxy-terminal group of Ile10 and OH group of Tyr3 β-OH fatty acids 96 Chapter 1 Fengycin A L-Glu-D-Orn-D-Tyr-D-a Thr-L-Glu-D-Ala-L-Pro-L-Gln-L-Tyr-L-Ile aC15, iC16, nC16 Volpon et al., 2000; Schneider et al., 1999 Fengycin B L-Glu-D-Orn-D-Tyr-D-a Thr-L-Glu-D-Val-L-Pro-L-Gln-L-Tyr-L-Ile aC15, iC16, nC16, C17 Volpon et al., 2000; Schneider et al.,1999 Plipastatin A L-Glu-D-Orn-D-Tyr-D-a Thr-L-Glu-D-Ala-L-Pro-L-Gln-D-Tyr-L-Ile nC16, aC17 Nishikiori et al., 1986; Volpon et al. 2000 Plipastatin B L-Glu-D-Orn-D-Tyr-D-a Thr-L-Glu-D-Val-L-Pro-L-Gln-L-Tyr-L-Ile nC16, aC17 Nishikiori et al., 1986 Kurstakin family Heptapeptide with a lactone ring between carboxy-terminal group of Gln 7 and OH group of Ser4 β-OH fatty acid chain Kurstakin D-Thr-Gly-D-Ala-Ser-His-D-Gln-Gln iC 11 , nC 12 , iC 12 , iC 13 Hathout et al., 2000 97 Chapter 1 Each LPBSs family comprises several variants, which can differ in their fatty acid chain and their peptide moiety (Figure 13). Moreover, the existence of several different compounds with the same molecular weight emphazises the need to a precise characterization of their chemical structure. Figure 13. Detailed structure of some representative biosurfactants produced by Bacillus spp. 6.3.1. Surfactin family The family of surfactin encompasses about 20 different LPBSs (also named surfactin-like compounds) (Bonmatin et al., 2003). With exception of esperin (Thomas & Ito, 1969), all of them present a common structural trait, which include a heptapeptide with a chiral sequence “LLDLLDL” interlinked with a β-hydroxy fatty acid (C14 to C17) and with a D-Leu in position 3 and 6 and L-Asp in position 4. Amino acid residues present in position 2, 4 and 7 belong to the aliphatic group, and include Val, Leu and Ile (Bonmatin et al., 1995; Itokawa et al., 1994; Peypoux et al., 1991). 98 Chapter 1 Moreover, LPBSs are usually a mixture of compounds with different lengths and types of fatty acid (FA), β-hydroxy FA (FA-β-OH), β-amino FA (FA-β-NH2) or guanidylated-β-OH FA (gFA-β-OH), in which the β-OH or β-NH2 group of FA forms an ester or a peptide bond with the carboxyl group of the C-terminal amino acid. The presence of these variants is a result of the biosynthesis mechanism of these compounds that involve non-ribosomal peptide synthetases (NRPSs), a process detailed in sub-section 6.4. In the literature it is established that lipopeptides which present a Glu in position 1 are named surfactin, while those that possess a Gln are named lichenysin, since it was discovered from a B. licheniformis isolate (Horowitz et al., 1990). This surfactin-like compound is at least 2-fold more efficient than surfactin, reducing the surface tension of water from 72 to 27 mN/m at a CMC of 25–220 mg/L depending on its variants and determined conditions (Grangemard et al., 1999; Arima et al., 1968). Moreover, the occurrence of an Asn in position 4 was first mentioned in the structure described for lichenysin A by Yakimov et al. (Yakimov et al., 1995). In addition, other surfactin-like compound, constituted by Leu4, Val7 or Ile7 surfactin, was isolated from a B. pumilus and was named pumilacidin (Morikawa et al., 1992). Esperin differs from the surfactin by a lactone ring involving the β-carboxyl of Asp in position 4, instead of the α-carboxyl of the terminal Leu. The β-hydroxy fatty acid chain linked to these different peptide moieties can contain 12 - 16 C atoms and exhibit n, iso and anteiso configurations. The main fatty acid chains are usually C14 and C15 (SoberónChávez, 2010). Bamylocin A, a recently described lipopeptide, was isolated from B. amyloliquefaciens (Lee et al., 2007). The peptide chain of this molecule is Glu-Leu-Met-Leu-Pro-Leu-LeuLeu and the molecular weight of the C13 form differs by less than 0.1 mass unit from the standard surfactin C14 isoform. This new result demonstrates the need of a precise mass spectrometry analysis of surfactin molecules to precisely confirm their primary structure. For fengycin, circulocins, fusaricidin, and kurstakin, the carboxyl group of the C-terminal amino acid is lactonised with the hydroxyl group of Tyr3, Thr1, Thr1, and Ser4, respectively (Roongsawang et al., 2010). 99 Chapter 1 6.3.2. Iturin family Iturin A, the best well studied lipopeptide from the iturin family, is a heptapeptide interlinked with a β-amino acid fatty acid, that present a length from C14 to C17 (Peypoux et al., 1978). Six other members of the iturin family were also recognized, which include iturin C, bacillomycin D, F, L and Lc and also mycosubtilin (Bonmatin et al., 2003). Generally, all iturins present the same “LDDLLDL” chiral sequence with a common part in the peptidic portion, β-amino acid L-Asx-D-Tyr-D-Asn (Soberón-Chávez, 2010). With the exception of Iturin C, the first amino acid of the peptide chain is L-Asn. Moreover, Volpon et al. (Volpon et al., 2007) confirmed the presence of L-Asn in position 1 of bacillomycin L instead of L-Asp as initially described, which demonstrates that bacillomycin Lc and bacillomycin L present the same structure. In opposition to some surfactin variants, members of the iturin family probably result from different synthetases. Additionally, the main length of the fatty acid chain differs on its members, of which FA of iturin A, iturin C, bacillomycin D and L presented a length of C14, C16 (Winkelmann et al., 1983) and C15, while C16 and C17 are representatives of bacillomycin F and mycosubtilin (SoberónChávez, 2010). 6.3.3. Fengycin family This family includes lipodecapeptides (as fengycins), which differ by their amino acid residue in position 6: Ala (present in form A) or Val (form B) (Soberón-Chávez, 2010). They present an internal lactone ring in the peptidic moiety between the carboxyl terminal amino acid (Ile), and the hydroxyl group, in the side chain of the tyrosine residue (position 3). Different β-hydroxy fatty acid chains (C14 to C18) can be linked with an amide bond to the N-terminal amino acid residue (Glu) (Nishikiori et al., 1986; Vanittanakom et al., 1986), although representative fatty acid chains are C15, C16 and C17. With the exception of a single lipopeptide isolated from the supernatant of B. thuringiensis, which present a fatty acid chain with one double bond between carbons 13 and 14 (Kim et al., 2004), all of them are saturated (Soberón-Chávez, 2010). Two differences were initially identified in fengycin and plipastatin molecules, which included a Gln instead of a Glu in position 8, and the L and D forms of tyrosine, which are presented in position 3 and 9, respectively, for plipastatins and 9 and 3 for fengycins (Soberón-Chávez, 2010). 100 Chapter 1 6.3.4. Other Lipopeptide Compounds: Kurstakins Kurstakins are a novel class of lipopeptides which include several lipoheptapeptide with the same amino acid sequence: Thr-Gly-Ala-Ser-His-Gln-Gln (Hathout et al., 2000). Different fatty acyl chains (isoC11, nC12, isoC12 and isoC13) are linked by an amide bond to the N-terminal amino acid residue. Moreover, each lipopeptide has a lactone linkage between the carboxyl terminal amino acid and the hydroxyl group on the side chain of the serine residue. The L and D forms of the amino acid residues are not yet characterized. However, the recent identification of the genes involved in the biosynthesis of such or similar compounds indicated that amino acids in positions 1 and 6 could be in presented D-form (Bumpus et al., 2009; Abderrahmani et al., 2011). 6.4. Biosynthesis of LPBSs: from genes to biomolecules Non-ribosomal peptide synthetases (NRPSs) are responsible for the biosynthesis of most LPBSs from Bacillus spp. by a thiotemplate process although polyketide synthetases (PKS) as well as fatty acid synthetases are also involved (Soberón-Chávez, 2010). Moreover, analysis of metabolic profiles of Bacillus species shows that single strains can simultaneously produce representatives of different lipopeptide familes, but also multiple structural analogues of one particular lipopeptide. 6.4.1. The non-ribosomal peptide synthesis machinery Non-ribosomal peptide synthesis involves large multienzymatic proteins organized in modules, named non-ribosomal peptide synthetases (NRPSs), which are multi-modular enzymes that recognize, activate, modify and link the amino acid intermediates to the product peptide (Koglin & Walsh, 2009; Sieber & Marahiel, 2005). Additionally, they are capable of synthesizing peptides that incorporate unusual amino acids, including D-, βand hydroxyor N-methylated amino acids. a) Primary catalytic domains The catalytic domains within the NRPSs modules can be classified into several different types. The four main types of domains include the adenylation (A), the thiolation (T) or peptidyl carrier protein (PCP), the condensation (C), and the thioesterase (TE), which 101 Chapter 1 together comprise the minimal set of domains required for a fully working NRPS, creating a linear assembly (Figure 14). Figure 14. Overview of NRPS relationship between genes, modules and domains. Each gene is related with a single protein, which can then be prepared through one of more modules. These modules are organized into domains, represented here by colors (A, adenylation domain; C, condensation domain; PCP, peptidyl carrier protein and E, epimerisation domain). (Adapted from: Jenke-Kodama & Dittmann, 2009). The Adenylation (A) domain is responsible for the amino acid recognition and ATPdependent activation of an amino acid to form an acyl-adenylate intermediate. Then, the adenylated amino acid binds covalently to a phosphopantetheine carrier of the adjacent thiolation (T) or peptidyl carrier protein (PCP) domain. In addition, peptide bond formation of two consecutively amino acids is catalysed by the condensation (C) domain and modification domains such as the epimerization (E) catalyse the conversion of L-amino acids to D-isomers. Finally, cyclization and release of the product peptide are carried out by C-terminal thioesterase (Te) domain, which is associated with a termination module (Roongsawang et al., 2010). An example of this structured multi-modular complex is shown in Figures 14, 15 and 16. 102 Chapter 1 Figure 15. General representation of biosynthesis and structure of surfactin (srf) operon. Assembly line of surfactin biosynthesis with condensation domains are colored in gray, adenylation domains in red and peptidyl carrier proteins in green. Epimerization domains are shown in blue, the thioesterase domain in orange. (Adapted from: Kraas et al., 2010). Figure 16. Surfactin biosynthesis by the Modular Peptide Synthetase. (A) The srf operon (top) presente three genes srfA-A, srfA-B, and srfA-C, which coded for surfactin synthetase subunits, shown below the genes. Bars indicate the module position within the protein, whereas the individual domains are shown as colored balls: A, adenylation domain; PCP, peptidyl carrier protein domain; C, condensation domain; E, epimerization domain; TE, thioesterase domain. 4′-phosphopantetheinyl cofactors with active thiol groups were shown with the corresponding peptides attached at their current synthesis states. The growing peptide chain is passed from left to right, until the linear product at the last PCP domain is cyclized to the lipopeptide by the TE domain. (B) The SNAC (S-N103 Chapter 1 acetyl cysteamine) acyl peptide can be cyclized by the genetically excised SrfTE domain. The native peptide (R = DLeu) and the soluble substrate (R = DOrn) are illustrated. (Adapted from: Bruner et al., 2002) b) Secondary catalytic domains A second thioesterase domain can be also encoded within the NRPS gene cluster, and can be called type II thioesterases (TEII). It presents only 10% of sequence identity with the type I thioesterases and can be involved in the regeneration of carrier proteins that have been mis-acylated. In fact, as A-domains are specific to a substrate, there is a chance, depending on the selectivity of the A-domain, to load the wrong amino acid onto the carrier protein. If this occurs, as had been shown within the gene clusters of some NRPS such as surfactin (TEIIsr f) and bacitracin (TEIIbac), TEII are responsible for the hydrolysis of the misacylated thiol groups of the 4´-phosphopantetheine arm (Schwarzer et al., 2002). 6.4.2. Diversity of NRPSs in Bacillus spp. Several gene clusters encoding NRPS for the biosynthesis of LPBSs in Bacillus spp., have been cloned and characterized (Figure 17). They present similarities in the modular architecture of their repetitive catalytic units and assembly-line mechanism, although some distinct unique features can be recognized. Characteristics of these LPBSs families and corresponding NRPSs are detailed in subsequent sections. 104 Chapter 1 Figure 17. Multidomain organization of the representative gene clusters encoding NRPSs in Bacillus spp. genes encompassing each peptide synthetase operon, including their sizes and organization within the modules. Surfactin, liquenysin, fengycin/plipastatin, mycosubtilin, Iturin A and bacillomycin D are represented. (Adapted from: Roongsawang et al., 2010). 105 Chapter 1 particular molecules in biofilm formation may be distinct (de Bruijn et al., 2008; Neu, 1996). Surfactin can also act as a signalling molecule in triggering cannibalism and matrix formation in biofilms (López et al., 2009a; López et al., 2009b). Thus, biofilm formation was stimulated by surfactin and also by other molecules that cause potassium leakage, whereas other compounds as iturin failed to induce multicellularity (López et al., 2009a). Moreover, Lopez et al. (López et al., 2009b) showed that the cannibal/matrix-producing can also be created in response to antimicrobial compounds produced by other microorganisms. Based on these findings, they hypothesized that other organisms, which produce compounds that mimic the action of surfactin, may activate toxin production and biofilm formation in B. subtilis as a defense mechanism (López et al., 2009b). These compounds may also adversely influence the attachment to surfaces and biofilm formation by other microorganisms, suggesting that lipopeptides may unfavorably affect the early stages of biofilm formation and, in some cases, can suppress existing biofilms of other microorganisms (Fracchia et al., 2012). 6.5.5. Chelation of metal ions and degradation of xenobiotics Chelation of metal ions has been described for several biosurfactants, including lipopeptides and rhamnolipids. Actually, the capacity for lipopeptides chelation can be affected by structural changes. For example, for surfactin, it was shown that when the leucine at position 2 was substituted by isoleucine, a threefold increase in affinity for Ca2+ occurred, possibly by an increase in accessibility of the acidic side chains and carboxylate groups that organize the calcium-binding site (Grangemard et al., 1997). Additionally, binding of surfactin to Ca2+ resulted in a conformational change of the peptide moiety allowing its incorporation into a phospholipid bilayer (Maget-Dana & Ptak, 1995). The actual functions of LPBSs in relation to metal chelation and degradation of xenobiotics are not clear, and even the benefits to the producing bacteria need to be clarified. Possible hypothesis are: i) a protective role due to chelation of metal ions; ii) bacteria use the biosurfactants for sequestering specific metal ions as micronutrients. With respect to the degradation of xenobiotics, lipopeptides and other biosurfactants may provide access to persistent aromatic compounds with low solubility in water and use these compounds as carbon and/or nitrogen sources (Ron & Rosenberg, 2002). 112 Chapter 1 6.6. Applications of LPBSs 6.6.1. Lipopeptides in pharmaceutical industry The rapid increase and incidence of bacterial infections, particularly of multidrug resistant (MDR) bacteria, renewed the interest in the development of novel antibiotics for infection control purposes (Mandal et al., 2013). Nature and its huge biodiversity seems to be an endless source of compounds containing unique chemical structures, some of them having been exploited for this purpose. However, their commercial exploitation depends largely on a cost-effective production and demonstration of low toxicity. The most wellstudied biosurfactant (surfactin) has been highlighted for different applications (Figure 19). Figure 19. Application sector and exploited properties of surfactin. 113 Chapter 1 A high number of scientific publications and patents have been developed but its application as an antibacterial agent is slowered by high production costs and also the suspected toxicity (From et al., 2007). Thus, this section will focus on the application of biosurfactant agents produced by Bacillus spp. in a pharmaceutical overall perspective. Lipopeptides present potent antibacterial activity due to the ability of these molecules to self-associate and form a pore-bearing channel or micellar aggregate inside the lipid membrane (Deleu et al., 2008). Previously identified lipopeptides and their presumptive activity for microbial infection control are summarized in Table 8 and Table 9. Surfactin, in particular, acts non-specifically causing membrane disruptions on both Grampositive and Gram-negative bacteria (Lu & Mosier, 2007), which has recently been suggested as the next generation of antibiotics (Rodrigues & Teixeira, 2010). Similar bioactive fractions from the marine B. circulans, present antimicrobial activity against various Gram-positive and Gram negative pathogens, including Micrococcus flavus, B. pumilis, Mycobacterium smegmatis, Acinetobacter calcoaceticus, Bordetella bronchiseptica and several Enterobacteriaceae species (Das et al., 2008). In addition to surfactin, B. subtilis strains produce other bioactive peptides (fengycin and iturin families) with great potential of antimicrobial activity, namely iturin A (Besson et al., 1978; Peypoux et al., 1978; Peypoux et al., 1984), mycosubtilin (Peypoux et al., 1986) and bacillomycin (Peypoux et al., 1984), all of which membrane-active agents. Lichenysin, pumilacidin and polymyxin B (Landman et al., 2008; Grangemard et al., 2001; Yakimov et al., 1995; Naruse et al., 1990; ) are other examples of antimicrobial lipopeptides produced by B. licheniformis, B. pumilus and B. polymyxa, respectively. Polymyxin B, in particular, due to its high affinity to the lipid moieties of lipopolysaccharide, presented antibacterial activities against a wide range of Gramnegative pathogens (Landman et al., 2008). Being a cationic agent, it binds to the anionic bacterial outer membrane, leading to a detergent effect that disrupts membrane integrity. Another promising example of an antimicrobial lipopeptide that is under commercial use is daptomycin (Cubicin®), with applications in the treatment of skin infections (Seydlová & Svobodová, 2008). Moreover, Daptomycin, produced by Streptomyces roseosporus has a potent activity against clinically-relevant methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), glycopeptide-intermediatesusceptible S. aureus (GISA), coagulase-negative Staphylococci (CNS), and penicillinresistant Streptococcus pneumoniae (PRSP) pathogens (Tally et al., 1999). In addition, 114 Chapter 1 glycolipids, both rhamnolipids (Benincasa et al., 2004) and sophorolipids (Van Bogaert et al., 2007) have also shown remarkable antimicrobial activities particularly against B. subtilis (Benincasa et al., 2004), B. cereus, S. aureus, Micrococcus luteus, Mucor miehei and Neurospora crassa (Nitschke et al., 2010) and also MRSA (Mimee et al., 2009). Studies also showed that surfactin has an anti-mycoplasma activity allowing the specific inactivation of mycoplasmas contaminating mammalian cells, without significantly damaging effects on cell metabolism (Fassi et al., 2007; Kumar et al., 2007; Vollenbroich et al., 1997). It has also been shown that this compound exhibit a synergistic effect in combination with enrofloxacin, which resulted in mycoplasma-killing activity of about two orders of magnitude greater when compared with the molecules used separately (Fassi et al., 2007). Caspofungin (Arendrup et al., 2012; Letscher-Bru & Herbrecht, 2003), pneumocandin (Schwartz et al., 1988), aculeacin (Mizuno et al., 1977) and other related compounds have been associated with antifungal activities (Hino et al., 2001). Other lipopeptides presenting this function include ciclic lipopetides (surfactin, iturin and fengycin) (Chen et al., 2009; Kim et al., 2010; Snook et al., 2009) and glycopeptides (cellobiose lipids and rhamnolipids) (Banat et al., 2010). Surfactin and its analogues also present a strong antiviral activity (Naruse et al., 1990), especially against enveloped viruses (retroviruses and herpes viruses), suggesting that this activity may be due to the formation of ion channels on the virus envelope, and consequently the lost of viral proteins (Seydlová & Svobodová, 2008; Jung et al., 2000). Additionally, in vitro experiments showed that surfactin and fengycin produced by a B. subtilis strain were able to inactivate cell-free virus of porcine parvovirus, pseudorabies, newcastle disease and bursal disease and, which could effectively inhibit its infection and replication (Huang et al., 2006). In addition, few peptides are already marketed as vaccines. In fact, most of them are applied in HIV immunization, combining an HIV derived peptide and a lipid tail (Rizos et al., 2007; Durier et al., 2006). Moreover, Monash Institute of Pharmaceutical Sciences has recently developed a novel lipopeptide antibiotic to target Gramnegative multi-drug-resistant bacteria, promising to be less apoptopic than polymyxins and with a longer half-life (patent no. PCT/AU2010/000568). 115 Chapter 1 Different studies have also demonstrated that particular lipopeptides (mainly surfactins) have anti-inflamatory, anti-tumoral or immuno-modulatory properties (Cao et al., 2010; Park et al., 2010; Park & Kim, 2009; ). The anti-inflamatory activity has been associated with oxid nitric induction and reduction of pro-inflammatory cytokines, including tumor necrosis factor-α and interleukin (IL)-1β, IL-6 and IL-12 (Byeon et al., 2008; Park et al., 2010). The anti-tumoral activity is related with the involvement of lipopeptides in signal transduction, cell differentiation and cell immune responses, having been demonstrated that surfactin induces apoptosis in human breast cancer (Cao et al., 2011; Osada, 1998). Moreover, Park et al. (2009) also demonstrated a potent immunosuppressive capability for surfactin, which suggests an important therapeutic implication to transplantation and also to autoimmune diseases, including allergy, arthritis and diabetes (Park & Kim, 2009). The colonization of medical or prostheses with microbial biofilms is a hazardous occurrence, particularly if the bacteria become highly resistant to antibiotic, since they can seriously compromise antimicrobial therapy (Morikawa, 2006). Several reports have suggested that, in addition to their direct action against pathogens, biosurfactants are able to interfere with biofilm formation, modulating microbial interaction with interfaces (Rodrigues et al., 2007; Rasmussen & Givskov, 2006; Rodrigues et al., 2006b; Rodrigues et al., 2006c; Merk et al., 2005; Federle & Bassler, 2003; Neu, 1996; ), which seems to be an attractive alternative to more conservative approaches (coating of medical surfaces with antimicrobial agents) (Basak et al., 2009; von Eiff et al., 2005). Indeed, biosurfactants have the advantages of precisely target biofilm growth, while causing no adverse toxicity in the environment. Surfactin, for example, has shown to be an important biofilm controlling agent, able to inhibit biofilm formation of Salmonella Typhimurium, Salmonella enterica, E. coli and Proteus mirabilis in polyvinyl chloride wells, as well as vinyl urethral catheters (Mireles et al., 2001). A recent study showed that two lipopeptide biosurfactants produced by B. subtilis V9T14 and B. licheniformis V19T21, evidenced the capacity to selectively inhibit biofilm formation of S. aureus ATCC 29213 and E. coli CFT073 on polystyrene surfaces (Rivardo et al., 2009). Chemical characterization of V9T14 lipopeptide biosurfactant carried out by Liquid Chromatography – Electrospray Tandem Mass Spectrometry (LC/ESI-MS/MS) revealed that it was composed of 77% of surfactin and of 23% of fengycin (Pecci et al., 2010). In another study, the V9T14 biosurfactant in association with some antibiotics led to a synergistic increase in the efficacy of antibiotics against E. coli CFT073 biofilm inhibition and, in some combinations, to the total eradication of the uropathogenic strain biofilm producer (Rivardo et al., 2011). 116 Chapter 1 Moreover, bioproducts recovered from marine B. pumilus and B. indicus significantly inhibited the initial attachment process and biofilm formation of mature biofilms of Vibrio spp. strains (Nithya & Pandian, 2010). Thus, anti-adhesive activity of biosurfactants against several pathogens highlights their potential applicability as coating agents for medical insertional materials, which can contribute to a reduction in a large number of nosocomial infections avoiding the use of synthetic drugs and/or chemicals. Lipopetides can also be used in the context of drug delivery due to their properties of detergency, emulsification and foaming (Faivre & Rosilio, 2010). Recently, fengycin and surfactin were successfully used in the increasing of aciclovir concentration in the epidermis (Nicoli et al., 2010). Moreover, microemulsions of water/oil/surfactant are being explored as liquid vehicles for future drug delivery systems due to its long-term stability, easy preparation and high solubilization capacity (Date & Nagarsenker, 2008). Gene transfection into the cells is a fundamental technology not only for molecular and cellular biology processes but also for clinical gene therapy (Mulligan et al., 2014). Although several methods for gene transfection have been investigated (Mulligan et al., 2014), More efficient and safe systems are desirable for gene transfection (Ueno et al., 2007), and lipofection (based on the use of liposomes) is considered a promising method for introduction of a foreign gene into the targeted cells (Nakanishi, 2003; Inoh et al., 2001). In fact, glycolipids have received particular attention in this area, when compared with lipopepides produced by Bacillus spp. (Ueno et al., 2007; Igarashi et al., 2006; Inoh et al., 2001) reported that MEL-A promoted DNA transfection efficiently by induction of membrane fusion between the target cells and the cationic liposomes. In addition to the known antimicrobial agents comprising low molecular weight antimicrobial substances and bacteriocins, probiotics have long been known for their ability to interfere with the adhesion and formation of pathogen biofilms to epithelial cells of urogenital and intestinal tracts (Reid et al., 2001) by releasing of surface active molecules (Gudiña et al., 2010; Rodrigues et al., 2006b; Rodrigues et al., 2006c). Several studies pointed out that probiotic microorganisms (mainly Streptococcus thermophilus and Lactobacillus spp. strains) and their biosurfactants may antagonize the growth and the development of potentially pathogenic microorganisms including S. aureus, S. epidermidis, Streptococcus spp., E. faecalis, Candida albicans, Candida tropicalis (van Hoogmoed et al., 2000; Busscher et al., 1997). Thus, considering their importance for human health and their recognized safety, probiotic organisms may represent a safe and effective intervention for infection control purposes. 117 Chapter 1 Probiotics themselves or their products (biosurfactants), could be applied to patient care equipment, such as tubes or catheters, with the aim of decreasing the colonization of these sites by nosocomial pathogens and potentially impede a central step in the pathogenesis of nosocomial infections (Falagas & Makris, 2009). Table 8. List of identified active lipopeptides produced by Bacillus, which can be used for infection control purposes. Lipopeptides Source Activity Reference Surfactin B. subtilis Enterococcus faecalis ATCC 2912 Lactococcus garviae KCCM 40698 Streptococcus parauberis DSM 6631 Flexibacter tractuosus ATCC 23168 Vibrio harveyi ATCC 14126 Kim et al., 2009 Mycosubtilin Micrococcus luteus Peypoux et al., 1979 Fusaricidin A MRSA S. aureus S. epidermidis Vancomycin-resistant E. faecium Stawikowski & Cudic, 2009 Lichenycin Corynebacterium variabiliss Acinetobacterr sp. Nerurkar, 2010 Bacillomycin D Antifungal (Sclerotinia sclerotiorum) Kumar et al., 2012 Iturin A Antifungal (Fusatium oxysporum) Thimon et al., 1995 Yuan et al., 2011 118 Chapter 1 Table 9. Recent patents on lipopeptide as infection control agents. Patent no. Year Patent Name Source Inventors US 2011/0224129 A1 2011 Lipopeptide compounds and their use Modified friulimicin Boyce et al. US 2011/0030103 A1 2011 Lipopeptides and lipopeptide synthetases Engineered lipopeptide synthetase polypeptide Reznik et al. US 7868135 B2 2011 Composition of lipopeptide antibiotic derivatives and methods of use thereof Amphomycin or aspartocin derivative Cameron et al. US 7795207 B2 2010 Lipopeptide compositions Cyclodextrin derivatives Labischinski et al. US 2010/0184649 A1 2010 Novel antibacterial agents for the treatment of Gram-positive infections Isolated combined compounds MetCalf,III et al. US 7671011 B2 2010 Antimicrobial and anticancer lipopeptides Conjugate lipopeptide Shai et al. US 7655623 B2 2010 Dab9 derivatives of lipopeptide antibiotics and methods of making and using the same Synthetic amphomycintype Fardis et al. US 2009/0233870 A1 2009 Antimicrobial peptides Synthetic Blondelle et al. US 2009/0202519 A1 2009 Compositions and methods for treating Gram positive bacterial infection in a mammalian subject Synthetic Beutler et al. US 7408025 B2 2008 Lipopeptides as antibacterial agents Synthetic Hill et al. US 6911525 B2 2005 Lipopeptides as antibacterial agents Synthetic Hill et al. US 6750199 B2 2004 Antimicrobial sulfonamide derivatives of lipopeptide antibiotics Lipopeptide derivatives Curran et al. US 6696412 B1 2004 High purity lipopeptides, lipopeptides micelles and processes for preparing same Daptomycin Kelleher et al. US 6624143 B1 2003 Calcium salts of lipopeptide antibiotics, method for producing same and their use Synthetic Vértesy et al. US 6511962 B1 2003 Derivatives of laspartomycin and preparation and use thereof Laspartomycin Borders et al. 119 Chapter 1 6.6.2. Lipopeptides in cosmetic industry In the last few decades, several lipopeptides have been extensively used in the cosmetics industry due to their exceptional surface properties, presenting anti-wrinkle and moisturizing activities on human skin (Kanlayavattanakul & Lourith, 2010). Moreover, they are also characterized by their capacity of stimulate the producing new collagen and elastin and their antioxidant properties (Singh & Cameotra, 2004b) delaying lipid peroxidation, in addition to the healthy skin physiology support, with potential application in types of facial cosmetics and lotions. Nevertheless, few cosmetic companies have already developed and marketed their lipopeptide-formulated products that directly help to stimulate collagen and elastin production as anti-aging agents (Kanlayavattanakul & Lourith, 2010) due to their high production cost. Surfactin has received greatest attention due to its low CMC value and consequent suitability for topical dermatological application (Kanlayavattanakul and Lourith, 2010). In fact, these compounds have attested safe application and minor irritation to human skin being used in dermatological products and in cleansing cosmetics (Kanlayavattanakul & Lourith, 2010). To expand biotechnology to a profitable industry, in the future, genetic manipulation of organisms should be essential for high yield and high quality production of lipopeptides. 6.6.3. Lipopeptides in food industry In the food industry, lipopeptides are mainly used as emulsifiers in the processing of raw materials and as preservatives during processing step to avoid microbial pathogenic spoilage. Among bio-preservatives, several antimicrobial compounds (more than 500) have been accepted to effectively control food pathogens (Stein, 2005; Ricca et al., 2004). Nevertheless, the use of these antimicrobial compounds as food preservatives, proteins and/or peptides, are limited due to their sensitivity to proteases. Iturins, fengycins and surfactins families are known as protease resistant, exhibiting strong growth inhibition of a wide range of pathogens (Touré et al., 2004). Notwithstanding, in the baking industry, surfactins and rhamnolipids are also used to maintain stability, texture, and volume, and also to help in the emulsification of fat tissue in order to control fat globule agglomeration (Campos et al., 2013). 120 Chapter 1 Additionally, biosurfactants can have also benefit on its use as pre-treatment of material surfaces found in food-processing environments. In fact, pathogenic bacteria implicated in food-borne illness outbreaks are able to form biofilms on food contact surfaces that are more resistant to sanitation than free-living cells (Kim et al., 2006; Stepanovic et al., 2004; Kalmokoff et al., 2001). Thus, pre-conditioning surfaces using microbial surfaceactive compounds may be an interesting strategy for preventing the adhesion of food-borne pathogens to solid surfaces. Recently, Nitschke et al., (Nitschke et al., 2009) investigated the effect of rhamnolipid and surfactin biosurfactants on the adhesion of the food pathogens Chronobacter, Listeria monocytogenes and S. enteritidis to polypropylene and stainless steel surfaces (Nitschke et al., 2009) and concluded that preconditioning with surfactin, rather than rhamnolipid, caused a reduction in the number of adhering cells particularly of L. monocytogenes and Chronobacter on stainless steel. Thus, lipopeptides satisfy several characteristics of beneficial additives for emulsifying, antiadhesive, and antimicrobial activities which suggest their application as multipurpose ingredients, additives or even in developing strategies to prevent or delay microbiological colonization of industrial plant surfaces used in foodstuffs preparation. 6.6.4. Lipopeptides in biotech and nanotech industry In a market where biosurfactants production is highly expensive, efforts have been conducted with special emphasis on the utilization of various cheap agro-industrial substrates and large-scale production (Mulligan et al., 2014; Winterburn & Martin, 2012) for their management of cost-effective production. In this context, some efforts have been made to genetically manipulate or alter the lipopeptide structure to selectively enhance antimicrobial activities (Koglin et al., 2010) and reduce their production cost. Lipopeptides can also be used in the synthesis of metal-bound nanoparticles as an alternative environmentally friendly technology (Sharma et al., 2009; Ariga et al., 2007; Shimizu et al., 2005) due to their ability to self-assemble into hierarchically ordered structures. Reddy et al. (Reddy et al., 2009) successfully synthesized surfactin-mediated gold and silver nanoparticles using B. subtilis. According to the authors, nanoparticles were stabilized by the surface-active molecules i.e., surfactin or other biomolecules released into the solution by B. subtilis. Surfactin produced by B. amyloliquefaciens KSU-109 was also used for the synthesis of cadmium sulfide nanoparticles which remained stable up to six months without compromising their functionality (Singh et al., 2011). This kind of molecules works as 121 Chapter 1 Caboche S, Pupin M, Leclère V, Fontaine A, Jacques P, Kucherov G. NORINE: a database of nonribosomal peptides. Nucleic Acids Research. 2008;36(suppl 1):D326-D31. Callegan MC, Cochran DC, Kane ST, Ramadan RT, Chodosh J, McLean C, et al. Virulence Factor Profiles and Antimicrobial Susceptibilities of Ocular Bacillus Isolates. Current Eye Research. 2006;31(9):693-702. Calza L, Manfredi R, Chiodo F. Infective endocarditis: a review of the best treatment options. Expert Opinion on Pharmacotherapy. 2004;5(9):1899-916. Cameotra S, Makkar R, Kaur J, Mehta SK. Synthesis of Biosurfactants and Their Advantages to Microorganisms and Mankind. In: Sen R, editor. Biosurfactants. Advances in Experimental Medicine and Biology. 672: Springer New York; 2010. p. 261-80. Campos JM, Montenegro Stamford TL, Sarubbo LA, de Luna JM, Rufino RD, Banat IM. Microbial biosurfactants as additives for food industries. Biotechnology Progress. 2013;29(5):1097-108. Cao XH, Wang AH, Wang CL, Mao DZ, Lu MF, Cui YQ, et al. Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway. Chemico-Biological Interactions. 2010;183(3):357-62. Cao XH, Zhao SS, Liu DY, Wang Z, Niu LL, Hou LH, et al. ROS-Ca(2+) is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis. Chemico-Biological Interactions. 2011;190(1):16-27. Cascales E, Buchanan SK, Duché D, Kleanthous C, Lloubès R, Postle K, et al. Colicin Biology. Microbiology and Molecular Biology Reviews. 2007;71(1):158-229. Castagnola E, Fioredda F, Barretta MA, Pescetto L, Garaventa A, Lanino E, et al. Bacillus sphaericus bacteraemia in children with cancer: case reports and literature review. The Journal of Hospital Infection. 2001;48(2):142-5. CDC (Center for Disease Control and Prevention). Antibiotic Resistance Threats in the United States, 2013 - http://www.cdc.gov/drugresistance/threat-report-2013/ pdf/ar-threats-2013-508.pdf. USA: CDC; 2013. Chan J, Halachev M, Loman N, Constantinidou C, Pallen M. Defining bacterial species in the genomic era: insights from the genus Acinetobacter. BMC Microbiology. 2012;12:302. Chan W, Bycroft B, Leyland M, Lian L-Y, Roberts G. A novel post-translational modification of the peptide antibiotic subtilin: isolation and characterization of a natural variant from Bacillus subtilis A.T.C.C. 6633. Biochemistry Journal. 1993(291):23–7. 128 Chapter 1 Chehimi S, Delalande F, Sablé S, Hajlaoui M-R, Van Dorsselaer A, Limam F, et al. Purification and partial amino acid sequence of thuricin S, a new anti-Listeria bacteriocin from Bacillus thuringiensis. Canadian Jjournal of Microbiology. 2007;53(2):284-90. Chen G, Qiao M, Zhang H, Zhu H. Sorption and Transport of Naphthalene and Phenanthrene in Silica Sand in the Presence of Rhamnolipid Biosurfactant. Separation Science and Technology. 2005;40(12):2411-25. Chen P, Lu Y, Harrington PB. Biomarker Profiling and Reproducibility Study of MALDI-MS Measurements of Escherichia coli by Analysis of Variance−Principal Component Analysis. Analytical Chemistry. 2008;80(5):1474-81. Chen S-T, Chen S-Y, Tu C-C, Chiou S-H, Wang K-T. Physicochemical properties of alkaline serine proteases in alcohol. Journal of Protein Chemistry. 1995;14(4):205-15. Chen XH, Koumoutsi A, Scholz R, Borriss R. Production of Antibiotics and Other Secondary Metabolites by Bacillus amyloliquefaciens FZB42. Journal of Molecular Microbiology and Biotechnology. 2009;16:14-24. Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, et al. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotech. 2007;25(9):1007-14. Chen XH, Koumoutsi A, Scholz R, Schneider K, Vater J, Süssmuth R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. Journal of Biotechnology. 2009;140(1–2):27-37. Chen X-H, Vater J, Piel J, Franke P, Scholz R, Schneider K, et al. Structural and Functional Characterization of Three Polyketide Synthase Gene Clusters in Bacillus amyloliquefaciens FZB 42. Journal of Bacteriology. 2006;188(11):4024-36. Cherif A, Chehimi S, Limem F, Hansen BM, Hendriksen NB, Daffonchio D, et al. Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9. Journal of Applied Microbiology. 2003;95(5):9901000. Cherif A, Ouzari H, Daffonchio D, Cherif H, Ben Slama K, Hassen A, et al. Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil. Letters in Applied Microbiology. 2001(32):243–7. 129 Chapter 1 Cherif A, Rezgui W, Raddadi N, Daffonchio D, Boudabous A. Characterization and partial purification of entomocin 110, a newly identified bacteriocin from Bacillus thuringiensis subsp. entomocidus HD110. Microbiological Research. 2008;163(6):684-92. Chollet-Imbert M, Gancel F, Slomianny C, Jacques P. Differentiated pellicle organization and lipopeptide production in standing culture of Bacillus subtilis strains. Archives of Microbiology. 2009;191(1):63-71. Clark A, Kaleta E, Arora A, Wolk D. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clinical Microbiology Reviews 2013;26(3):547-603. Claydon MA, Davey SN, Edwards-Jones V, Gordon DB. The rapid identification of intact microorganisms using mass spectrometry. Nature Biotechnology. 1996;14(11):1584-6. Cohn F. Untersuchungen über Bakterien. Breslau, Germany; 1872. Conway GC, Smole SC, Sarracino DA, Arbeit RD, Leopold PE. Phyloproteomics: species identification of Enterobacteriaceae using matrix-assisted laser desorption}ionization time-of-flight mass spectrometry. Jornal Molecular Microbiololgy Biotechnology 2001;3:103-12. Cosmina P, Rodriguez F, de Ferra F, Grandi G, Perego M, Venema G, et al. Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis. Molecular Microbiology. 1993;8(5):821-31. Cotter PD, Hill C, Ross RP. Bacteriocins: developing innate immunity for food. Nature Reviews Microbiology 2005(3):777-88. Dahrazma B, Mulligan CN. Investigation of the removal of heavy metals from sediments using rhamnolipid in a continuous flow configuration. Chemosphere. 2007;69(5):705-11. Dai L, Wu C-M, Wang M-G, Wang Y, Wang Y, Huang S-Y, et al. First Report of the Multidrug Resistance Gene cfr and the Phenicol Resistance Gene fexA in a Bacillus Strain from Swine Feces. Antimicrobial Agents and Chemotherapy. 2010;54(9):3953-5. Danhorn T, Fuqua C. Biofilm formation by plant-associated bacteria. Annual Review of Mcrobiology. 2007;61:401-22. Das P, Mukherjee S, Sen R. Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans. Journal of Applied Microbiology. 2008;104(6):1675-84. Date AA, Nagarsenker MS. Parenteral microemulsions: An overview. International Journal of Pharmaceutics. 2008;355(1–2):19-30. 130 Chapter 1 de Bruijn I, de Kock MJ, de Waard P, van Beek TA, Raaijmakers JM. Massetolide A biosynthesis in Pseudomonas fluorescens. Journal of Bacteriology. 2008;190(8):2777-89. Deegan LH, Cotter PD, Hill C, Ross P. Bacteriocins: Biological tools for bio-preservation and shelflife extension. International Dairy Journal. 2006;16(9):1058-71. Delcambe L, Peypoux F, Besson F, Guinand M, Michel G. Structure of iturin and iturin-like substances [proceedings]. Biochemical Society Transactions. 1977;5(4):1122-4. Deleu M, Paquot M, Nylander T. Effect of Fengycin, a Lipopeptide Produced by Bacillus subtilis, on Model Biomembranes. Biophysical Journal. 2008;94(7):2667-79. Delsuc F, Brinkmann H, Philippe H. Phylogenomics and the reconstruction of the tree of life. Nature Reviews Genetics. 2005;6(5):361-75. Demain A, Fang A. The Natural Functions of Secondary Metabolites. In: Fiechter A, editor. History of Modern Biotechnology I. Advances in Biochemical Engineering/Biotechnology. 69: Springer Berlin Heidelberg; 2000. p. 1-39. Desai JD, Banat IM. Microbial production of surfactants and their commercial potential. Microbiology and Molecular Biology Reviews. 1997;61(1):47-64. Devirgiliis C, Barile S, Perozzi G. Antibiotic resistance determinants in the interplay between food and gut microbiota. Genes & Nutrition. 2011;6(3):275-84. Dickinson DN, La Duc MT, Haskins WE, Gornushkin I, Winefordner JD, Powell DH, et al. Species Differentiation of a Diverse Suite of Bacillus Spores by Mass Spectrometry-Based Protein Profiling. Applied and Environmental Microbiology. 2004;70(1):475-82. Diddens H, Dorgerloh M, Zahner H. Metabolic products of microorganisms 176. On the transport of small peptide antibiotics in bacteria. Journal of Antibiotics. 1979;32:87-90. Diep DB, Håvarstein LS, Nissen-Meyer J, Nes IF. The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system. Applied and Environmental Microbiology. 1994;60(1):160-6. Diep DB, Skaugen M, Salehian Z, Holo H, Nes IF. Common mechanisms of target cell recognition and immunity for class II bacteriocins. Proceedings of the National Academy of Sciences. 2007;104(7):2384-9. Donoghue HD. Properties and Comparative Starch-gel Electrophoresis of Megacins from Several Bacillus megaterium Strains. Journal of General Microbiology. 1972;72(3):473-83. 131 Chapter 1 Donohue MJ, Smallwood AW, Pfaller S, Rodgers M, Shoemaker JA. The development of a matrixassisted laser desorption/ionization mass spectrometry-based method for the protein fingerprinting and identification of Aeromonas species using whole cells. Journal of Microbiological Methods. 2006;65(3):380-9. Drosinos EH, Mataragas M, Nasis P, Galiotou M, Metaxopoulos J. Growth and bacteriocin production kinetics of Leuconostoc mesenteroides E131. Journal of Applied Microbiology. 2005;99(6):1314-23. Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, Saenger W, et al. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proceedings of the National Academy of Sciences. 1999;96(23):13294-9. Duitman EH, Wyczawski D, Boven LG, Venema G, Kuipers OP, Hamoen LW. Novel Methods for Genetic Transformation of Natural Bacillus subtilis Isolates Used To Study the Regulation of the Mycosubtilin and Surfactin Synthetases. Applied and Environmental Microbiology. 2007;73(11):3490-6. Durier C, Launay O, Meiffredy V, Saidi Y, Salmon D, Levy Y, et al. Clinical safety of HIV lipopeptides used as vaccines in healthy volunteers and HIV-infected adults. Aids. 2006;20(7):1039-49. Dybwad M, van der Laaken AL, Blatny JM, Paauw A. Rapid Identification of Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS). Applied and Environmental Microbiology. 2013;79(17):5372-83. Earl AM, Losick R, Kolter R. Ecology and genomics of Bacillus subtilis. Trends in Microbiology. 2008;16(6):269-75. EFSA (European Food Safety Authority). Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal, 2012; 10(6):2740. EFSA (European Food Safety Authority). Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. The EFSA Journal: 2007. EFSA (European Food Safety Authority). Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2013 update). EFSA Journal, 2013; 11(11):3449. 132 Chapter 1 EFSA (European Food Safety Authority). Technical Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition. 2011; 9(11):2445. Ehling-Schulz M, Fricker M, Grallert H, Rieck P, Wagner M, Scherer S. Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1. BMC Microbiology. 2006;6(1):20. Ehling-Schulz M, Guinebretiere M-H, Monthán A, Berge O, Fricker M, Svensson B. Toxin gene profiling of enterotoxic and emetic Bacillus cereus. FEMS Microbiology Letters. 2006;260(2):23240. Ehling-Schulz M, Messelhaeusser U. Bacillus 'next generation' diagnostics: Moving from detection towards sub-typing and risk related strain profiling. Frontiers in Microbiology. 2013;4. Ehling-Schulz M, Svensson B, Guinebretiere M-H, Lindbäck T, Andersson M, Schulz A, et al. Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains. Microbiology. 2005;151(1):183-97. Ehling-Schulz M, Vukov N, Schulz A, Shaheen R, Andersson M, Märtlbauer E, et al. Identification and Partial Characterization of the Nonribosomal Peptide Synthetase Gene Responsible for Cereulide Production in Emetic Bacillus cereus. Applied and Environmental Microbiology. 2005;71(1):105-13. Emonet S, Shah HN, Cherkaoui A, Schrenzel J. Application and use of various mass spectrometry methods in clinical microbiology. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2010;16(11):1604-13. Eng JK, McCormack AL, Yates Iii JR. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry. 1994;5(11):976-89. European Comission (EC). Opinion of the Scientific Committee on Animal Nutrition on the safety of use of Bacillus species in animal nutrition. 2000. European Comission (EC). Opinion of the Scientific Committee on Animal Nutrition on the criteria for assessing the safety of microorganism resistant to antibiotics of human clinical and veterinary importance. Health and Consumer Protection Directorate, European Commission, Brussels, Belgium., 2002. Fagerlund A, Lindbäck T, Storset AK, Granum PE, Hardy SP. Bacillus cereus Nhe is a poreforming toxin with structural and functional properties similar to the ClyA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia. Microbiology. 2008;154(3):693-704. 133 Chapter 1 Faheem F, Saeed S, Rasool SA. Studies on brevicin AF01: a bacteriocin like inhibitory substance active against methicillin resistant Staphylococcus aureus. Pakistan Journal of Botany. 2007;39:1293–302. Faivre V, Rosilio V. Interest of glycolipids in drug delivery: from physicochemical properties to drug targeting. Expert Opinion on Drug Delivery. 2010;7(9):1031-48. Falagas ME, Makris GC. Probiotic bacteria and biosurfactants for nosocomial infection control: a hypothesis. The Journal of Hospital Infection. 2009;71(4):301-6. Farfour E, Leto J, Barritault M, Barberis C, Meyer J, Dauphin B, et al. Evaluation of the Andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing Gram-positive bacilli. Journal of Clinical Microbiology. 2012;50(8):2702-7. Fassi FL, Wroblewski H, Blanchard A. Activities of antimicrobial peptides and synergy with enrofloxacin against Mycoplasma pulmonis. Antimicrobial Agents and Chemotherapy. 2007;51(2):468-74. Favret ME, Yousten AA. Thuricin: The bacteriocin produced by Bacillus thuringiensis. Journal of Invertebrate Pathology. 1989;53(2):206-16. Federle MJ, Bassler BL. Interspecies communication in bacteria. The Journal of Clinical Investigation. 2003;112(9):1291-9. Felsenstein J. phylip: Phylogenetic Inference Package, version 3.5. Seattle: University of Washington. 1993. Fenselau C, Demirev PA. Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrometry Reviews. 2001;20(4):157-71. Fernandez-No IC, Bohme K, Diaz-Bao M, Cepeda A, Barros-Velazquez J, Calo-Mata P. Characterisation and profiling of Bacillus subtilis, Bacillus cereus and Bacillus licheniformis by MALDI-TOF mass fingerprinting. Food Microbiology. 2013;33(2):235-42. Fickers P. Antibiotic Compounds from Bacillus: Why are they so Amazing? American Journal of Biochemistry and Biotechnology. 2012;8(1):38-43. Filip Z, Herrmann S, Kubat J. FT-IR spectroscopic characteristics of differently cultivated Bacillus subtilis. Microbiological Research. 2004;159(3):257-62. Forrester JB, Valentine NB, Su Y-F, Johnson TJ. Chemometric analysis of multiple species of Bacillus bacterial endospores using infrared spectroscopy: Discrimination to the strain level. Analytica Chimica Acta. 2009;651(1):24-30. 134 Chapter 1 Fracchia L, Cavallo M, Martinotti MG, Banat IM. Biosurfactants and Bioemulsifiers Biomedical and Related Applications – Present Status and Future Potentials. In: Ghista DN, editor. Biomedical Science, Engineering and Technology: InTech; 2012. Freitas D, Reis M, Lima-Bittencourt C, Costa P, Assis P, Chartone-Souza E, et al. Genotypic and phenotypic diversity of Bacillus spp. isolated from steel plant waste. BMC Research Notes 2008(1):92. From C, Hormazabal V, Granum PE. Food poisoning associated with pumilacidin-producing Bacillus pumilus in rice. International Journal of Food Microbiology. 2007;115(3):319-24. From C, Hormazabal V, Hardy SP, Granum PE. Cytotoxicity in Bacillus mojavensis is abolished following loss of surfactin synthesis: Implications for assessment of toxicity and food poisoning potential. International Journal of Food Microbiology. 2007;117(1):43-9. From C, Pukall R, Schumann P, Hormazábal V, Granum PE. Toxin-Producing Ability among Bacillus spp. Outside the Bacillus cereus Group. Applied and Environmental Microbiology. 2005;71(3):1178-83. Gaggìa F, Mattarelli P, Biavati B. Probiotics and prebiotics in animal feeding for safe food production. International Journal of Food Microbiology. 2010;141, Supplement(0):S15-S28. Galanos J, Perera S, Smith H, O'Neal D, Sheorey H, Waters MJ. Bacteremia Due to Three Bacillus Species in a Case of Munchausen's Syndrome. Journal of Clinical Microbiology. 2003;41(5):22478. Gálvez A, López RL, Abriouel H, Valdivia E, Omar NB. Application of Bacteriocins in the Control of Foodborne Pathogenic and Spoilage Bacteria. Critical Reviews in Biotechnology. 2008;28(2):12552. Gálvez A, Maqueda M, Martínez-Bueno M, Lebbadi M, Valdivia E. Isolation and physico-chemical characterization of an antifungal and antibacterial peptide produced by Bacillus licheniformis A12. Applied Microbiology and Biotechnology. 1993;39(4-5):438-42. Garcia-Junco M, Gomez-Lahoz C, Niqui-Arroyo J-L, Ortega-Calvo J-J. Biosurfactantand Biodegradation-Enhanced Partitioning of Polycyclic Aromatic Hydrocarbons from NonaqueousPhase Liquids. Environmental Science & Technology. 2003;37(13):2988-96. Gebhardt K, Schimana J, Müller J, Fiedler H-P, Kallenborn HG, Holzenkämpfer M, et al. Screening for biologically active metabolites with endosymbiotic bacilli isolated from arthropods. FEMS Microbiology Letters. 2002;217(2):199-205. 135 Chapter 1 Gevers D, Dawyndt P, Vandamme P, Willems A, Vancanneyt M, Swings J, et al. Stepping stones towards a new prokaryotic taxonomy. Philosophical Transactions of the Royal Society B: Biological Sciences. 2006;361(1475):1911-6. Gevers D, Masco L, Baert L, Huys G, Debevere J, Swings J. Prevalence and Diversity of Tetracycline Resistant Lactic Acid Bacteria and their tet Genes Along the Process Line of Fermented Dry Sausages. Systematic and Applied Microbiology. 2003;26(2):277-83. Glaser R, Harder J, Lange H, Bartels J, Christophers E, Schroder J-M. Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat Immunol.6(1):1529-2908. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. International Journal of Systematic and Evolutionary Microbiology. 2007;57(1):81-91. Gottheil O. Botanische beschreibung einiger bodenbakterien. Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene. Abteilung II. 1901;7(680-691). Grangemard I, Bonmatin JM, Bernillon J, Das BC, Peypoux F. Lichenysins G, a novel family of lipopeptide biosurfactants from Bacillus licheniformis IM 1307: production, isolation and structural evaluation by NMR and mass spectrometry. The Journal of Antibiotics. 1999;52(4):363-73. Grangemard I, Peypoux F, Wallach J, Das B, Labbé H, Caille A, et al. Lipopeptides with improved properties: structure by NMR, purification by HPLC and structure-activity relationships of new isoleucyl-rich surfactins. Journal of Peptide Science. 1997;3(2):145-54. Grangemard I, Wallach J, Maget-Dana R, Peypoux F. Lichenysin: a more efficient cation chelator than surfactin. Applied Biochemistry and Biotechnology. 2001;90(3):199-210. Gray EJ, Di Falco M, Souleimanov A, Smith DL. Proteomic analysis of the bacteriocin thuricin 17 produced by Bacillus thuringiensis NEB17. FEMS Microbiology Letters. 2006;255(1):27-32. Gray EJ, Lee KD, Souleimanov AM, Di Falco MR, Zhou X, Ly A, et al. A novel bacteriocin, thuricin 17, produced by plant growth promoting rhizobacteria strain Bacillus thuringiensis NEB17: isolation and classification. Journal of Applied Microbiology. 2006;100(3):545-54. Green DH, Wakeley PR, Page A, Barnes A, Baccigalupi L, Ricca E, et al. Characterization of Two Bacillus Probiotics. Applied and Environmental Microbiology. 1999;65(9):4288-91. Gross H. Strategies to unravel the function of orphan biosynthesis pathways: recent examples and future prospects. Applied Microbiology and Biotechnology. 2007;75:267–77. 136 Chapter 1 Gudiña EJ, Teixeira JA, Rodrigues LR. Isolation and functional characterization of a biosurfactant produced by Lactobacillus paracasei. Colloids and Surfaces B: Biointerfaces. 2010;76(1):298-304. Guinebretière M-H, Auger S, Galleron N, Contzen M, De Sarrau B, De Buyser M-L, et al. Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning. International Journal of Systematic and Evolutionary Microbiology. 2013;63(Pt 1):31-40. Gupta RS, Griffiths E. Critical Issues in Bacterial Phylogeny. Theoretical Population Biology. 2002;61(4):423-34. Gustafson K, Roman M, Fenical W. The macrolactins, a novel class of antiviral and cytotoxic macrolides from a deep-sea marine bacterium. Journal of the American Chemical Society. 1989;111(19):7519-24. Haft D. A strain-variable bacteriocin in Bacillus anthracis and Bacillus cereus with repeated CysXaa-Xaa motifs. Biology Direct. 2009;4(1):15. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews Microbiology. 2004;2(2):95-108. Hammami I, Rhouma A, Jaouadi B, Rebai A, Nesme X. Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains. Letters in Applied Microbiology. 2009;48(2):253-60. Hansen DB, Bumpus SB, Aron ZD, Kelleher NL, Walsh CT. The Loading Module of Mycosubtilin: An Adenylation Domain with Fatty Acid Selectivity. Journal of the American Chemical Society. 2007;129(20):6366-7. Harshey RM. Bacterial motility on a surface: many ways to a common goal. Annual Review of Microbiology. 2003;57:249-73. Hathout Y, Ho Y-P, Ryzhov V, Demirev P, Fenselau C. Kurstakins: A New Class of Lipopeptides Isolated from Bacillus thuringiensis. Journal of Natural Products. 2000;63(11):1492-6. Hayashi K, Kensuke T, Kobayashi K, Ogasawara N, Ogura M. Bacillus subtilis RghR (YvaN) represses rapG and rapH, which encode inhibitors of expression of the srfA operon. Molecular Microbiology. 2006;59(6):1714-29. Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 Stress-Responsive Regulator PerR Positively Regulates srfA Expression in Bacillus subtilis. Journal of Bacteriology. 2005;187(19):6659-67. 137 Chapter 1 Laber B, Gerbling K-P, Harde C, Neff K-H, Nordhoff E, Pohlenz H-D. Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors. Biochemistry. 1994;33(11):341323. Lai Q, Liu Y, Shao Z. Bacillus xiamenensis sp. nov., isolated from intestinal tract contents of a flathead mullet (Mugil cephalus). Antonie van Leeuwenhoek. 2014;105(1):99-107. Lai Y, Gallo RL. AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends in Immunology. 2009;30(3):131-41. Landman D, Georgescu C, Martin DA, Quale J. Polymyxins revisited. Clinical Microbiology Reviews. 2008;21(3):449-65. Lartigue MF. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for bacterial strain characterization. Infection, genetics and evolution. Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases. 2013;13:230-5. Lasch P, Beyer W, Nattermann H, Stämmler M, Siegbrecht E, Grunow R, et al. Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks. Applied and Environmental Microbiology. 2009;75(22):7229-42. Lazzarini A, Cavaletti L, Toppo G, Marinelli F. Rare genera of actinomycetes as potential producers of new antibiotics. Antonie van Leeuwenhoek. 2000;78:388–405. Le Marrec C, Hyronimus B, Bressollier P, Verneuil B, Urdaci MC. Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by Bacillus coagulans I4. Applied and Environmental Microbiology. 2000;66(12):5213-20. Leclère V, Marti R, Béchet M, Fickers P, Jacques P. The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties. Archives of Microbiology. 2006;186(6):475-83. Lee H, Churey JJ, Worobo RW. Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced by Bacillus thuringiensis SF361. FEMS Microbiology Letters. 2009;299(2):205-13. Lee H, Kim H-Y. Lantibiotics, Class I Bacteriocins from the Genus Bacillus. Journal of Microbiology and Biotechnology. 2011;21(3):229-35. 144 Chapter 1 Lee H, Williams SKR, Wahl KL, Valentine NB. Analysis of Whole Bacterial Cells by Flow Field-Flow Fractionation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Analytical Chemistry. 2003;75(11):2746-52. Lee S-C, Kim S-H, Park I-H, Chung S-Y, Choi Y-L. Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oilemulsifying activity. Archives of Microbiology. 2007;188(4):307-12. Lehman LJ, Mccoy RJ, Messenger BJ, Manker DC, Orjala JE, Lindhard D, et al. Strain of Bacillus pumilus for controlling plant diseases caused by fungi. U.S. patent 6245551; 2001. Lehman LJ, Mccoy RJ, Messenger BJ, Manker DC, Orjala JE, Lindhard D, et al. A strain of Bacillus pumilus for controlling plant diseases. Google Patents; 2013. Letscher-Bru V, Herbrecht R. Caspofungin: the first representative of a new antifungal class. The Journal of Antimicrobial Chemotherapy. 2003;51(3):513-21. Leuschner R. Bacillus: Occurrence. Encyclopedia of Food Sciences and Nutrition2003. p. 355-8. Li J, Luo Y, Lee J-K, Zhao H. Cloning and characterization of a type III polyketide synthase from Aspergillus niger. Bioorganic & Medicinal Chemistry Letters. 2011;21(20):6085-9. Li Y-M, Haddad NA, Yang S-Z, Mu B-Z. Variants of Lipopeptides Produced by Bacillus licheniformis HSN221 in Different Medium Components Evaluated by a Rapid Method ESI-MS. International Journal of Peptide Research and Therapeutics. 2008;14(3):229-35. Lin S, C.,, Minton M, A.,, Sharma MM, Georgiou G. Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2. Applied and Environmental Microbiology. 1994;60(1):31-8. Liu Y, Lai Q, Dong C, Sun F, Wang L, Li G, et al. Phylogenetic Diversity of the Bacillus pumilus Group and the Marine Ecotype Revealed by Multilocus Sequence Analysis. PLoS ONE 2013;8(11):e80097. López D, Fischbach MA, Chu F, Losick R, Kolter R. Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis. Proceedings of the National Academy of Sciences. 2009;106(1):280-5. López D, Vlamakis H, Losick R, Kolter R. Cannibalism enhances biofilm development in Bacillus subtilis. Molecular Microbiology. 2009;74(3):609-18. Lu Y, Mosier NS. Biomimetic catalysis for hemicellulose hydrolysis in corn stover. Biotechnology Progress. 2007;23(1):116-23. 145 Chapter 1 Lücking G, Dommel MK, Scherer S, Fouet A, Ehling-Schulz M. Cereulide synthesis in emetic Bacillus cereus is controlled by the transition state regulator AbrB, but not by the virulence regulator PlcR. Microbiology. 2009;155(3):922-31. Lund T, De Buyser M-L, Granum PE. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular Microbiology. 2000;38(2):254-61. Lund T, Granum PE. Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiology Letters. 1996;141(2-3):151-6. Mäder U, Antelmann H, Buder T, Dahl M, Hecker M, Homuth G. Bacillus subtilis functional genomics: genome-wide analysis of the DegS-DegU regulon by transcriptomics and proteomics. Molecular Genetics and Genomics. 2002;268(4):455-67. Maget-Dana R, Ptak M. Interactions of surfactin with membrane models. Biophysical Journal. 1995;68(5):1937–43. Magnuson R, Solomon J, Grossman AD. Biochemical and genetic characterization of a competence pheromone from B. subtilis. Cell. 1994;77(2):207-16. Mandal SM, Barbosa AEAD, Franco OL. Lipopeptides in microbial infection control: Scope and reality for industry. Biotechnology Advances. 2013;31(2):338-45. Marlow VL, Haag AF, Kobayashi H, Fletcher V, Scocchi M, Walker GC, et al. Essential Role for the BacA Protein in the Uptake of a Truncated Eukaryotic Peptide in Sinorhizobium meliloti. Journal of Bacteriology. 2009;191(5):1519-27. Martinotti MG, Allegrone G, Cavallo M, Fracchia L. Biosurfactants. Sustainable Development in Chemical Engineering Innovative Technologies: John Wiley & Sons, Ltd; 2013. p. 199-240. Martirani L, Varcamonti M, Naclerio G, De Felice M. Purification and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis. Microbial Cell Factories. 2002;1(1):1. Mattiuzzo M, Bandiera A, Gennaro R, Benincasa M, Pacor S, Antcheva N, et al. Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides. Molecular Microbiology. 2007;66(1):151-63. Mazza P. The use of Bacillus subtilis as an antidiarrhoeal microorganism. Bollettino chimico farmaceutico. 1994;133(1):3-18. 146 Chapter 1 McCarter L, Silverman M. Surface-induced swarmer cell differentiation of Vibrio parahaemolyticus. Molecular Microbiology. 1990;4(7):1057-62. McClerren AL, Cooper LE, Quan C, Thomas PM, Kelleher NL, van der Donk WA. Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic. Proceedings of the National Academy of Sciences. 2006;103(46):17243-8. Mellmann A, Bimet F, Bizet C, Borovskaya AD, Drake RR, Eigner U, et al. High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria. Journal of Clinical Microbiology. 2009;47(11):3732-4. Menkhaus M, Ullrich C, Kluge B, Vater J, Vollenbroich D, Kamp RM. Structural and functional organization of the surfactin synthetase multienzyme system. Journal of Biological Chemistry. 1993;268(11):7678-84. Merk K, Borelli C, Korting HC. Lactobacilli - bacteria-host interactions with special regard to the urogenital tract. International Journal of Medical Microbiology. 2005;295(1):9-18. Meylheuc T, Methivier C, Renault M, Herry J-M, Pradier C-M, Bellon-Fontaine MN. Adsorption on stainless steel surfaces of biosurfactants produced by gram-negative and gram-positive bacteria: Consequence on the bioadhesive behavior of Listeria monocytogenes. Colloids and Surfaces B: Biointerfaces. 2006;52(2):128-37. Mietke H, Beer W, Schleif J, Schabert G, Reissbrodt R. Differentiation between probiotic and wildtype Bacillus cereus isolates by antibiotic susceptibility test and fourier transform infrared spectroscopy (FT-IR). International Journal of Food Microbiology. 2010;140(1):57-60. Mikkola R, Andersson MA, Teplova V, Grigoriev P, Kuehn T, Loss S, et al. Amylosin from Bacillus amyloliquefaeiens, a K+ and Na+ channel-forming toxic peptide containing a polyene structure. Toxicon. 2007(49):1158-71. Mills S, Stanton C, Hill C, Ross RP. New Developments and Applications of Bacteriocins and Peptides in Foods. Annual Review of Food Science and Technology. 2011;2(1):299-329. Mimee B, Pelletier R, Bélanger RR. In vitro antibacterial activity and antifungal mode of action of flocculosin, a membrane-active cellobiose lipid. Journal of Applied Microbiology. 2009;107(3):98996. Mireles JR, 2nd, Toguchi A, Harshey RM. Salmonella enterica serovar Typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation. Journal of Bacteriology. 2001;183(20):5848-54. 147 Chapter 1 Mizuno K, Yagi A, Satoi S, Takada M, Hayashi M. Studies on aculeacin. I. Isolation and characterization of aculeacin A. The Journal of Antibiotics. 1977;30:297–302. Morikawa M, Ito M, Imanaka T. Isolation of a new surfactin producer Bacillus pumilus A-1, and cloning and nucleotide sequence of the regulator gene, psf-1. Journal of Fermentation and Bioengineering. 1992;74(5):255-61. Morikawa M. Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species. Journal of Bioscience and Bioengineering. 2006;101(1):1-8. Moura H, Woolfitt AR, Carvalho MG, Pavlopoulos A, Teixeira LM, Satten GA, et al. MALDI-TOF mass spectrometry as a tool for differentiation of invasive and noninvasive Streptococcus pyogenes isolates. FEMS Immunology & Medical Microbiology. 2008;53(3):333-42. Moyne AL, Cleveland TE, Tuzun S. Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiology Letters. 2004;234(1):43-9. Msadek T, Kunst F, Klier A, Rapoport G. DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. Journal of Bacteriology. 1991;173(7):2366-77. Mukherjee AK, Das K. Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat. FEMS Microbiology Ecology. 2005;54(3):479-89. Mulligan CN, Gibbs BF. Types, production and applications of biosurfactants. Proc Indian Nat Sci Acad. 2004;1:31–55. Mulligan CN, Sharma SK, Mudhoo A. Biosurfactants: Research Trends and Applications: Taylor & Francis; 2014. Mulligan CN, Yong RN, Gibbs BF. Heavy metal removal from sediments by biosurfactants. Journal of Hazardous Materials. 2001;85(1–2):111-25. Mulligan CN, Yong RN, Gibbs BF. Surfactant-enhanced remediation of contaminated soil: a review. Engineering Geology. 2001;60(1–4):371-80. Mulligan CN, Yong RN. Natural attenuation of contaminated soils. Environment International. 2004;30(4):587-601. Mulligan CN. Environmental applications for biosurfactants. Environmental Pollution. 2005;133(2):183-98. 148 Chapter 1 Mulligan CN. Recent advances in the environmental applications of biosurfactants. Current Opinion in Colloid & Interface Science. 2009;14(5):372-8. Naclerio G, Ricca E, Sacco M, De Felice M. Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus. Applied and Environmental Microbiology. 1993;59(12):4313-6. Naes TI, T; Fearn, T; Davies, T. A user Friendly guide to Multivariate Calibration and Classification. .Chichester UK: NIR Publications; 2002. Nakanishi M. New strategy in gene transfection by cationic transfection lipids with a cationic cholesterol. Current Medicinal Chemistry. 2003;10(14):1289-96. Nakano C, Ozawa H, Akanuma G, Funa N, Horinouchi S. Biosynthesis of Aliphatic Polyketides by Type III Polyketide Synthase and Methyltransferase in Bacillus subtilis. Journal of Bacteriology. 2009;191(15):4916-23. Nakano S, Nakano MM, Zhang Y, Leelakriangsak M, Zuber P. A regulatory protein that interferes with activator-stimulated transcription in bacteria. Proceedings of the National Academy of Sciences. 2003;100(7):4233-8. Nakatsuji T, Gallo R. Antimicrobial peptides: old molecules with new ideas. Journal of Investigative Dermatology. 2012;132:887-95. Naruse N, Tenmyo O, Kobaru S, Kamei H, Miyaki T, Konishi M, et al. Pumilacidin, a complex of new antiviral antibiotics. Production, isolation, chemical properties, structure and biological activity. The Journal of Antibiotics. 1990;43(3):267-80. Naumann D, Helm D, Labischinski H. Microbiological characterizations by FT-IR spectroscopy. Nature. 1991;351(6321):81-2. Neilson JW, Artiola JF, Maier RM. Characterization of lead removal from contaminated soils by nontoxic soil-washing agents. Journal of Environmental Quality. 2003;32(3):899-908. Nerurkar AS. Structural and molecular characteristics of lichenysin and its relationship with surface activity. Advances in Experimental Medicine and Biology. 2010;672:304-15. Nes I, Diep D, Håvarstein L, Brurberg M, Eijsink V, Holo H. Biosynthesis of bacteriocins in lactic acid bacteria. Antonie van Leeuwenhoek. 1996;70(2-4):113-28. Nes I, Yoon S-S, Diep D. Ribosomally synthesiszed antimicrobial peptides (bacteriocins) in lactic acid bacteria: a review. Food Science and Biotechnology. 2007;16:675–90. 149 Chapter 1 Neu TR. Significance of bacterial surface-active compounds in interaction of bacteria with interfaces. Microbiological Reviews. 1996;60(1):151-66. Nicoli S, Eeman M, Deleu M, Bresciani E, Padula C, Santi P. Effect of lipopeptides and iontophoresis on aciclovir skin delivery. Journal of Pharmacology and Pharmacotherapeutics. 2010;62(6):702-8. Nielsen P, Fritze D, Priest FG. Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Microbiology. 1995;141(7):1745-61. Nieminen T, Rintaluoma N, Andersson M, Taimisto AM, Ali-Vehmas T, Seppälä A, et al. Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk. Veterinary Microbiology. 2007;124(3–4):329-39. Nishikiori T, Naganawa H, Muraoka Y, Aoyagi T, Umezawa H. Plipastatins: new inhibitors of phospholipase A2, produced by Bacillus cereus BMG302-fF67. II. Structure of fatty acid residue and amino acid sequence. The Journal of Antibiotics. 1986;39(6):745-54. Nithya C, Pandian SK. The in vitro antibiofilm activity of selected marine bacterial culture supernatants against Vibrio spp. Archives of Microbiology. 2010;192(10):843-54. Nitschke M, Araújo LV, Costa SGVAO, Pires RC, Zeraik AE, Fernandes ACLB, et al. Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces. Letters in Applied Microbiology. 2009;49(2):241-7. Nitschke M, Costa SGVAO. Biosurfactants in food industry. Trends in Food Science & Technology. 2007;18(5):252-9. Nitschke M, Costa SVAO, Contiero J. Structure and Applications of a Rhamnolipid Surfactant Produced in Soybean Oil Waste. Applied Biochemistry and Biotechnology. 2010;160(7):2066-74. Novais A, Sousa C, J. C, A. F-O, J. L, MH. R, et al. Discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131) and D (ST69, ST405, ST393) by MALDI-TOF mass spectrometry. . European Journal of Clinical Microbiology and Infectious Diseases. 2014. Novak KN, Davis E, Wehnes CA, Shields DR, Coalson JA, Smith AH, et al. Effect of supplementation with an electrolyte containing a Bacillus-based direct-fed microbial on immune development in dairy calves. Research in Veterinary Science. 2012;92(3):427-34. Novotny JF, Perry JJ. Characterization of bacteriocins from two strains of Bacillus thermoleovorans, a thermophilic hydrocarbon-utilizing species. Applied and Environmental Microbiology. 1992;58(8):2393-6. 150 Chapter 1 Oberreuter H, Seiler H, Scherer S. Identification of coryneform bacteria and related taxa by Fouriertransform infrared (FT-IR) spectroscopy. International Journal of Systematic and Evolutionary Microbiology. 2002;52(1):91-100. Ogura M, Fujita Y. Bacillus subtilis rapD, a direct target of transcription repression by RghR, negatively regulates srfA expression. FEMS Microbiology Letters. 2007;268(1):73-80. Ohsawa T, Tsukahara K, Sato T, Ogura M. Superoxide Stress Decreases Expression of srfA through Inhibition of Transcription of the comQXP Quorum-Sensing Locus in Bacillus subtilis. Journal of Biochemistry. 2006;139(2):203-11. Økstad O, Kolstø A-B. Genomics of Bacillus Species. In: Wiedmann M, Zhang W, editors. Genomics of Foodborne Bacterial Pathogens. Food Microbiology and Food Safety: Springer New York; 2011. p. 29-53. Ongena M, Jourdan E, Adam A, Paquot M, Brans A, Joris B, et al. Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environmental Microbiology. 2007;9(4):1084-90. Osada H. Bioprobes for investigating mammalian cell cycle control. The Journal of Antibiotics. 1998;51(11):973-82. Oscáriz JC, Cintas L, Holo H, Lasa Íñ, Nes IF, Pisabarro AG. Purification and sequencing of cerein 7B, a novel bacteriocin produced by Bacillus cereus Bc7. FEMS Microbiology Letters. 2006;254(1):108-15. Oscáriz JC, Pisabarro AG. Characterization and mechanism of action of cerein 7, a bacteriocin produced by Bacillus cereus Bc7. Journal of Applied Microbiology. 2000;89(2):361-9. Ouoba LII, Diawara B, Amoa-Awua Wk, Traoré AS, Møller PL. Genotyping of starter cultures of Bacillus subtilis and Bacillus pumilus for fermentation of African locust bean (Parkia biglobosa) to produce Soumbala. International Journal of Food Microbiology. 2004;90(2):197-205. Ozkocaman V, Ozcelik T, Ali R, Ozkalemkas F, Ozkan A, Ozakin C, et al. Bacillus spp. among hospitalized patients with haematological malignancies: clinical features, epidemics and outcomes. The Journal of Hospital Infection. 2006;64(2):169-76. Paik HD, Bae SS, Park SH, Pan JG. Identification and partial characterization of tochicin, a bacteriocin produced by Bacillus thuringiensis subsp tochigiensis. Journal of Industrial Microbiology and Biotechnology. 1997;19(4):294-8. 151 Chapter 1 Paik SH, Chakicherla A, Hansen JN. Identification and Characterization of the Structural and Transporter Genes for, and the Chemical and Biological Properties of, Sublancin 168, a Novel Lantibiotic Produced by Bacillus subtilis 168. Journal of Biological Chemistry. 1998;273(36):2313442. Park SY, Kim Y. Surfactin inhibits immunostimulatory function of macrophages through blocking NK-kappaB, MAPK and Akt pathway. International Immunopharmacology. 2009;9(7-8):886-93. Park SY, Kim YH, Kim EK, Ryu EY, Lee SJ. Heme oxygenase-1 signals are involved in preferential inhibition of pro-inflammatory cytokine release by surfactin in cells activated with Porphyromonas gingivalis lipopolysaccharide. Chemico-biological Interactions. 2010;188(3):437-45. Parte AC. List of Prokaryotic names with Standing in Nomenclature, http://www.bacterio.net. 2013. Patel P, Huang S, Fisher S, Pirnik D, Aklonis C, Dean L, et al. Bacillaene, a novel inhibitor of procaryotic protein synthesis produced by Bacillus subtilis: production, taxonomy, isolation, physico-chemical characterization and biological activity. The Journal of Antibiotics. 1995;48(9):997-1003. Pattnaik P, Kaushik JK, Grover S, Batish VK. Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffalo. Journal of Applied Microbiology. 2001;91(4):636-45. Pecci Y, Rivardo F, Martinotti MG, Allegrone G. LC/ESI-MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain. Journal of mass spectrometry : JMS. 2010;45(7):772-8. Pedersen PB, Bjørnvad ME, Rasmussen MD, Petersen JN. Cytotoxic Potential of Industrial Strains of Bacillus sp. Regulatory Toxicology and Pharmacology. 2002;36(2):155-61. Peltola J, Andersson MA, Haahtela T, Mussalo-Rauhamaa H, Rainey FA, Kroppenstedt RM, et al. Toxic-Metabolite-Producing Bacteria and Fungus in an Indoor Environment. Applied and Environmental Microbiology. 2001;67(7):3269-74. Pérez-García A, Romero D, de Vicente A. Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture. Current Opinion in Biotechnology. 2011;22(2):187-93. Perkins DN, Pappin DJC, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999;20(18):355167. 152 Chapter 1 Peypoux F, Besson F, Michel G, Delcambe L. Preparation and antibacterial activity upon Micrococcus luteus of derivatives of iturin A, mycosubtilin and bacillomycin L, antibiotics from Bacillus subtilis. The Journal of Antibiotics. 1979;32(2):136-40. Peypoux F, Besson F, Michel G, Delcambe L. Structure of Bacillomycin D, a New Antibiotic of the Iturin Group. European Journal of Biochemistry. 1981;118(2):323-7. Peypoux F, Besson F, Michel G. Structure of iturin C from Bacillus subtilis. Tetrahedron Letters. 1978;34:1147-52. Peypoux F, Bonmatin J-M, LabbÉ H, Das BC, Ptak M, Michel G. Isolation and characterization of a new variant of surfactin, the [Val7]surfactin. European Journal of Biochemistry. 1991;202(1):101-6. Peypoux F, Bonmatin JM, Wallach J. Recent trends in the biochemistry of surfactin. Applied Microbiology and Biotechnology. 1999;51(5):553-63. Peypoux F, Marion D, Maget-Dana R, Ptak M, Das BC, Michel G. Structure of bacillomycin F, a new peptidolipid antibiotic of the iturin group. European Journal of Biochemistry. 1985;153(2):33540. Peypoux F, Pommier M, Das B, Besson F, Delcambe L, Michel G. Structures of bacillomycin D and bacillomycin L peptidolipid antibiotics from Bacillus subtilis. The Journal of Antibiotics. 1984;37(12):1600-4. Peypoux F, Pommier MT, Marion D, Ptak M, Das BC, Michel G. Revised structure of mycosubtilin, a peptidolipid antibiotic from Bacillus subtilis. The Journal of Antibiotics. 1986;39(5):636-41. Phelps RJ, McKillip JL. Enterotoxin Production in Natural Isolates of Bacillaceae outside the Bacillus cereus Group. Applied and Environmental Microbiology. 2002;68(6):3147-51. Pinchuk IV, Bressollier P, Sorokulova IB, Verneuil B, Urdaci Maria C. Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats. Research in Microbiology. 2002;153(5):269-76. Price NPJ, Rooney AP, Swezey JL, Perry E, Cohan FM. Mass spectrometric analysis of lipopeptides from Bacillus strains isolated from diverse geographical locations. FEMS Microbiology Letters. 2007;271(1):83-9. Pueyo M, Bloch C, Jr., Carmona-Ribeiro A, Mascio P. Lipopeptides Produced by a Soil Bacillus megaterium Strain. Microbial Ecology. 2009;57(2):367-78. 153 Chapter 2 27. Fernández-No, I. C.; Böhme, K.; Díaz-Bao, M.; Cepeda, A.; Barros-Velázquez, J.; Calo-Mata, P. Characterization and profiling of Bacillus subtilis, Bacillus cereus and Bacillus licheniformis by MALDI-TOF mass fingerprinting. Food Microbiol. 2013, 33, 23542. 28. Arnold, R.J.; Reilly, J.P. Observation of Escherichia coli ribosomal proteins and their posttranslational modifications by mass spectrometry. Anal. Biochem. 1999, 269, 105– 112. 29. Driks, A. The Bacillus subtilis spore coat. Microbiol. Mol. Biol. Rev. 1999, 63, 1–20. 30. Driks, A. Proteins of the spore core and coat. In Bacillus subtilis and its Closest Relatives: from Genes to Cells, Sonenshein, A. L.; Hoch, J. A.; Losick, R., Eds; American Society for Microbiology, Washington D C, 2002, p. 527–535. 31. Lai, E. M.; Phadke, N. D.; Kachman, M. T.; Giorno, R.; Vazquez, S.; Vazquez, J.A.; Maddock, J.R.; Driks, A. Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis. J. Bacteriol. 2003, 185, 1443–1454. 32. Branquinho, R.; Pintado, M. E.; Peixe, L. Clonality and protein diversity of Bacillus pumilus isolates from different sources and geographic regions, In: Journal of Medicinal Plant and Natural Product Research – 78 - PA5, New York, 2012. DOI: 10.1055/s-00321320320. 33. Matarante, A.; Baruzzi, F.; Cocconcelli, P. S.; Morea, M. Genotyping and Toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages. Appl. Environ. Microbiol. 2004, 70, 5168–5176. 34. Jetiyanon, K. Interaction between PGPR and Cucumber during Induced Systemic Resistance: Recognition and Early Host Defense Responses. Ph.D. dissertation, Auburn University, Auburn, AL, USA, 1997. 35. Ouoba, L.; Diawara, B.; Amoa-Awua, W. K.; Traore, A. S.; Moller, P. L. Genotyping of starter cultures of Bacillus subtilis and Bacillus pumilus for fermentation of African locust bean (Parkiabiglobosa) to produce Soumbala. Int. J. Food Microbiol. 2004, 90, 197-205. 256 Chapter 2 Table 1. Origins of Bacillus spp. isolates (n=27) included in this study. Isolate Origin Year/Location References Bacillus pumilus Bp ATCC14884 Reference strain Bp ATCC 7061T Type Strain Bp7 Medicine´s (n=3) 2005/Portugal1 [32] Bp11 2005/Portugal1 Bp15 2005/Portugal1 Bacillus safensis Bs1 Animals Gastropods (n=3) 2004/Portugal1 [32] Bs2 2005/Portugal1 Bs3 2007/Portugal1 Bs13 Medicine´s (n=3) 2005/Portugal1 Bs16 2005/Portugal1 Bs17 2005/Portugal1 Bs5 Cosmetic´s (n=4) 2002/Portugal1 Bs18 2002/Portugal1 Bs19 2002/Portugal1 Bs27 2002/Portugal1 Bs24 Food´s /salame (n=3) 2004/Italy2 [33] Bs25 2004/Italy2 Bs33 2004/Italy2 Bs22 Plant Growth Promoters (PGPR) (n=2) 1997/USA3 [34] Bs23 1997/USA3 Bs31 Food/beans (n=1) 2003/Africa4 [35] Bs FO-36bT Clean-room/ air particulate (n=1) 1999/USA5 Type Strain, [21] Bs35 Clean-room/floor (n=1) 2001/USA5 [2] Bs36 Clean-room/cabinet top (n=1) 2001/USA5 Bs37 Clean-room/Mars Odyssey spacecraft surface (n=2) 2001/USA5 Bs38 Bs42 Clean-room/anteroom (n=1) 2001/USA5 257 Chapter 2 1Isolates obtained from the Quality Control Department (INFARMED), Lisbon, Portugal. 2Isolates FEL 55 from salame felino, UNG22 from salame ungherese and MIL46 from salame milano obtained from the Istituto di Scienze delle Produzioni Alimentari (ISPA), Bari, Italy. 3Isolates SE 49 (AP3) and SE 52 (AP7) from cucumber roots obtained from the Culture collection of the Department of Entomology and Plant Pathology, Auburn University, Alabama, USA. 4Isolates Bs31 from African locust beans for Soumbala production obtained from Ouagadougou, Africa. 5Isolates F036-b, SAFN-027, SAFN-037, KL-052, 51-3C and 82-2C from spacecraft and assembly-facility surfaces obtained from California Institute of Technology, California, USA. 258 Chapter 2 Table 2. Species-specific ion peaks values (average) of B. pumilus and B. safensis isolates. Experimental average m/z values* B. pumilus B. safensis B. pumilus 2069.5 2170 3060 3608.5 5271 6122 B. safensis 2287.5 2399.5 2511.5 3692.5 3821.5 4305.5 5948.5 6704 6793.5 7415 2063.5 2623.5 2730 3049 3396 5288 5568 6094 6413 * average m/z values (±2Da) 259 Chapter 2 Table 3. Overview of biomarkers tentative assignment of MALDI-TOF/MS mass signals of B. pumilus group species. Protein identity was determined by the TagIdent software and compared with ribosomal subunit proteins developed by Hotta et al [23] described in Materials and Methods section. Specie(s) Observed Mass (Da) Predicted Mass (Da) UniProt Accession ID Protein Description Peptide sequence Organismc B. pumilus 2069.5 NA Unassigned NA NA 2170 NA Unassigned NA NA 3060 NA Unassigned NA NA 3608.5 NA Unassigned NA NA 5271 5270 A8FJG4 50S bRP subunit L34 MKRTFQPNNRKRSKVHGFRSRMSSKNGRLVLKRRRSKGRKKLSA B. pumilus SAFR-032 5266.89 A8FDQ9 SASP O MTKRKANHVINGMNAAKSQGNGAGYIEDDQLVLTAEQRQNNKKRKKNQ B. pumilus SAFR-032 6122 6114 C0H3U0 Uncharacterized membrane protein YyzG MQTNRVILLAVMICLVSAITVFLLNGCKVDFLDIGGTIIGCFLGIFVVVRIQKKQS B. subtilis subsp. subtilis str. 168 6126 P0C8M5 Transcriptional regulator SlrA MKTHVKKDLDKGWHMLIQEARSIGLGIHDVRQFLESETASRKKNHKKTVRQD B. subtilis subsp. subtilis str. 168 B. safensis 2063.5 NA Unassigned NA NA 2623.5 NA Unassigned NA NA 2730 NA Unassigned NA NA 3049 NA Unassigned NA NA 3396 NA Unassigned NA NA 5288 5299.88* A8FHD4 SASP J MSFFQKDKKAKSEKDHKQVDQLLEEASKELAGDPLQEAVQKKKNNDQ B. pumilus SAFR-032 5568 5544 A8FDQ8 SASP P MTNKNTGKDIRQNSPKEHQSGQPEPLSGSKKVKNRNHTRQKHNSHHDM B. pumilus SAFR-032 6094 NA Unassigned NA NA 6413 6411.7* A8FCW7 bRP subunit L32 MAVPFRRTSKMKKRLRRTHFKLQVPGMVACPECGEMKISHRVCKSCGTYKGKDVKSN B. pumilus ATCC 7061 T B. pumilus and B. safensis 2287.5 NA Unassigned NA NA 2399.5 NA Unassigned NA NA 2511.5 NA Unassigned NA NA 3692.5 3698 C0H3V1 UPF0752 membrane MSGYSNGGGYGGISSFALIVVLFILLIIVGTAFVGGF B. subtilis subsp. 260 Chapter 2 protein YczN subtilis str. 168 3821.5 NA Unassigned NA NA 4305.5 4305 A8F9A9 50S bRP subunit L36 MKVRPSVKPICEKCKVIRRKGKVMVICENPKHKQKQG B. pumilus SAFR-032 5948.5 5932 A8FF72 50S bRP subunit L33 2 MRVNITLACTECGERNYITKKNKRNNPDRVEFKKYCSRDKKQTVHRETK B. pumilus SAFR-032 6704 6691 C0H3Z1 Uncharacterized protein YjzG MMKNGFAYKNGKLVNIFCGKEELYNELKAFLVKTFSINVKEVSRPSIYRRTKSKQLE B. subtilis subsp. subtilis str. 168 6793.5 6793* B4AE40 50S bRP subunit L28 MARKCVITGRKTKAGNNRSHAMNSTKRTWGANLQKVRILVDGKPKRVYVSARALKSGKVERV B. pumilus ATCC 7061T 7415 7410.8* B4AM90 50S bRP subunit L35 MPKMKTHRGSAKRFKKTGSGKLKRSHAYTSHLFANKSTKQKRKLRKSAIVSAGDFKRIKQQLANIK B. pumilus ATCC 7061T aSASP - Small, acid-soluble spore protein. bRP – Ribosomal protein. cOrganism – bacterial strain where the protein was described. NA – not applicable. *Predicted Mw proposed by Hotta et al [23], considering “N-end rule” [24] where N-terminal methionine is cleaved from specific penultimate amino acid residues such as glycine, alanine, serine, proline, valine, threonine and cysteine. B. pumilus and B. safensis 261 Chapter 2 Table 4. Candidate species-specific biomarkers assignments of B. pumilus and B.safensis. Peak masses are presented as m/z values. The presence/absence of a peak in each spectral group is represented by +/-, respectively. Species Ion peaks (m/z) B. pumilus B. safensis 4305.5 + + 5271 + - 5288 - + 5568 - + 5948.5 + + 6122 + - 6413 - + 6793.5 + + 7415 + + 262 Chapter 2 Figure 1. Mean mass spectra obtained by MALDI-TOF/MS of (a) B. pumilus (5 spectra), (b) B. safensis (22 spectra) in the range 2000 to 12000 m/z. Spectra were obtained by averaging the respective experimental mass spectra from all isolates (see text for details). 263 Chapter 2 Figure 2. Score plot of the PLSDA regression model (a) and respective dendrogram (b), with B. pumilus and B. safensis isolates. Legend:  B. pumilus isolates and  B. safensis isolates. Unfiled symbols correspond to the type strains of both species. 264 Chapter 2 Figure 3. Representative description of the evaluation of molecular masses of speciesspecific peaks using MALDI-TOF/MS in linear mode (a) and the correspondent reflector positive mode (b) of B. pumilus ATCC 7061T and B. safensis F036-bT spectra. 265 Chapter 2 2. Material and methods 2.1. Bacterial isolates Four B. pumilus isolates and twenty B. safensis previously identified by phenotypic and genotypic (16S rRNA, gyrB and rpoB gene sequences) methods were studied. This collection comprises isolates of diverse PFGE-types, recovered from different geographic terrestrial locations and sources mainly from food samples (Italy and Africa) (n=4), plants (USA) (n=2), gastropods (Portugal) (n=3), health (n=6) and cosmetic (n=4) products (Portugal) and clean room environments from Mars Odyssey (USA) (n=3). The type strains B. pumilus ATCC 7061T and B. safensis FO-36bT were also included (table 1). Details about the collection are provided in table1. 2.2. FTIR-ATR experiments Bacillus isolates were grown on Mueller Hinton agar (37ºC, 16h) and colonies were directly transferred from the agar plates to the ATR crystal. FTIR-ATR spectra were acquired using a PerkinElmer Spectrum BX FTIR System spectrophotometer with a PIKE Technologies Gladi ATR accessory from 4000-600 cm-1 with a resolution of 4 cm-1 and 32 scan co-additions. For each isolate, three instrumental replicates (obtained in the same day) and two biological replicates (obtained in two different days from different agar plates) were obtained and analysed corresponding to a total of six spectra for each isolate. Between each isolate measurement, a background was acquired. 2.3. Chemometric analysis FTIR-ATR spectra were analysed by partial least squares discriminant analysis (PLSDA) after being processed with standard normal variate (SNV) (Næs et al., 2002) followed by the application of a Savitzky-Golay filter (7 smoothing points, 2nd order polynomial and first derivative) (Savitzky & Golay, 1964) and mean-centred. PLSDA is a supervised chemometric method based on the PLS regression method (Geladi & Kowalsky, 1986; Alsberg et al., 1998) and it requires a previous knowledge of assigned classes (species) for all isolates tested. All chemometric models were performed in Matlab version 6.5 272 Chapter 2 release 13 (MathWorks, Natick, MA) and the PLS Toolbox version 3.5 for Matlab (Eigenvector Research, Manson, WA). 273 Chapter 2 3. Results and Discussion In this work, we demonstrate for the first time that FTIR-ATR spectroscopy combined with PLSDA is a reliable, quick and low cost method to accurately discriminate B. pumilus and B. safensis isolates. Some previous studies used FTIR to discriminate Bacillus isolates at genus (Whittaker et al. 2003; Maity et al. 2013) and species (Mietke et al. 2010; Ammann & Brandl 2011; Lücking et al. 2013) levels; however none of them included the closely related species and highly relevant B. pumilus and B. safensis. Infrared spectra of the isolates included in this study revealed the presence of bands associated with bacterial components such as lipids (3000-2800 cm-1), proteins/amides I and II (1700-1500 cm-1), phospholipids/DNA/RNA (1500-1185 cm-1), polysaccharides (1185-900 cm-1) and the fingerprint region (900-600 cm-1) (Naumann et al. 1991). A high degree of spectral similarity was observed among both species (Figure 1) remarking their phylogenetics (Liu et al., 2013; Satomi et al., 2006). Similarly to previous works (Coutinho et al., 2009; Sousa et al., 2012; Vaz et al., 2013; Sousa et al., 2014), the main spectral differences (marked with black arrows in Figure 1) were found in the phospholipids/DNA/RNA and the polysaccharides regions (1500-900cm-1), which were chosen for further comparisons. Those differences reflects the cellular composition dissimilarity between both species which mainly corresponds to vibrations associated with C−H bending of alkanes (1461 cm1); (CH3)3N+ symmetric bending (lipids) and C−H bending of alkenes =CH2 (1414 cm-1); C−O stretching of RNA ribose (1165 cm-1); alcoholic C−OH vibration of polysaccharide fraction and >PO2 - stretching (1070 cm-1); S=O stretching of organic sulfoxides (1052 cm1), P−O−P stretching (phospholipids, ribose phosphate chain pyrophosphate and glycipeptides) and C−O stretching of nucleic acids (1030-980 cm-1). In addition, a PLSDA of the infrared spectra in the selected region revealed two perfectly distinct clusters, each one containing isolates of one single species (Figure 2). Moreover, model statistics (data not shown) revealed that 98.05% of the isolates perfectly fit the developed model, being the first three latent variables (that encompass 52.97% of spectral variability) responsible for species discrimination. Although the results obtained herein only include two species of the B. pumillus group they strongly indicated that FTIR-ATR possess a high potential in the discrimination of Bacillus spp. Moreover, FTIR-ATR seems to possess the same discriminatory power as other high throughput techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (unpublished results) with the advantage of having a smaller cost, concerning equipment and analysis 274 Chapter 2 and be totally free of reagents. The reliability of this technique for species discrimination should be further tested in a higher number of species of the B. pumillus group as B. altitudinis, B. stratosphericus, B. aerophilus and the newly described B. xiamenensis (Lai et al. 2014) as well as in a higher number of isolates of B. pumillus and B. safensis. At this moment one doesn’t possess conveniently characterized isolates of B. altitudinis and B. xiamenensis and it was not possible to obtain B. stratosphericus and B. aerophilus in the type culture collections neither with the authors responsible for their first description. 275 Chapter 2 4. Conclusions We demonstrated that species-specific cellular components of B. pumilus and B. safensis isolates detected by FTIR-ATR can differentiate these closed species. This achievement is of high relevance due to the possibility to reliably differentiate isolates belonging to these closely related species rapidly and with low cost, which is of interest for quality control purposes in different industrial production units. Furthermore, this study unveils molecular signatures (e.g. carbohydrates and phospholipids) of these species that could be further explored to improve the overall knowledge of these bacteria. 276 Chapter 2 Acknowledgements This study was supported by Fundação para a Ciência e Tecnologia grants (PEstC/EQB/LA0006/2011, POCI/AMB/61814/2004-FSE/FEDER). Clara Sousa was supported by a post-doctoral grant (SFRH/BPD/70548/2010) and Raquel Branquinho by a PhD grant (SFRH/BD/61410/2009) from Fundação para a Ciência e Tecnologia. 277 Chapter 2 References 1. Alsberg BK, Kell DB, Goodacre R. 1998 Variable selection in discriminant partial least-squares analysis. Anal Chem. 70:4126-33. 2. Ammann AB, Brandl H. 2011 Detection and differentiation of bacterial spores in a mineral matrix by Fourier transform infrared spectroscopy (FTIR) and chemometrical data treatment. BMC Biophysics. 4: 14. 3. Argyri AA, Panagou EZ, Tarantilis PA, Polysiou M, Nychas GJE. 2010 Rapid qualitative and quantitative detection of beef fillets spoilage based on Fourier transform infrared spectroscopy data and artificial neural networks. Sens Actuators B Chem. 145:146e154. 4. Aunpad R, Na-Bangchang K. 2007 Pumilicin 4, a novel bacteriocin with anti-MRSA and anti-VRE activity produced by newly isolated bacteria Bacillus pumilus strain WAPB4. Curr Microbiol. 55:308–313. 5. Blum M, Harald J. 2012 Historical perspective and modern applications of Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR). Drug Test Anal. 4:298-302. 6. Branquinho R, Pintado ME, Peixe L. 2012 Clonality and protein diversity of Bacillus pumilus isolates from different sources and geographic regions, In: Journal of Medicinal Plant and Natural Product Research – 78 - PA5, New York, DOI: 10.1055/s-00321320320. 7. Coutinho CP, Sá-Correia I, Lopes JA. 2009 Use of Fourier transform infrared spectroscopy and chemometrics to discriminate clinical isolates of bacteria of the Burkholderia cepacia complex from different species and ribopatterns. Anal Bioanal Chem. 394(8): 2161-2171. 8. Coorevits A, De Jonghe V, Vandroemme J, Reekmans R, Heyrman J, Messens, W, De Vos P, Heyndrickx M. 2008 Comparative analysis of the diversity of aerobic sporeforming bacteria in raw milk from organic and conventional dairy farms. Syst Appl Microbiol. 31(2):126-40. 9. Davis R, Burgula Y, Deering A, Irudayaraj J, Reuhs BL, Mauer LJ. 2010 Detection and differentiation of live and heat-treated Salmonella enterica serovars inoculated onto chicken breast using Fourier transform infrared (FT-IR) spectroscopy. J Appl Microbiol. 109:2019e2031. 10. De Clerck E, Vanhoutte T, Hebb T, Geerinck J, Devos J, De Vos P. 2004 Isolation, characterization, and identification of bacterial contaminants in semifinal gelatin extracts. Appl Environ Microbiol. 70(6):3664-72. 278 Chapter 2 11. European Food Safety Authority (EFSA). Technical guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition EFSA panel on additives and products or substances used in animal feed (FEEDAP) 2011 EFSA Journal. 9: 11-2445. 12. From C, Hormazabal V, Granum P. 2007 Food poisoning associated with pumilacidin-producing Bacillus pumilus in rice. Int J Food Microbiol. 115:319-24. 13. Geladi P, Kowalsky BR. 1986 Partial least-squares regression: a tutorial. Anal Chim Acta. 185:1-17. 14. Jetiyanon K. 1997 Interaction between PGPR and Cucumber during Induced Systemic Resistance: Recognition and Early Host Defense Responses. Ph.D. dissertation, Auburn University, Auburn, AL, USA. 15. Lai Q, Liu Y, Shao Z. 2014 Bacillus xiamenensis sp. nov., isolated from intestinal tract contents of a flathead mullet (Mugil cephalus). Antonie Van Leeuwenhoek. 105(1):99-107. 16. Lehman LJ, McCoy RJ, Messenger BJ, Manker DC, Orjala JE, Lindhard D, Marrone PG. 2001 Strain of Bacillus pumilus for controlling plant diseases caused by fungi. U.S. Patent. 6:245-551. 17. Liu Y, Lai Q, Dong C, Sun F, Wang , Li G, Shao Z. 2013 Phylogenetic diversity of the Bacillus pumilus group and the marine ecotype revealed by Multilocus Sequence Analysis. PLoS ONE. 8:1-11. 18. Lücking G, Stoeckel M, Atamer Z, Hinrichs J, Ehling-Schulz M. 2013 Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage. Int J Food Microbiol. 166(2):270-9. 19. Maquelin K, Kirschner C, Choo-Smith LP, van den Braak N, Endtz HP, Naumann D, Puppels GJ. 2002 Identification of medically relevant microorganisms by vibrational spectroscopy. J Microbiol Methods 51:255e271. 20. Matarante A, Baruzzi F, Cocconcelli PS, Morea M. 2004 Genotyping and Toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages. Appl Environ Microbiol. 70:5168-76. 21. Maity JP, Kar S, Lin CM, Chen CY, Chang YF, Jean JS, Kulp TR. 2013 Identification and discrimination of bacteria using Fourier transform infrared spectroscopy. Spectrochim Acta A Mol Biomol Spectrosc. 116:478-84. 22. Mietke H, Beer W, Schleif J, Schabert G, Reissbrodt R. 2010 Differentiation between probiotic and wild-type Bacillus cereus isolates by antibiotic susceptibility test and Fourier transform infrared spectroscopy (FT-IR). Int J Food Microbiol 140:57:60. 23. Næs T, Isaksson T, Fearn T, Davis T. 2002 A user-friendly guide to multivariate calibration and classification. Chichester, UK: NIR Publications. 279 Chapter 2 24. Naumann D, Helm D, Labischinski H. 1991 Microbiological characterizations by FT-IR spectroscopy. Nature 351:81-2. 25. Ouoba L, Diawara B, Amoa-Awua WK, Traore AS, Moller PL. 2004 Genotyping of starter cultures of Bacillus subtilis and Bacillus pumilus for fermentation of African locust bean (Parkiabiglobosa) to produce Soumbala. Int J Food Microbiol. 90:197-205. 26. Pirttijärvi TS Graeffe TH, Salkinoja-Salonen MS. 1996. Bacterial contaminants in liquid packaging boards: assessment of potential for food spoilage. J Appl Bacteriol. 81(4):445-58. 27. Satomi M, La Duc MT, Venkateswaran K. 2006 Bacillus safensis sp. nov., isolated from spacecraft and assembly-facility surfaces. Int J Syst Evol Microbiol. 56:1735-40. 28. Savitzky A, Golay MJE. 1964 Smoothing and Differentiation of Data by Simplified Least Squares Procedures. Anal Chem. 36(8):1627-39. 29. Slepecky RA, Hemphill HE. 2006 The Genus Bacillus-Nonmedical. Prokaryotes. 4:530–562. 30. Sousa C, Grosso F, Meirinhos-Soares L, Peixe L, Lopes JA. 2012 Identification of carbapenem-resistant Acinetobacter baumannii clones using infrared spectroscopy. J. biophotonics; DOI:10.1002/jbio.201200075. 31. Sousa C, Silva L, Grosso F, Nemec A, Lopes J, Peixe L. 2014 Discrimination of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex species by Fourier transform infrared spectroscopy. Eur J Clin Microbiol Infect Dis. DOI: 10.1007/s10096014-2078-y. 32. Stecchini ML, Del Torre M, Polese P. 2013 Survival strategies of Bacillus spores in food. Indian J Exp Biol. 51(11):905-9. 33. Sutherland AD, Murdoch R. 1994 Seasonal occurrence of psychrotrophic Bacillus species in raw milk, and studies on the interactions with mesophilic Bacillus sp. Int J Food Microbiol. 21(4):279-92. 34. Van Doren JM, Neil KP, Parish M, Gieraltowski L, Gould LH, Gombas KL 2013 Foodborne illness outbreaks from microbial contaminants in spices, 1973-2010. Food Microbiol. 36(2):456-64. 35. Vaz M, Meirinhos-Soares L, Sousa C Ramirez M, Melo-Cristino J, Lopes JA. 2013 Serotype discrimination of encapsulated Streptococcus pneumoniae strains by Fouriertransform infrared spectroscopy and chemometrics. J. Microbiol. Meth. 93:102-107. 36. Whittaker P, Mossoba M M, Al-Khaldi S, Fry F S, Dunkel V C, Tall B D, Yurawecz M P. 2003 Identification of foodborne bacteria by infrared spectroscopy using cellular fatty. 280 Chapter 2 Table 1. Features of Bacillus pumilus and B. safensis included in this study. Isolate PFGEa Origin Year/Location References Bacillus pumilus ATCC 7061 T NA ---- ---- Type Strain Bp7 Ip Health products (n=3) 2005/Portugal Branquinho et al., 2012 Bp11 2005/Portugal Bp15 2005/Portugal Bacillus safensis Bs1 Vs Animals Gastropods (n=3) 2004/Portugal Branquinho et al., 2012 Bs2 2005/Portugal Bs3 2007/Portugal Bs13 VIIs Health products (n=3) 2005/Portugal Bs16 IIIs 2005/Portugal Bs17 IVs 2005/Portugal Bs5 XIs Cosmetic´s (n=4) 2002/Portugal Bs18 VIs 2002/Portugal Bs27 IIs 2002/Portugal Bs24 XVs Food´s /salame (n=3) 2004/Italy Matarante et al., 2004 Bs25 VIIIs 2004/Italy Bs33 Non typeable 2004/Italy Bs22 IXs Plant Growth Promoters (PGPR) (n=2) 1997/USA Jetiyanon et al., 1997 Bs23 Is 1997/USA Bs31 Xs Food/beans (n=1) 2003/Africa Ouoba et al., 2004 FO-36b T XIIIs Clean-room/ air particulate (n=1) 1999/USA Satomi et al., 2006 (Type Strain) Bs35 XIVs Clean-room/floor (n=1) 2001/USA Satomi et al., 2006 Bs37 XIIIs Clean-room/Mars Odyssey spacecraft surface (n=2) 2001/USA Bs38 Bs42 Non typeable Clean-room/anteroom (n=1) 2001/USA aPFGEPulsed Field Gel Electrophoresis; NA - not applicable. 281 Chapter 2 The biotechnological and pharmaceutical relevance of Bacillus subtilis group members is mainly supported by their ability to produce compounds with different biological activities, namely antibacterial (Mannanov and Sattarova 2001; Seydlová et al 2011; Balciunas et al., 2013), antifungal (Gupte et al., 2002; Zhao et al., 2013), insecticide (Revathi et al., 2013), biosurfactant (Fracchia et al., 2013; Martinotti et al., 2013), probiotic and plant growth promoters (USEPA 2004; Pérez-García et al., 2011). In order to be used as feed additives, biotechnological products containing Bacillus species need to attain the Qualified Presumption of Safety (QPS) status (EFSA, 2013). For this purpose, EFSAFEEDAP guidelines state that microorganisms cannot be resistant to antibiotics of human and veterinary importance, neither possess acquired antibiotic resistance genes and toxin encoding genes (EFSA, 2011). In this study, we determined the susceptibility and presence of resistance genes to several antimicrobials, β-lactamase activity and presence of genes encoding for entero-, emeticand cyto-toxins in a collection of Bacillus subtilis complex isolates, contributing for the establishment of their safety profile. Forty-three Bacillus subtilis complex isolates (27 B. safensis, 9 B. altitudinis, 4 B. pumilus and 3 B. subtilis) previously identified by phenotypic and genotypic (16S rRNA, gyrB and rpoB gene sequences) methods, were tested (Branquinho et al., 2014) (Table S1). Minimum inhibitory concentrations (MIC) to different antibiotics were determined by broth microdilution method and interpreted according to clinical breakpoints (CLSI M45-A, 2006) or microbiological cut-offs (Table 1) (EFSA, 2012). All MIC experiments were performed in duplicate. β-lactamase activity was assessed through the nitrocefin test (Antunes et al., 2004). Transferable genes encoding resistance to tetracylines, macrolides, aminoglycosides, glycopeptides, phenicols and oxazolidinones, and toxins usually present in B. cereus (cytotoxin K, non-hemolytic enterotoxins, haemolysin and emetic toxins) were screened by PCR using primers and conditions outlined in Table S2. All isolates presented MICs to the different antibiotics lower than the established microbiological cut-offs for the detection of acquired antibiotic resistance genes, suggesting their absence, which was further confirmed by the absence of resistance genes. Remarkably, isolates were clinically intermediate or resistant to cefotaxime, except for B. subtilis isolates. Moreover one B. subtilis presented MIC levels to penicillin that would prevent the therapeutical success of this antibiotic. In addition, nitrocefin test revealed that 69.7% of the isolates produced β-lactamases (18 B. safensis, 8 B. altitudinis and 4 B. pumilus). Furthermore, no amplification for toxins encoding toxins was obtained. 288 Chapter 2 According to the FEEDAP guidelines, this collection fulfills the antibiotic resistance parameters for the QPS, as none of the isolates would apparently constitute a reservoir for mobile antibiotic resistance genes, and thus could be of potential used for industrial purposes, including as feed additives. Nevertheless, the identification of isolates with intermediate or clinical resistance to cefotaxime or penicillin (both antibiotics recommended for testing in CLSI-M45 but not required by the FEEDAP guidelines), should be considered in the strains safety evaluations due to the therapeutic failure that would be observed with their use. Despite the absence of toxins usually encoded by B. cereus, the assessment in these isolates of surfactin-like compounds production should be further studied. It is widely known that Bacillus spp. can harbor intrinsic β-lactamases (Andrews and Wise, 2002), however their clinical importance has been poorly studied. Our results show that βlactamase production is not correlated with higher MIC values to β-lactams, as the same MIC for ampicillin is verified either for β-lactamase producers and non-producers. Despite these observations, we should also consider these β-lactamases: i) probably possess additional/unknown factors required for their activity; and ii) might have high substrate specificity. Besides, Penicilin-Binding-Proteins (PBP) variants with different β-lactam affinities might lead to variable susceptibility behavior to these antibiotics, as previously observed for other Gram positive bacteria (Leclercq et al., 2013). In conclusion, this work demonstrates the absence of antibiotic and virulence features required for the usage of B. subtilis complex isolates in the food sector, also enabling their safe application for other relevant biotechnological purposes. In addition, future QPS assessments should take into consideration the assessment of susceptibility to clinically relevant antibiotics, such as cefotaxime and penicillin in Bacillus spp. 289 Chapter 2 Table 1. Susceptibility profile of Bacillus spp. tested. Antibiotic Clinical breakpointa (µg/mL) (S/I/R) Microbiologicalb cut-off (µg/mL) Species (n) MIC (µg/mL) range MIC50 ( µ g/mL) MIC90 ( µ g/mL) Nº of I and R Isolates Penicillin <0.12 / - / ≥0.25 NA B. safensis (n=27) <0.03 <0.03 <0.03 0 B. altitudinis (n=9) <0.03 <0.03 <0.03 0 B. pumilus (n=4) <0.03 <0.03 <0.03 0 B. subtilis (n=3) <0.03->64 <0.03 >64 1 Oxacillin NA B. safensis (n=27) <0.06-0.125 0.125 0.125 NA B. altitudinis (n=9) <0.06-0.125 <0.06 0.125 B. pumilus (n=4) <0.06-0.125 0.125 0.125 B. subtilis (n=3) <0.06 <0.06 <0.06 Ampicillin ≤0.25 / - / ≥0.5 NA B. safensis (n=27) <0.07 <0.07 <0.07 0 B. altitudinis (n=9) <0.07 <0.07 <0.07 0 B. pumilus (n=4) <0.07 <0.07 <0.07 0 B. subtilis (n=3) <0.07 <0.07 <0.07 0 Amoxicillin NA B. safensis (n=27) <0.06 <0.06 <0.06 NA B. altitudinis (n=9) <0.06 <0.06 <0.06 B. pumilus (n=4) <0.06 <0.06 <0.06 B. subtilis (n=3) <0.06 <0.06 <0.06 Piperacillin NA B. safensis (n=27) 0.5-2 1 1 NA B. altitudinis (n=9) <0.5-1 0.5 1 B. pumilus (n=4) 1 1 1 B. subtilis (n=3) 0.25-0.5 0.25 0.5 Cefotaxime ≤8 / 16-32 / ≥64 NA B. safensis (n=27) 16-64 32 32 27 B. altitudinis (n=9) 8-64 32 64 8 B. pumilus (n=4) 16-32 32 32 4 B. subtilis (n=3) <0.125 <0.125 <0.125 0 Cefoxitin NA B. safensis (n=27) <0.125-4 2 2 NA B. altitudinis (n=9) 1-4 1 1 B. pumilus (n=4) 1-2 2 2 B. subtilis (n=3) <0.125-1 <0.125 1 Ertapenem NA B. safensis (n=27) 0.06-0.125 0.125 0.125 NA B. altitudinis (n=9) 0.03-0.125 0.03 0.06 B. pumilus (n=4) 0.125-0.25 0.125 0.25 B. subtilis (n=3) <0.016-0.03 0.03 0.03 Imipenem ≤4 / 6 / ≥16 NA B. safensis (n=27) <0.07 <0.07 <0.07 0 B. altitudinis (n=9) <0.07 <0.07 <0.07 0 B. pumilus (n=4) <0.07 <0.07 <0.07 0 290 Chapter 2 B. subtilis (n=3) <0.07 <0.07 <0.07 0 Vancomycin ≤4 / - / - 4 B. safensis (n=27) <0.07-0.25 0.125 0.125 0 B. altitudinis (n=9) <0.07-0.25 0.125 0.25 0 B. pumilus (n=4) 0.125 0.125 0.125 0 B. subtilis (n=3) 0.125 0.125 0.125 0 Gentamicin ≤4 / 8 / ≥16 4 B. safensis (n=27) <0.07 <0.07 <0.07 0 B. altitudinis (n=9) <0.07 <0.07 <0.07 0 B. pumilus (n=4) <0.07 <0.07 <0.07 0 B. subtilis (n=3) <0.07 <0.07 <0.07 0 Amikacin ≤16 / 32 / ≥64 NA B. safensis (n=27) <0.06-0.5 0.125 0.125 0 B. altitudinis (n=9) <0.06-0.25 <0.06 0.125 0 B. pumilus (n=4) <0.06-0.125 0.125 0.125 0 B. subtilis (n=3) 0.25-0.5 0.25 0.5 0 Kanamycin NA 8 B. safensis (n=27) 0.125-0.5 0.25 0.25 0 B. altitudinis (n=9) 0.125-0.5 0.25 0.25 0 B. pumilus (n=4) 0.125-0.25 0.25 0.25 0 B. subtilis (n=3) 0.25-0.5 0.25 0.5 0 Streptomycin NA 8 B. safensis (n=27) 1-2 1 2 0 B. altitudinis (n=9) 1-2 2 2 0 B. pumilus (n=4) 2 2 2 0 B. subtilis (n=3) 1-4 1 4 0 Erythromycin ≤0.5 / 1-4 / ≥8 4 B. safensis (n=27) <0.016-0.06 0.03 0.06 0 B. altitudinis (n=9) <0.016-0.03 <0.016 <0.016 0 B. pumilus (n=4) 0.03-0.06 0.03 0.06 0 B. subtilis (n=3) <0.016-0.03 0.03 0.03 0 Tetracycline ≤4 / 8 / ≥16 8 B. safensis (n=27) <0.06 <0.06 <0.06 0 B. altitudinis (n=9) <0.06 <0.06 <0.06 0 B. pumilus (n=4) <0.06 <0.06 <0.06 0 B. subtilis (n=3) <0.06 <0.06 <0.06 0 Chlortetracycline NA B. safensis (n=27) <0.125 <0.125 <0.125 NA B. altitudinis (n=9) <0.125 <0.125 <0.125 B. pumilus (n=4) <0.125 <0.125 <0.125 B. subtilis (n=3) <0.125 <0.125 <0.125 Ciprofloxacin ≤1 / 2 / ≥4 NA B. safensis (n=27) <0.03-0.06 <0.03 <0.03 0 B. altitudinis (n=9) <0.03 <0.03 <0.03 0 B. pumilus (n=4) <0.03 <0.03 <0.03 0 B. subtilis (n=3) <0.03 <0.03 <0.03 0 Norfloxacin NA B. safensis (n=27) 0.125-1 0.25 0.5 NA B. altitudinis (n=9) 0.125-0.5 0.25 0.5 B. pumilus (n=4) 0.25-0.5 0.25 0.5 291 Chapter 2 B. subtilis (n=3) <0.07-0.125 <0.07 0.125 Florphenicol NA B. safensis (n=27) 1-2 2 2 NA B. altitudinis (n=9) 1-2 1 2 B. pumilus (n=4) 2 2 2 B. subtilis (n=3) 1 1 1 Tiamulin NA B. safensis (n=27) >128 >128 >128 NA B. altitudinis (n=9) 1->128 >128 >128 B. pumilus (n=4) >128 >128 >128 B. subtilis (n=3) >128 >128 >128 Rifampin ≤1 / 2 / ≥4 NA B. safensis (n=27) <0.016 <0.016 <0.016 0 B. altitudinis (n=9) <0.016 <0.016 <0.016 0 B. pumilus (n=4) <0.016 <0.016 <0.016 0 B. subtilis (n=3) <0.016 <0.016 <0.016 0 a CLSI - Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria – Standard M45-A 2006; bFEEDAP - Scientific opinion on guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance; NA – not applicable 292 Chapter 2 Acknowledgments Dr. Kasthuri Venkateswaran, Dr. Irène Ouoba, Dr. Joseph W. Kloepper, Dr. Cecilie From and Dr. Maria Morea are gratefully acknowledged for providing isolates.. This study was funded by Fundação para a Ciência e a Tecnologia (FCT), which belongs to the Ministry of Education and Science from Portugal, through grant no. PEst-C/EQB/LA0006/2011. Raquel Branquinho and Manuela Amorim were supported by PhD fellowships, SFRH/BD/77518/2011 and SFRH/BD/81901/2011, respectively. 293 Chapter 2 References Aarestrup FM, Agerso Y, Gerner-Smidt P, Madsen M, Jensen LB. Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagn Microbiol Infect Dis 2000; 37: 127-137. Andrews JM, Wise R. Susceptibility testing of Bacillus species. J Antimicrob Chemother 2002; 49(6): 1040-1042. Antunes P, Machado J, Sousa JC, Peixe L. Dissemination amongst humans and food products of animal origin of a Salmonella Typhimurium clone expressing an integronborne OXA-30 β-lactamase. J Antimicrob Chemother 2004; 54: 429-434. Balciunas EM, Martinez F, Todorov S, Franco B, Converti A, Oliveira R. Novel biotechnological applications of bacteriocins: A review. Food Control 2013; 32(1):134-142. Branquinho R, Meirinhos-Soares L, Carriço JA, Pintado M, Peixe LV. Phylogenetic and clonality analysis of Bacillus pumilus isolates uncovered a highly heterogeneous population of different closely related species and clones. FEMS Microb Ecol 2014 (submitted). Branquinho R, Pintado ME, Peixe L. Clonality and protein diversity of Bacillus pumilus isolates from different sources and geographic regions. Planta Med 2012; 78: PA5. doi: 10.1055/s-0032-1320320. Clinical and Laboratory Standards Institute (CLSI) – Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria – Standard M45-A 2006; Wayne, PA: Clinical and Laboratory Standards Institute. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 1995; 33: 24-27. Ehling-Schulz M, Fricker M, Scherer S. Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay FEMS Microbiol Let 2004; 232 (2) 189–195. 294 Chapter 2 European Food Safety Authority (EFSA). Scientific opinion on guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J 2012; 10: 2740. European Food Safety Authority (EFSA). Technical Guidance on the assessment of the toxigenic potential of Bacillus. EFSA J 2011; 9: 2445. European Food Safety Authority (EFSA). Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2013 update). EFSA J 2013; 11: 3449. Fracchia L., Cavallo M., Martinotti MG, Banat IM 2012. Biosurfactants and Bioemulsifiers Biomedical and Related Applications – Present Status and Future Potentials. Ghista DN (Ed.). Biomedical Science, Engineering and Technology, InTech 2012; DOI: 10.5772/23821. From C, Hormazabal V, Granum P. Food poisoning associated with pumilacidinproducing Bacillus pumilus in rice. Int J Food Microbiol 2007; 115: 319-324. Guerra B, Junker E, Miko A, Helmuth R, Mendoza MC. Characterization and localization of drug resistance determinants in multidrug-resistant, integron-carrying Salmonella enterica serotype Typhimurium strains. Microb Drug Resist 2004; 10(2): 83-91. Gupte M, Kulkarni P, Ganguli B. Antifungal antibiotics. Appl Microbiol Biotechnol 2002; 58(1): 46-57. Jetiyanon K. Interaction between PGPR and Cucumber during Induced Systemic Resistance: Recognition and Early Host Defense Responses. USA: Auburn University, Auburn; 1997. Kehrenberg C, Schwarz S. Distribution of florfenicol resistance genes fexA and cfr among chloramphenicol-resistant Staphylococcus isolates. Antimicrob Agents Chemother 2006; 50(4): 1156-1163. Leclercq R, Cantón R, Brown DFJ, Giske CG, Heisig ., MacGowan AP, Mouton JW, Nordmann P, Rodloff AC, Rossolini GM, Soussy C-J, Steinbakk M, Winstanley TG, Kahlmeter G. EUCAST expert rules in antimicrobial susceptibility testing. Clinl Microbiol Infect 2013; 19: 141–160. 295 Chapter 2 Mannanov RN, Sattarova RK. Antibiotics Produced by Bacillus Bacteria. Chem NatCompd 2001; 37(2):117-123. Martinotti MG, Allegrone G, Cavallo, M. and Fracchia, L. Biosurfactants, in Sustainable Development in Chemical Engineering Innovative Technologies. Piemonte V, De Falco M, Basile A (Eds), John Wiley & Sons, Ltd, Chichester, UK 2013; doi: 10.1002/9781118629703.ch9. Matarante A, Baruzzi F, Cocconcelli PS, Morea M. Genotyping and toxigenic potential of Bacillus subtilius and Bacillus pumilus strains occurring in industrial and artisanal cured sausages. Appl Environ Microbiol 2004; 70: 5168–5176. Ouoba LI, Cantor MD, Diawara B, Traoré AS, Jakobsen M. Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala, a fermented African locust bean condiment. J Appl Microbiol 2003; 95(4): 868-73. Pérez-García A, Romero D, de Vicente A. Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture. Cur Opin Biotec 2011; 22(2): 187-193. Revathi K, Chandrasekaran R, Thanigaivel A, Kirubakaran SA, Sathish-Narayanan S, Senthil-Nathan S. Effects of Bacillus subtilis metabolites on larval Aedes aegypti L Pesticide. Biochem Physiol 2013; 107(3): 369-376. Satomi M, La Duc MT, Venkateswaran K. Bacillus safensis sp. nov., isolated from spacecraft and assembly-facility surfaces. Int J Syst Evol Microbiol 2006; 56: 1735–1740. Seydlová, G, Čabala, R and Svobodová, J (2011). Surfactin - Novel Solutions for Global Issues, Biomedical Engineering, Trends, Research and Technologies, Sylwia Olsztynska (Ed.), ISBN: 978-953-307-514-3, InTech, DOI: 10.5772/13015. Available from: http://www.intechopen.com/books/biomedical-engineering-trends-research-andtechnologies/surfactin-novel-solutions-for-global-issues. Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 1996; 40: 2562-6. U. S. Environmental Protection Agency (USEPA). Biopesticides Registration Action Document, Bacillus pumilus strain QST 2808 (PC Code 006485). 2004; Office of Pesticide Programs, Washington, DC: Government Printing Office. 296 Chapter 2 Vakulenko SB, Donabedian SM, Voskresenskiy AM, Zervos MJ, Lerner SA, Chow JW. Multiplex PCR for detection of aminoglycoside resistance genes in enterococci. Antimicrob Agents Ch 2003; 47: 1423-6. Wei G, Kloepper JW, Tuzun S. Induced systemic resistance to cucumber diseases and increased plant growth by plant growth-promoting rhizobacteria under field conditions. Phytopathology 1996; 86: 221–224. Zhao X, Zhou Z, Han Y, Wang Z-Z, Fan J, Xiao H-Z. Isolation and identification of antifungal peptides from Bacillus BH072, a novel bacterium isolated from honey Microbiol Res 2013; 168(9):598-606. 297 304 CHAPTER 3 Characterization of the antibacterial compound produced by Bacillus safensis Bs1, the medicine-slug isolate Chapter 3 3.1. Characterization of the antibacterial compound produced by Bacillus safensis Bs1 Publication: Characterization of a new antimicrobial peptide with anti-MRSA activity produced by Bacillus safensis Bs1 Results disclosed from this work proved the hypothesis proposed for this thesis, in which clonal lineage adapted to an Arion ater, Bacillus safensis Bs1, revealed the production of a potentially novel cationic peptide (48-residues) with anti-staphylococci activity. 307 Chapter 3 Characterization of a new antimicrobial peptide with anti-MRSA activity produced by Bacillus safensis Bs1 Raquel Branquinho1, Hugo Osório2,3, Pedro Pereira4, Manuela E. Pintado5, Luísa V. Peixe1* 1REQUIMTE. Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade Farmácia, Universidade Porto, Porto, Portugal; 2IPATIMUP. Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal; 3Faculdade de Medicina, Universidade do Porto, Porto, Portugal; 4IBMC, Instituto de Biologia Molecular e Celular da Universidade do Porto, Porto, Portugal; 3CBQF, Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, 5Universidade Católica Portuguesa, Porto, Portugal. Running title: Antimicrobial peptides produced by Bacillus safensis Bs1 Keywords: Bacillus safensis, Antimicrobial peptide, SDS-PAGE, MALDI-TOF MS-MS, N-terminal sequencing, CD, AMPA *Corresponding author: Luísa Vieira Peixe, REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal Phone: +351-222078946; FAX: +351-222003977; E-mail: [email protected] Manuscript in preparation Patentability under evaluation 309 Chapter 3 Abstract We previously demonstrated the, in vitro, anti-staphylococci activity of a Bacillus safensis strain (Bs1) recovered from a slug used in the treatment of cutaneous infections. In the present work the characterization of an antimicrobial peptide produced by B. safensis Bs1 is presented. The Bs1 antimicrobial peptide was recovered and purified by a combination of SDSPAGE, ultrafiltration, ammonium sulfate precipitation and chromatographic approaches. The integrity of the purified peptide was verified by mass spectrometry and N-terminal sequencing and prediction of its antimicrobial stretches was also attempted, using AMPA and CAMP databases. The performed characterization revealed a new cationic peptide (48-residues) with antimicrobial activity against Gram-positive bacteria, including (Methicillin-resistant Staphylococcus aureus) MRSA. No remarkably amino acid sequence homologies were detected with other classes of antimicrobial peptides previously described. Further studies should be conducted in order to confirm the proposed amino acid composition and also its structure elucidation. Moreover, its antimicrobial property against a panel of clinical isolates expressing different mechanisms of resistance should also be extended. 310 Chapter 3 1. Introduction Due to the decline in antibiotic discovery, illustrated by a low number of new registered entities and a dramatic lack of new antibiotic classes, the increasing of multi-drug resistant bacteria causing infection is a serious concern (Carlet et al., 2012). Staphylococcus aureus is one of the most disturbing and common human pathogens, being the most frequent agent of skin and soft tissues infections, osteomyelitis and bacteremia (Elsayed et al., 2004; Weigelt et al., 2005). Higher morbidity and mortality in this species is associated with methicillin-resistant S. aureus (MRSA) which often are also resistant to diverse antibiotic classes (Appelbaum et al., 2007). In fact, in the last decades several pre-existing molecules have been modified in new derivatives, in an attempt to circumvent bacterial resistance mechanisms. Nevertheless, bacteria have demonstrated the potential to create new mechanisms of resistance allowing its surviving due to mutations or acquisition of resistance genes (Martínez and Baquero, 2002). It was only recently that linezolid, belonging to oxazolidinones class, was introduced in therapeutic use. Afterward, daptomycin and tygecicline also with activity upon MRSA, were introduced in therapeutic armamentarium. Nevertheless, and despite their recent introduction, linezolid and tygecycline resistant isolates have already emerged during treatment of S. aureus infections (Hentschke et al., 2008) being therefore imperative the recognition of therapeutic alternatives. Therapeutic applications of organic compounds from terrestrial microorganisms have an extensive past and present use in the treatment of diseases and serve as agents of interest both in their natural form and as templates for synthetic modification. In fact, secondary metabolites from natural sources and related drugs are used to treat 87% of all categorized human diseases, including antibacterial, anticancer, anticoagulant, antiparasitic, and immunosuppressive agents (Chin et al., 2006; Raja et al., 2010). The great number of antibiotics occurs as a result of secondary metabolic pathways and can be produced by different microorganisms with a diverse chemical nature, and such diversity is reflected in its mode of action (Stein, 2005). Among the most thoroughly studied secondary metabolites with remarkable therapeutic effects are antimicrobial peptides (AMPs) agents. Although their sequences vary, in general, they present an overall positive charge, an amphipathic structure and comprised of 10–50 amino acids, among which cysteine, lysine, proline or arginine are key compositions (Agerberth et al., 1991; Sahl et al., 2005; Groot et al., 2006; Lesmes et al., 311 Chapter 3 2009; Scocchi et al., 2011). Moreover, under hydrophobic environment, AMPs can fold into four classes of structures, including α-helix, β-sheets, extended structures, and loops (Brown and Hancock 2006; Peters et al., 2010). Related to its mechanisms of action, it is well known that most of them interact with bacterial membranes (Melo et al., 2009; Peters et al., 2010). In the case of Bacillus spp., AMPs compounds are subdivided into different classes, comprising bacteriocins (Oscáriz and Pisabarro 2000; Bizani and Brandelli 2002; Cherif et al., 2001, 2003 and 2008; Abriouel et al., 2011), bacteriocin-like inhibitory substances (BLIs) (Korenblum et al., 2005; Cladera-Oliveira et al., 2004; Aunpad and Na-Bangchang 2007; Xie et al., 2009), polyketides (PKs) (Chen et al., 2007; Arguelles-Arias et al., 2009), nonribosomal peptides (NRPs) (Naruse et al., 1990; Peypoux et al., 1999; Hathout et al., 2000; Bonmatin et al., 2002; Lee et al., 2007; Caboche et al., 2008; Rodrigues and Teixeira 2010; Nerurkar et al., 2010), miscellaneous antibiotics (Tamehiro et al., 2002; Pinchuk et al., 2002; Inaoka and Ochi 2007) and unusual peptides (Kino et al., 2009 and 2010; Borisova et al., 2010) Therefore, and considering the enormous diversity of Bacillus spp. in nature and the abundance of chemical structure compounds that they are able to produce, reflected also on its biological activities (Abriouel et al., 2011), they clearly represent an interesting challenge to explore. During several decades, in some regions of Portugal, a post mortem terrestrial black slug was used as traditional medicine in the treatment of cutaneous infections, including wounds and abscesses. Recently, we proved in vitro, a high anti-staphylococci activity, including in MRSA, of this slug (Branquinho, 2007). Preliminary data suggests the contribution of a peptide compound produced by a Bacillus safensis isolate (Branquinho et al 2014) for the observed antimicrobial activity. The high anti-MRSA activity and the therapeutic success reported for this traditional medicine together with the absence of description of secondary effects justifies the characterization of the bioactive compound(s) associated with this traditional medicine. Although some peptides with antibacterial activity have been described among B. pumillus group members, none was yet attributed to B. safensis. Thus, in the present work, throughout the use of advanced analytical and molecular methodologies we characterized the anti-bacterial compound produced by B. safensis Bs1 in a perspective of applicability to the pharmaceutical, biomedical and/or food sectors. 312 Chapter 3 2. Material and Methods 2.1. Bacterial strain B. safensis isolate Bs1 recovered from a black slug normal flora, which was collected from Northern of Portugal, was evaluated in its potential to produce antimicrobial compounds. Species identification was assessed by phenotypic, biochemical and genotypic (16S rRNA, gyrB and rpoB gene sequences) analysis (Branquinho et al 2014). Bacterium was maintained as stock culture at − 80 °C in Tryptic Soy Broth (TSB) (BD, Germany) supplemented with 20% (v/v) glycerol. 2.2. Production of AMP´s Pre-inoculum of B. safensis Bs1 isolate was prepared from Tryptic Soy Agar (TSA, BD, Germany) plates incubated overnight at 37 ºC. Their subculture was prepared under agitation at 250 rpm in 100 mL of TSB (TSB, BD, Germany) at pH=7.0. Then, for the inoculum preparation, 10 mL of pre-inoculum culture was transferred to 500 mL of TSB previously diluted (1:2) with sterile water and incubated for 24 h at 37 ºC, under agitation conditions (250 rpm). Finally, cells were separated by centrifugation at 8000 rpm during 20 min at 4 °C. Supernatants were then filtered through 0.45 µm pore-size filter (Millipore Corp., Bedford MA) to obtain a cell-free supernatant (CFS) for evaluation of the activities in the further tasks. 2.3. Screening for antimicrobial activity CFS was screened for its antimicrobial activity, by the agar well-diffusion method, against indicator strains summarized in Table 1. Briefly, indicator strains suspensions adjusted at 0.5 McFarland were swabbed in Mueller Hinton (MH) agar cation adjusted (bioMérieux, France) plates, where 5 mm diameter wells were cut and filled with 50 µL of CFS. Plates were then incubated at the optimal temperature and incubation times and inhibition zone diameters measured. Un-inoculated TSB broth was used as negative control. The experiment was conducted in triplicate on separate conditions. 313 Chapter 3 amino acid compositions and correspondent structures elucidation was poorly explored, which hinders comparisons. 3.5. Antimicrobial activity of the FPLC and HPLC purified fractions In order to separate the active Bs1 peptide, a combination of chromatographic techniques were applied and the fractions obtained tested for its antimicrobial activity against S. aureus ATCC 29213 using well diffusion assays. In both FPLC and HPLC analysis, the presence of active peptide in the different Bs1 CFS extracts was initially confirmed. Resulting chromatograms (Figure S1 and S2) beside the active peptide, seems to present a large number of other peptides and proteins. FPLC elution profiles analysis of ultrafiltered fractions with 10 kDa and 3 kDa membranes, followed by ammonium sulfate precipitation at 40% and 60% of saturation (Figure S1), showed the presence of an additional peak (when compared with the negative control), in the retention time between 50 and 60 min, which possibly corresponds to the previously associated active peptide with 5 kDa. After fractions collection, a lyophilization step was preceded and then the antimicrobial activity tested. Unfortunately, no fraction exhibited antimicrobial activity against the strain indicator tested. HPLC chromatograms analysis of the Bs1 active extracts (Figure S2) revealed the presence of distinct peaks (when compared with the negative control), after 35 min of retention time. Moreover, and under these conditions different inactive peptides seem to be also precipitated (Figure S2 (d) and (e)). All fractions derived from ammonium sulfate precipitation were collected, lyophilized and antimicrobial activity of each individual peak was assessed. Similar to FPLC, no fraction presented antimicrobial activity against the indicator organism tested. Thus, in order to evaluate the presence of the active peptide in the correspondent prepurified steps prior to structural characterization, fractions derived from these steps were also purified by C18 ZipTip and the single collected fraction tested for its antimicrobial activity. The resulting purified peptide from both 40% and 60% of ammonium sulfate precipitation revealed activity against S. aureus ATCC 29213, with inhibition zones of 18 mm detected by well diffusion assay. These results prompt us to suggest that possibly the chromatographic separation induced a structural change in the active peptide. Thus, the absence of antibacterial activity in the 320 Chapter 3 fractions recovered from FPLC and HPLC can be probably associated to the combination of some essential peaks, which cooperatively functionalize the activity of peptide and actively changed during HPLC and FPLC separation. Chromatographic separations can cause irreversible denaturation of peptide, thereby reducing the potential of their recovery in a biologically active form (Aguilar, 2003). Indeed, the nature of the organic solvent can influence the peptide conformation and also, the use of longer gradient times, despite provides improved separation, can also increase the residence time of the peptide at the sorbent surface, which may then result in an increase of the peptide denaturation degree (Aguilar 2003). Therefore, this hypothesis could possible justify the results obtained, since they can confer an impact on the level of recovery of biologically active material or impose important changes in peptide structure. 3.6. Characterization of the purified AMP 3.6.1. Mass spectrometry and PMF Mass spectrometry analysis of the excised SDS-PAGE gel bands, resolved with 40% and 60% of ammonium sulfate saturation, which revealed the presence of an antimicrobial peptide with approximately 5 kDa, demonstrate the presence of two ion peaks at m/z of 5372.6 and 5502.5 (Figure 4). This difference probably corresponds to the Met losses (- 131 Da) in the m/z 5372.6. Further, primary structure elucidation of these m/z peaks, conducted using their tryptic digestion, was accesses by MS/MS. The resulting digestion revealed the presence of two main fragment peptides (Figure 5) at m/z 1540.75 and 1991.87, corresponding respectively to SAPTCFLQGVGTFR and YYVDRDTGEICTSQER. PMF analysis was performed by Matrix Science Mascot UK software, and significant (P<0.05) result was obtained. After searching in the MASCOT protein database for amino acid sequences identity, the partially resulted sequence, matches the sequence of a hypothetical protein including 64 amino acids, which was derived from the genome of B. pumilus ATCC 7061T (gi 489307730), whose complete sequence is MLDRFLNKMTNVQPAASRCILQKLVTSCKSAPTCFLQGVGTFRYYVDRDTGEICTSQERI RCGC. Our partial sequence identified, present 100% of homology with 30 amino acids of the previous one, from position 30 to 59 (SAPTCFLQGVGTFRYYVDRDTGEICTSQER). Except for the amino sequence, no any other information such as function, genetic location or its relation with any antimicrobial peptide produced by bacteria was never been 321 Chapter 3 assigned. The predicted molecular weight of the total protein (7225.42 Da) did not match with the observed molecular weights (5372.6 and 5502.5 Da) (Figure 4). In addition, MALDI-TOF of the BS1 active peptide allows ascertaining the molecular mass predicted by the correspondent SDS-PAGE with higher reliability. One possible explanation which can be speculated is the fact that our Bs1 peptide is a result of peptide maturation where the N-terminal extension of precursor peptide is cleaved off by the action of a certain enzyme that possible cleaves at a Met residue when the adjacent residues are non-bulky amino acids (Giglione et al., 2004). 3.6.2. N-terminal amino acid sequencing and analysis Bs1 was subjected to automated Edman degradation to determine their N-terminal amino acid sequence. Their analysis provided the identification of a partial sequence A(N,V,I,L)- A(Q,P,I)-?(P,A,R,Q,D)-R(L,F,G,H)-?-I(T,Q,M,G)-L(Q)-Q(K)-K-L(V)-V-T(V,S)-S(T)-?-K(F), which present some uncertain positions in the assigned amino acids in brackets. However, a database search revealed homology with the same hypothetical protein as determined by MS/MS approach, namely gi 489307730 from B. pumilus ATCC 7061T. Therefore, custom synthesis was performed and synthetic peptide (MLDRFLNKMTNVQPAASRCILQKLVTSCKSAPTCFLQGVGTFRYYVDRDTGEICTSQER IRCGC) was evaluated on its antibacterial activity against S. aureus. Results evidenced that linear structure of this peptide have not any effect in the growth of S. aureus. The absence of cationic amino acids in the N-terminal sequence of this 64 amino acid peptide, reported as a crucial factor for antimicrobial activity, could be a possible justification for this result. In addition, comparison of resulting N-terminal sequence and peptide gi 489307730 seems to suggest a N-terminal staggered from position 11 or 15. In this sense, further partial custom syntheses of the partial sequence SRCILQKLVTSCKSAPTCFLQGVGTFRYYVDRDTGEICTSQERIRCGC should be performed and antibacterial activity tested. 322 Chapter 3 3.6.3. Secondary structure prediction Circular dichroism (CD) spectroscopy was employed in an attempt to elucidate secondary structure integrity of the 5kDa purified peptide. The linear conformation of the synthesized synthetic peptide was used for comparison. CD spectra suggested that Bs1 peptide presented a random coil, e.g. unstructured nature in solution with a characteristic minimum at approximately 200 nm (Figure 6 and 7). Most naturally antimicrobial peptides occurring in nature are cationic (i.e., the net charge at neutral pH varies from + 2 to + 9) and amphipathic molecules, which enables the peptides to interact with and disrupt lipid membranes. Moreover, they are very short in length, containing 5 to 40 amino acid residues, while others containing more than 40 residues. Amino acid sequences of different AMPs are highly heterogeneous, nevertheless, positively charged residues such as Lys and Arg and substantial hydrophobic residues (∼ 30% or more) are commonly found in these peptides class. In addition, a great variation in their secondary structures has also been reported, such as α-helices, βsheets, or extended polyproline-like helices, due to the amphipathicity of these molecules which enhanced their antimicrobial mechanism of action. Nevertheless, most of the linear AMPs are unstructured in solution, with some exceptions, e.g. LL37, a human antimicrobial peptide (Wildman et al., 2003; Dürr et al., 2006) and cyclic peptides due to the presence of one or more Cys–Cys disulfide bonds which possibly the formation β-sheets (Dhople et al., 2006). Moreover, these unstructured (linear) cationic peptides present the possibility to embrace an amphipathic α-helical structure. In fact, while these peptides lack a secondary structure in solution, interactions with lipid bilayers in membranes induce an amphipathic α-helical structure that is believed to be a pre-requisite for its antimicrobial activity (Jin et al 2005) by the formation of pore-like structures (He et al., 1995; Matsuzaki et al., 1995) or a more general disruption (Oren et al., 1998). Therefore, and since the mechanism by which an AMP executes its function depends on a number of physicochemical properties, such as the amino acid sequence, net charge, amphipathicity, hydrophobicity, structural folding (includes secondary structure, dynamics and orientation) in membranes, oligomerization, peptide concentration, and membrane 323 Chapter 3 composition (Shai, 1999), further studies should be conducted in order to clarify the mechanisms of action of the unstructured Bs1 AMP to explain its antimicrobial activity. 3.6.4. Detection of possible post-translational modifications: glycosylation Using SDS–PAGE electrophoresis followed by PAS staining, we did not observe any positive reaction for the purified BS1 CFS (Figure 8 (a)). Nevertheless, since limitations of this method are known, namely in the nature and in the degree of protein glycosylation, this results and evaluation of other possible post-translational modifications, as the determination of the presence of lipid moieties, should be attempted. 3.6.5. Prediction of protein antimicrobial regions Due to the growing interest in AMPs, web applications as AMPA, based on algorithms, which provides the prediction of antimicrobial domains in proteins and can be used to propose short AMP, have been developed (Lata et al., 2007; Fjell et al., 2009; Torrent et al., 2011; Wang et al., 2011). This platform uses an antimicrobial propensity scale to generate an antimicrobial profile, for which antimicrobial IC50 values for all amino acid are determined. Therefore, from the IC50 data, an antimicrobial index is calculated, providing a tendency of such amino acid to be found within an AMPs sequence. Considering Bs1 sequence presenting N-terminal sequence starting in amino acid 15 from gi 489307730, SRCILQKLVTSCKSAPTCFLQGVGTFRYYVDRDTGEICTSQERIRCGC, AMPA detect an antimicrobial domain, presenting a high probability to pertain to an antimicrobial peptide (Figure 9). This antimicrobial stretch was found in amino acids 1 to 13 positions, SRCILQKLVTSCK, from the resulting peptide. Statistic results revealed a propensity value 0.226 (9 %) and a mean antimicrobial value of 0.243, which permit to infer with 91% of probability that this sequence pertain to an antimicrobial peptide, supported by a low antimicrobial index, which also favor to be part of an AMP. In fact, an adequate combination of hydrophobic and cationic amino acid residues, such as Arg, Lys, Gly, Cys, Trp and Val, underlies the action of antimicrobial regions, generally by conferring them an amphipathic secondary structure that favors interaction with microbial membranes, which can induces cell damage (Wimley, 2010). Therefore, the antimicrobial activity is usually confined to discrete stretches of the protein, often located 324 Chapter 3 at the Nor C-terminal of proteins, rather than spread over the entire sequence (Ramanathan et al., 2002). When gi 489307730 was analyzed by AMPA the stretch RCILQKLVTSCK is assigned to pertain to an antimicrobial peptide, with a propensity value 0.229 (12 %) and a mean antimicrobial value of 0.246. This results can confirm our previously assumption, that N-terminal of antimicrobial mature protein derived from gi 489307730 start in position 17. Moreover Collection of Antimicrobial Peptide (CAMP) database (http://www.camp.bicnirrh.res.in/) was also used to confirm the previous established antimicrobial region, using different statistical algorithms, such as Support Vector Machine (SVM), Random Forests (RF) and Artificial Neural Network (ANN) (Schaffer et al., 2001). For biological activity structural or physical parameters could be considered critical, namely size, sequence, charge, degree of structuring (helicity), hydrophobicity, amphipathicity and angles subtended by hydrophobic and hydrophilic faces of the formed helix (Faccone et al., 2014). Therefore, physicochemical properties as amino acid composition, net charge, hydrophobicity, aliphatic index, instability index and pI were determined and results summarized in table 2. Physicochemical properties of the 48 amino acids predicted antibacterial peptide, revealed a pI of 8.65, an aliphatic index of 68.96, a net charge of 3 (total number of negatively charged residues (Asp + Glu): 4; total number of positively charged residues (Arg + Lys): 7), a hydropathy of -0.15 and an instability index of 46.36 (which classifying the peptide as unstable are in agreement with CD data). Moreover, using antimicrobial peptides and proteins collections deposited in CAMP database, homologous sequences searches using BLAST were performed (Table S1). Nevertheless, no significant similarity was observed among our antimicrobial peptide with available AMP´s present in this database, suggesting that probably a new peptide can be involved in the reported activity, despite some reported antimicrobial proteins and peptides, including a short fraction of subtilin, present some similarity with our peptide. Therefore, our finding and predictions prompt us to suggest the presence of a new alternative antibiotic as a potential candidate to circumvent S. aureus infections and thus a good possibility for its inclusion in topical use. Further investigations should be carried out to confirm the proposed amino acid composition and its structure elucidation. Moreover, its antimicrobial activity against a panel of clinical isolates expressing different mechanisms of resistance should also be extended. 325 Chapter 3 Table 1. Strains used in antimicrobial activity assays. Isolates Origin Susceptibility profile Reference Gram positive Staphylococcus aureus ATCC 29213 NA ATCC Staphylococcus aureus MRSA FFUP 001 Methicillin resistant FFUPCC Staphylococcus epidermidis Ste1 Catheter - Portugal (2012) Methicillinand Linezolidresistant Barros et al., 2014 Staphylococcus epidermidis 55 Human commensal flora– Italy (2011) Susceptible Cavallo, 2011 Enterococcus faecium (VRE) E1 Hospital sewage – Portugal (2001) Vancomycin resistant Freitas et al., 2009 Bacillus subtilis BGA NA Spore suspension, Merck (Product number 1106490001) Bacillus pumilus ATCC 14884 Animal commensal flora– Portugal (2004) NA ATCC Bacillus safensis Bs1 Intermediate profile to Cefotaxime Branquinho et al., 2014 Bacillus cereus FFUP 95 NA FFUPCC Lysinibacillus fusiformis Animal commensal flora– Portugal (2004) Intermediate profile to Clindamycin FFUPCC Listeria monocytogenes FFUP 35 NA FFUPCC Gram negative Acinetobacter baumanni ATCC 19606 NA ATCC Escherichia coli ATCC 25922 NA ATCC Pseudomonas aeruginosa ATCC 27853 NA ATCC Salmonella Typhymurium F154/22 Food – Portugal (2004) Nitrofurantoin resistant FFUPCC; Antunes et al., 2006 Salmonella Enteritidis 62/02 Human – Portugal (2002) Susceptible INSA; Antunes et al., 2006 NA – not applied; ATCC - American Type Culture Collection; FFUPCC - Faculty of Pharmacy of Porto University Culture Collection; INSA - National Institute Dr. Ricardo Jorge Culture Collection 326 Chapter 3 Table 2. Prediction of antimicrobial regions accordingly CAMP database. Algorithm Class AMP Probability Artificial Neural Network (ANN) classifier AMP NA Discriminant Analysis classifier 0.884 Random Forest Classifier 0.7465 Support Vector Machine (SVM) 0.646 327 Chapter 3 Figure 1. Antibacterial activity against S. aureus ATCC 29213 of the purified peptide recovered from B. safensis Bs1, using well diffusion assay. Legend: (1) direct Bs1 CFS, (2) ultrafiltered fraction followed by peptide precipitation using 40% (w/v) and (3) 60% (w/v) of ammonium sulfate saturation. Bs1 peptide revealed 14, 16 and 17 mm of inhibition zones for S. aureus ATCC 29213, respectively. Similar results were obtained in the three individual experiments. 328 Chapter 3 Figure 2. SDS-PAGE of the precipitated proteins from CFS of B. safensis Bs1. Lane 1: direct CFS Bs1, Lane 2: fraction ultrafiltered throughout >10 kDa, Lane 3: fraction ultrafiltered followed by peptide(s) precipitation using 40% (w/v) ammonium sulfate saturation, Lane 4: fraction ultrafiltered followed by peptide(s) precipitation using 60% (w/v) ammonium sulfate saturation, Mw: Molecular weight marker. 329 Chapter 3 Figure 9. AMPA results from the antimicrobial chart profile of the Bs1 sequence peptide. On X-axis is reported the amino acid position in the peptide and on the Y-axis the antimicrobial score at that position. The probability values displayed correspond to the probability to find the predicted stretch in a non-antimicrobial protein. 336 Chapter 3 References Abriouel H, Franz CM, Ben Omar N, Gálvez A. Diversity and applications of Bacillus bacteriocins. FEMS Microbiol Rev 2011; 35(1): 201-32. Agerberth B, Lee JY, Bergman T, Carlquist M, Boman HG, Mutt V, Jörnvall H. Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of prolinearginine-rich antibacterial peptides. Eur J Biochem 1991; 202: 849–854. Aguilar M-A. Reversed-Phase High-Performance Liquid Chromatography, edited by Marie-Isabel Aguilar (HPLC of peptides and proteins: methods and protocols; Walker JM ed. Humana Press, Totowa, NJ, Methods in Molecular Biology 2003; 251: 9-22. Alam SI, Kamran M, Sohail M, Ahmad A, Khan SA. Partial characterization of bacteriocin like inhibitory suabtance from Bacillus subtilis BS15, a local soil isolate. Pak J Bot 2011; 43(4): 2195-2199. Appelbaum PC. Microbiology of Antibiotic Resistance in Staphylococcus aureus. Clin Infect Dis 2007; 45: S165-S170. Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, Fickers P. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microbl Cell Fact 2009; 8: 63. Aunpad R, Na-Bangchang K. Pumilicin 4, A Novel Bacteriocin with Anti-MRSA and AntiVRE Activity Produced by Newly Isolated Bacteria Bacillus pumilus Strain WAPB4. Curr Microbiol 2007; 55: 308-313. Baindara P, Mandal SM, Chawla N, Singh PK, Pinnaka AK, Korpole S. Characterization of two antimicrobial peptides produced by a halotolerant Bacillus subtilis strain SK.DU.4 isolated from a rhizosphere soil sample. AMB Express 2013; 3(1): 2. Barros M, Branquinho R, Grosso F, Peixe L, Novais C (2013) Emergence of Methicillin and Linezolid Resistant ST2/CC5 Staphylococcus epidermidis in Portugal: First Cases Report. Emerging Infectious Diseases (In Press). Bizani D, Brandelli A. Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A. J Appl Microb 2002; 93: 512-519. 337 Chapter 3 Bonmatin JM, Laprevote O, Peypoux F. Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents. Comb Chem High Throughput Screen 2003; 6: 541-556. Borisova SA, Circello BT, Zhang JK, van der Donk WA, Metcalf WW. Biosynthesis of Rhizocticins, Antifungal Phosphonate Oligopeptides Produced by Bacillus subtilis ATCC6633. Chem Biol 2010; 17: 28-37. Branquinho R, Meirinhos-Soares L, Carriço JA, Pintado M, Peixe LV. Phylogenetic and clonality analysis of Bacillus pumilus isolates uncovered a highly heterogeneous population of different closely related species and clones. FEMS Microbiol Ecol 2014 (submitted). Branquinho R. Searching and characterization of antimicrobial activity associated with terrestrial slug Arion ater. Porto: Faculty of Pharmacy, University of Porto - Department of Microbiology; 2007. Brown KL, Hancock RE. Cationic host defense (antimicrobial) peptides. Curr Opin Immunol 2006; 18: 24–30. Caboche S, Pupin M, Leclère V, Fontaine A, Jacques P, Kucherov G. NORINE: a database of nonribosomal peptides. Nucleic Acids Research 2008; 36. Carlet J, Jarlier V, Harbarth S, Voss A, Goossens H, Pittet D. Ready for a world without antibiotics? The Pensières Antibiotic Resistance Call to Action. Antim Resist Infect Contl 2012, 1:11. Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, Junge H, Voigt B, Jungblut PR, Vater J, Sussmuth R, Liesegang H, Strittmatter A, Gottschalk G, Borriss R. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotech 2007; 25: 1007-1014. Cherif A, Chehimi S, Limem F, Hansen BM, Hendriksen NB, Daffonchio D, Boudabous A. Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9. Journal of Applied Microbiology 2003; 95: 990-1000. 338 Chapter 3 Cherif A, Ouzari H, Daffonchio D, Cherif H, Ben Slama, K, Hassen A, Jaoua S Soudabous A. Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil. Lett Appl Microbiol 2001; 243–247. Cherif A, Rezgui W, Raddadi N, Daffonchio D, Boudabous A. Characterization and partial purification of entomocin 110, a newly identified bacteriocin from Bacillus thuringiensis subsp. entomocidus HD110. Microbiol Research 2008; 163: 684-692. Chin Y-W, Balunas MJ, Chai HB, Kinghorn AD. Drug discovery from natural sources. The AAPS Journal 2006; 8(2):28. Cladera-Olivera F, Caron GR, Brandelli A. Bacteriocin-like substance production by Bacillus licheniformis strain P40. Lett Appl Microbiol 2004; 38: 251-256. Compaoré CS, Nielsen DS, Ouoba LI, Berner TS, Nielsen KF, Sawadogo-Lingani H, Diawara B, Ouédraogo GA, Jakobsen M, Thorsen L. Co-production of surfactin and a novel bacteriocin by Bacillus subtilis subsp. subtilis H4 isolated from Bikalga, an African alkaline Hibiscus sabdariffa seed fermented condiment. Int J Food Microbiol 2013; 162(3): 297-307. Dhople V., Krukemeyer A., Ramamoorthy A. The human beta-defensin-3, an antibacterial peptide with multiple biological functions. BBA Biomembranes 2006; 1758: 1499–1512. Dürr UHN, Sudheendra US, Ramamoorthy A. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. BBA Biomembranes 2006; 1758: 1408–1425. Elsayed S, Laupland KB. Emerging gram-positive bacterial infections. Clin Lab Med 2004; 24(3):587-603. Faccone D, Veliz O, Corso A, Noguera M, Martínez M, Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, Saenger W, Bernhard F, Reinhardt R, Schmidt M, Ullrich C, Stein T, Leenders F, Vater J. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc. Natl. Acad. Sci USA 1999; 96:13294–13299. Fjell CD, Jenssen H, Hilpert K, Cheung WA, Panté N, Hancock REW, Cherkasov A. Identification of novel antibacterial peptides by chemoinformatics and machine learning. J Med Chem 2009; 52: 2006-2015. 339 Chapter 3 Giglione C, Boularot A, Meinnel T. Protein N-terminal methionine excision. Cellular and Molecular Life Sciences CMLS 2004; 61(12): 1455-1474. Greenfield N, Fasman GD. Computed Circular Dichroism Spectra for the Evaluation of Protein Conformation, Biochemistry 1996; 8(10): 4108. Groot F, Sanders RW, ter Brake O, Nazmi K, Veerman EC, Bolscher JGM, Berkhout B. Histatin 5-derived peptide with improved fungicidal properties enhances human immunodeficiency virus type 1 replication by promoting viral entry. J Virol 2006; 80: 9236– 9243. Hathout Y, Ho YP, Ryzhov V, Demirev P, Fenselau C. Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis. J Nat Prod 2000; 63: 1492–1496. He K, Ludtke SJ, Huang HW, Worcester DL. Antimicrobial peptide pores in membranes detected by neutron in-plane scattering. Biochemistry 1995; 34: 15614-15618. Hentschke M, Saager B, Horstkotte MA, Scherpe S, Wolters M, Kabisch H, Grosse R, Heisig P, Aepfelbacher M, Rohde H. Emergence of linezolid resistance in a methicillin resistant Staphylococcus aureus strain. Infection 2008; 36(1): 85-87. Inaoka T, Ochi K. Glucose Uptake Pathway-Specific Regulation of Synthesis of Neotrehalosadiamine, a Novel Autoinducer Produced in Bacillus subtilis. J Bacteriol 2007; 189: 65-75. Jin Y, Hammer J, Pate M, Zhang Y, Zhu F, Zmuda E, Blazyk J. Antimicrobial Activities and Structures of Two Linear Cationic Peptide Families with Various Amphipathic β-Sheet and α-Helical Potentials. Antimicrob Agents Chemother 2005; 49(12): 4957-64. Kino K, Kotanaka Y, Arai T, Yagasaki M. A Novel L-Amino Acid Ligase from Bacillus subtilis NBRC3134 Catalyzed Oligopeptide Synthesis. Bioscience, Biotechnology and Biochemistry 2009; 73: 901-907. Korenblum E, von Der Weid I, Santos ALS, Rosado AS, Sebastián GV, Coutinho CMLM, Magalhães FCM, de Paiva MM, Seldin L. Production of antimicrobial substances by Bacillus subtilis LFE-1, B. firmus H2O-1 and B. licheniformis T6-5 isolated from an oil reservoir in Brazil. J Appl Microbiol 2005; 98: 667-675. 340 Chapter 3 Lata S, Sharma BK, Raghava GP. Analysis and prediction of antibacterial peptides. BMC Bioinformatics 2007; 8:263. Lee SC, Kim SH, Park IH, Chung SY, Choi YL. Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity. Arch Microbiol 2007; 188: 307-312. Lesmes LP, Bohorquez MY, Carreno LF, Patarroyo ME, Lozano JM. A C-terminal cationic fragment derived from an arginine-rich peptide exhibits in vitro antibacterial and antiplasmodial activities governed by its secondary structure properties. Peptides 2009; 30: 2150–2160. Lisboa MP, Bonatto D, Bizani D, Henriques JAP, Brandelli A. Characterization of a bacteriocin-like substance produced by Bacillus amyloliquefaciens isolated from the Brazilian Atlantic Forest. Intl Microbiol 2006; 9: 111-118. Martínez JL, Baquero F. Interactions among strategies associated with bacterial infection: pathogenicity, epidemicity, and antibiotic resistance. Clin Microbiol Rev 2002; 15(4): 64779. Matsuzaki K, Murase O, Fujii N, Miyajima K. Translocation of a channel-forming antimicrobial peptide, magainin 2, across lipid bilayers by forming a pore. Biochemistry 1995; 34: 6521-6526. Melo MN, Ferre R, Castanho MA. Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations. Nat Rev Microbiol 2009; 7: 245–250. Naruse N, Tenmyo O, Kobaru S, Kamei H, Miyaki T, Konishi M, Oki T. Pumilacidin, a complex of new antiviral antibiotics. Production, isolation, chemical properties, structure and biological activity. J Antibiot (Tokyo) 1990; 43: 267 280. Nerurkar AS. Structural and molecular characteristics of lichenysin and its relationship with surface activity. Adv Exp Med Biol 2010;672:304–15. Oren Z, Shai Y. Mode of action of linear amphipathic α-helical antimicrobial peptides. Biopolymers 1998; 47: 451-463. 341 Chapter 3 Oscáriz JC, Pisabarro AG. Characterization and mechanism of action of cerein 7, a bacteriocin produced by Bacillus cereus Bc7. J Appl Microbiol 2000; 89: 361-369. Peters BM, Shirtliff ME, Jabra-Rizk MA. Antimicrobial peptides: primeval molecules or future drugs? PLoS Pathog 2010; 6: e1001067. Peypoux F, Bonmatin JM, Wallach J. Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol 1999; 51: 553-563. Pinchuk IV, Bressollier P, Sorokulova IB, Verneuil B, Urdaci MC. Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats. Res Microbiol 2002; 153: 269-276. Raja PG, Raja M. Drugs from the Natural Bio Sources for Human Disease. International Journal of Pharmacology 2010; 6: 360-363. Ramanathan B, Davis EG, Ross CR, Blecha F. Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity. Microbes Infect 2002; 4: 361-372. Rodrigues LR, Teixeira JA. Biomedical and therapeutic applications of biosurfactants Adv Exp Med Biol 2010; 672: 75-87. Sahl HG, Pag U, Bonness S, Wagner S, Antcheva N, Tossi A. Mammalian defensins: structures and mechanism of antibiotic activity. J Leukoc Biol 2005; 77: 466–475. Schaffer AA, Aravind L, Madden TL, Shavirin S, Spouge JL, Wolf YI, Koonin EV, Altschul SF. Improving the accuracy of PSI-BLAST protein database searches with compositionbased statistics and other refinements. Nucleic Acids Res 2001; 29: 2994-3005. Scocchi M, Tossi A, Gennaro R. Proline-rich antimicrobial peptides: converging to a nonlytic mechanism of action. Cell Mol Life Sci 2011; 68: 2317–2330. Shai Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta, 1999; 1462: 55–70. Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol 2005; 56: 845-857. 342 Chapter 3 Tamehiro N, Okamoto-Hosoya Y, Okamoto S, Ubukata M, Hamada M, Naganawa H, Ochi K. Bacilysocin, a Novel Phospholipid Antibiotic Produced by Bacillus subtilis 168. Antimicrobial Agents and Chemotherapy 2002; 46: 315-320. Thomas S, Karnik S, Barai RS, Jayaraman VK, Idicula-Thomas S. Camp: a useful resource for research on antimicrobial peptides. Nucleic Acids Res 2010; 38:D774–80. Torrent M, Tommaso PD, Pulido D, Nogués MV, Notredame C, Boix E, Andreu D. AMPA: an automated web server for prediction of protein antimicrobial regions. Bioinformatics 2012; 28(1): 130-131. Torrent M, Andreu D, Nogués VM, Boix E. Connecting peptide physicochemical and antimicrobial properties by a rational prediction model. PLoS One 2011; 6: e16968. Wang P, Hu L, Liu G, Jiang N, Chen X, Xu J, Zheng W, Li L, Tan M, Chen Z, Song H, Cai Y-D, Chou K-C. Prediction of antimicrobial peptides based on sequence alignment and feature selection methods. PLoS One 2011; 6: e18476. Weigelt J, Itani K, Stevens D, Lau W, Dryden M, Knirsch C. Linezolid versus Vancomycin in Treatment of Complicated Skin and Soft Tissue Infections. Antimicrob Agents Chemother 2005; 49(6): 2260-2266. Wildman KAH. The mechanism of lipid bilayer disruption by the human antimicrobial peptides. Michigan: University of Michigan; 2003. Wimley WC. Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS Chem Biol 2010; 5: 905-917. Xie J, Zhang R, Shang C and Y, G. Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112 that exhibits antimicrobial activity against domestic animal pathogens. Afr J Biotechnol 2009; 5611–5619. 343 Chapter 3 Supplementary data Characterization of a new antimicrobial peptide with anti-MRSA activity produced by Bacillus safensis Bs1 Raquel Branquinho1, Hugo Osório2,3, Pedro Pereira4, Manuela E. Pintado5, Luísa V. Peixe1* 1REQUIMTE. Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade Farmácia, Universidade Porto, Porto, Portugal; 2IPATIMUP. Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal; 3Faculdade de Medicina, Universidade do Porto, Porto, Portugal; 4IBMC, Instituto de Biologia Molecular e Celular da Universidade do Porto, Porto, Portugal; 3CBQF, Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, 5Universidade Católica Portuguesa, Porto, Portugal. Running title: Antimicrobial peptides produced by Bacillus safensis Bs1 344 Chapter 3 Table S1. Comparison resulting sequences producing significant alignment and blast results from homologous sequences using BLAST tool inside the CAMP database. CAMP ID UniProt ID Protein name Taxonomy Sequence Blast Score (bits) Values Expect Identities Bacteria CAMPSQ12 04 P10946 Lantibiotic subtilin Bacillus subtilis WKSESLCTPGCVTGALQTCFLQTLTCNCKISK 18.1 bits (35) 9.9 6/11 (55%) CAMPSQ32 88 P36961 Lactococcin G-a Lactococcus lactis LMGT2081 GTWDDIGQGIGRVAYWVGKALGNLSDVNQASRINRKKKH 18.5 bits (36) 8.1 6/17 (35%) Animalia CAMPSQ23 90 E1B2T7 Brevinin a) 2LF 2 Amphibians IMSTLKQFGISAIKGAAQNVLGVLSCKIAKTC 18.5 bits (36) 6.8 7/10 (70%) CAMPSQ42 90 K7ZGR3 Brevinin 2 a) AJ1 GLMSTFKRVGISAIKGAAKNVLDVLSCKIAKTC 18.5 bits (36) 7.0 7/10 (70%) CAMPSQ42 84 K7ZAL2 AJ2 GLMSTFKQVGISAIKGAAKNVLDVLSCKIAKTC 18.5 bits (36) 7.0 7/10 (70%) CAMPSQ42 89 K7Z432 AJ3 GLMSTFKQVGISAIKGAAQNVLGVLSCKLAKTC 18.5 bits (36 ) 7.0 7/10 (70%) CAMPSQ42 87 K7ZAL3 AJ7 GLVSTFKQVGISAIKGAAKNVLDVLSCKIAKTC 18.5 bits (36) 7.0 7/10 (70%) CAMPSQ42 83 K7ZJ56 AJ4 GFMSTFKQVGISAIKGAAKNVLDVLSCKIAKTC 18.5 bits (36) 7.0 7/10 (70%) CAMPSQ13 01 Q9U6U0 Antimicrobial peptide MGD2b Molluscs (Bivalvia) MKAVFVLLVVGLCIMMMDVATAGFGCPNNYACHQHCKSIRGY CGGYCASWFRLRCTCYRCGGRRDDVEDIFDIYDNVAVERF 19.6 bits (39) 4.7 7/14 (50%) a)antimicrobial peptide precursor Bs1 3 CILQKLVTSCK 13 C LQ L +CK Subtilin 19 CFLQTLTCNCK 29 Bs1 21 QGVGTFRYYVDRDTGEI 37 QG+G Y+V + G + Lactocin 8 QGIGRVAYWVGKALGNL 24 Bs1 9 VTSCKSAPTC 18 V SCK A TC Brevinin LF2 23 VLSCKIAKTC 32 Bs1 9 VTSCKSAPTC 18 V SCK A TC Brevinin 2AJ1 24 VLSCKIAKTC 33 Bs1 9 VTSCKSAPTC 18 V SCK A TC Brevinin 2AJ2 24 VLSCKIAKTC 33 Bs1 9 VTSCKSAPTC 18 V SCK A TC Brevinin 2AJ3 24 VLSCKLAKTC 33 Bs1 9 VTSCKSAPTC 18 V SCK A TC Brevinin 2AJ7 24 VLSCKIAKTC 33 Bs1 9 VTSCKSAPTC 18 V SCK A TC Brevinin 2AJ4 24 VLSCKIAKTC 33 Bs1 35 GEICTSQERIRCGC 48 G C S R+RC C MGD2b 44 GGYCASWFRLRCTC 57 345 Chapter 4 4.1. Species-specific surfactin-like biosurfactants combination within Bacillus pumilus group Publication: Species-specific surfactin-like combinations of biosurfactants within Bacillus pumilus group This work addresses the species-specific combinations of biosurfactants, namely surfactin-like compounds achieved in Bacillus safensis and Bacillus pumilus. Remarkably, B. safensis isolates revealed the production of a mixture of biosurfactants composed of pumilacidin variants and, in smaller amounts of a surfactin variant, whereas in B. pumilus only pumilacidin variants were detected. Moreover B. altitudinis isolates doesn´t revealed surface-active compounds production. In addition absence of correlation between detection of lipopeptides synthetase genes by a PCR approach and biosurfactant production was also detected. Results disclosed from this work highlight these species as further exploring sources of bioactive compounds, broadening their potential applications. 353 Chapter 4 Species-specific surfactin-like combinations of biosurfactants within Bacillus pumilus group Raquel Branquinho1, Luísa V. Peixe1, João Pires1, Manuela E. Pintado2, Maria Giovanna Martinotti3, Letizia Fracchia3, Gianna Allegrone3 1REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal; 2CBQFCentro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal; 3Department of Pharmaceutical Sciences (DSF), University of Eastern Piemont Amadeo Avogadro, Novara, Italy. Running title: Surfactin-like combinations within Bacillus pumilus group species biosurfactants. Key-words: pumilacidin, surfactin, biosurfactants, Bacillus pumilus, Bacillus safensis *Corresponding author: Gianna Allegrone, Department of Pharmaceutical Sciences (DSF), University of Eastern Piemont Amadeo Avogadro,Largo Donegani 2, 28100, Novara, Italy Phone: + 390321375755 ; Fax : + 390321375621; E-mail: [email protected] Manuscript submitted to: Bioresource Technology 355 Chapter 4 Abstract Assessing the diversity of biosurfactants produced by B. pumilus group species we verified surface-active properties compatible with biosurfactant compounds in most B. pumilus (n=3 of 4 isolates) and B. safensis (n=9 of 15) isolates tested, and their absence in B. altitudinis (n=9). Moreover, srf/lch (100%) and fen (52%) genes were frequently detected, although iturin family genes were not observed. Interestingly, LC/ESI-MS/MS analysis revealed a mixture of pumilacidin variants and surfactin compounds in B. safensis, whereas in B. pumilus only pumilacidin variants were detected. These data contrast with B. subtilis were surfactin and fengycin were detected. In conclusion, despite the frequent detection of surfactin and fengycin genes in B. pumilus group isolates, surface-active properties were only associated with surfactin-like combinations in B. safensis and B. pumilus. Interestingly, a species-specific content in biosurfactant homologues was observed. Further studies of these biosurfactant variants could unveil interesting effects, broadening potential applications of these compounds. 356 Chapter 4 1. Introduction Biosurfactants (i. e. surface-active compound of microbial origin), are particular amphipathic molecules that recently have emerged as a promising class of bioactive secondary metabolites with increasing scientific, therapeutic and biotechnological interest (Banat et al., 2010, Fracchia et al., 2012, Gudiña et al., 2013, Martinotti et al., 2013, Seydlová et al., 2011). Among the different current classes, lipopeptides, due to their structural novelty and versatility, represent one of the most important ones widely produced among Bacillus species (Fracchia et al., 2012, Kalinovskaya et al., 2002, Martinotti et al., 2013, SoberónChávez, 2011). Chemically, these compounds are constituted of 7–10 amino acids, which are cyclized by a lactone ring to a β-hydroxy fatty acid with distinct chain lengths. Moreover, they are classified into different families, encompassing surfactins (Arima et al., 1968), iturins (Delcambe et al., 1977), fengycins (Vanittanakom et al., 1986), bacillomycins (Roongsawang et al., 2002), mycosubtilin (Duitman et al., 1999) and kurstakins (Hathout et al., 2000). Surfactin family encompasses about 20 different lipopeptides biosurfactants (also named surfactin-like compounds), which have been reported as widely produced by members of Bacillus subtilis complex (Soberón-Chávez, 2011). Moreover, B. pumilus species, taxonomically classified into B. pumilus group, which belong to B. subtilis complex, have also been described as surfactin-like producers, especially for a biosurfactant named pumilacidin (Melo et al., 2009, Morikawa et al., 1992, Naruse et al., 1990). Nevertheless, until our knowledge, its production was not demonstrated in other members of this group, namely in B. safensis and B. altitudinis. Since the discovery of surfactin (Arima et al., 1968) and the characterization of its molecular structure as a macrolide lipopeptide, it has been recognized its remarkable surface-, interfaceand membrane-active properties, which have resulted in a high number of promising biological activities of great relevance in biotechnology (Fracchia et al., 2012, Seydlová et al., 2011), medicine (Banat et al., 2010, Fracchia et al., 2012, Gudiña et al., 2013, Martinotti et al., 2013, Seydlová et al., 2011) and environmental protection (Pacwa-Płociniczak et al., 2011). Nevertheless, the knowledge concerning surfactin-like biosurfactants as a class of molecules remains limited. In fact, there's a lack of a concerted effort to perform a comprehensive screening for biosurfactants producers 357 Chapter 4 and, in addition, the present knowledge in the surfactin-like biosurfactants has been developed around a relatively small number of well-characterized molecules. Lipopeptide biosurfactants share a common mode of assembly through the action of multienzymatic proteins organized in modules, named non-ribosomal peptide synthetases (NRPSs) by a thiotemplate process (Tapi et al., 2010). Although the genetics of surfactin biosynthesis and its control have been most extensively studied in B. subtilis (Tapi et al., 2010), genes homologous to their peptide synthetases have not yet been studied in B. pumilus group. Nevertheless, molecular approaches solely might not be useful for the screening of biosurfactant producers (Soberón-Chávez, 2011) thus methodologies for the detection of putative biosurfactant-like activity, such as surface tension analysis, should be incorporated for accurate detection/screening of these compounds. In this study we assessed the ability of species enclosed in Bacillus pumilus group to produce biosurfactants, screening for surface-active properties and representative lipopeptide family genes, in isolates collected from diverse sources and geographic locations. Moreover, structure and composition of the produced bioactive products was elucidated by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) analysis. 358 Chapter 4 2. Material and Methods 2.1. Bacterial isolates Fifteen B. safensis, nine B. altitudinis and four B. pumilus isolates previously identified by genotypic (16S rRNA, gyrB and rpoB gene sequences) methods were studied (Branquinho et al., 2014). They were recovered from different geographic terrestrial locations and sources, including food samples (Norway, Italy and Africa) (n=6), plants (USA) (n=4), gastropods (Portugal) (n=2), health (n=12) and cosmetic (n=4) products (Portugal). B. pumilus ATCC 14884 was also include in the analyses. Additionally, a B. subtilis isolate recovered from a health product contaminant was included for comparison (Table 1). Bacillus isolates were maintained on Luria Bertani-Miller (LB) agar (Oxoid, United Kingdom) for short-term storage and in LB broth supplemented with 40% (v/v) glycerol at - 80ºC for long-term storage. 2.2. Biosurfactant production and purification For biosurfactant production, a seed culture was prepared for each isolate by transferring a loop from a LB agar overnight culture into 10 mL of LB broth incubated for 4 h, at 28°C and 200 rpm, on an orbital shaker. Thereafter, 2 ml of these cultures were inoculated into 500 mL of LB broth and incubated at 28°C and 120 rpm for 24 h. A cell-free preparation from these cultures was then prepared by centrifuging at 8,000 g-1 for 30 min. This supernatant was used for qualitative measurement of surface tension. For biosurfactant extraction, the cell-free preparation was acidified to pH 2 with HCl (6 mol L-1), and left for 12h at 4ºC for precipitation. The precipitate was then removed by centrifugation at 8,000xg for 10 min at 4ºC and the resulting solution extracted twice with ethyl acetate/methanol (4:1 v/v) mixture according to the method described by Rivardo et al. (2009). The remaining water present in the organic phase was then removed by anhydrous sodium sulfate. Finally, the organic phase was evaporated to dryness under vacuum condition and acetone was added to recover raw biosurfactant. Acetone was, then, evaporated and biosurfactant collected, weighted and quantified. 359 Chapter 4 2.3. Evaluation of surface-active properties 2.3.1. Qualitative measurement of surface tension. Screening of surface-active properties of biosurfactants was firstly conducted on the supernatant obtained as described in section 2.2, using the oil spreading assay (Morikawa et al 2000). Briefly, 20 μl of Motor Oil 10 W-40 (Selenia) were deposited onto a surface of 20 mL of distilled water to form a thin layer. Subsequently, 20μl of the Bacillus spp. supernatants were gently put onto the center of the oil layer and the diameter of the oil displacement circle formed was measured to determine the presence of biosurfactant. 2.3.2. Quantitative measurement of surface tension. Surface active tension of all supernatants revealing a positive result in the oil spreading assay was determined using a Sigma 703D tensiometer (KSV) equipped with a Du Noy platinum ring at 35 ºC, on a 20 ml of a biosurfactant solution which was prepared in alkaline sterile demineralized water at 500 μg ml−1. Distilled water was used for calibration. Critical Micelle Concentration (CMC) was then determined on serially diluted biosurfactant solutions in alkaline distilled water and was estimated from the intercept of two straight lines extrapolated from the concentration-dependent and concentration-independent sections of a curve plotted between biosurfactant concentration and surface tension values (Rivardo et al., 2009) 2.3.3. Screening of bioemulsifier producers The capacity of these biosurfactants to emulsify a hydrophobic substance was also tested. Cell-free supernatants (5 mL) obtained in section 2.2 were mixed with 5 mL of hexadecane with a vortex mixer and allowed to stand for 24 h, 8 days and 3 months at room temperature. After this time the emulsifying index was determined. E24 index is defined as percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm) (Iqbal et al., 1995). 360 Chapter 4 2.4. Detection of lipopetide synthethase genes by PCR The presence of genes srf/lch, sfp, lchAA, lchAB, lchAC, fen, fenA, fenB, fenD, pps , lpa-14 and ituD, encoding lipopeptides families (surfactins/lichenysins, iturins and fengycins/plipastatins) previously reported in Bacillus spp., was searched by PCR, using degenerated and specific primers, and sequencing (Table 1) (Athukorala et al., 2009, Hsieh et al., 2004 and 2008, Nieminen et al., 2007, Tapi et al., 2010). Sequences obtained were compared on GenBank database using the BLAST search. 2.5 Characterization of lipopeptides by LC/ESI-MS/MS 2.5.1 Mass spectrometry analysis An aliquot of the biosurfactant extract was dissolved in methanol/acetonitrile (50/50 v/v) to obtain a 1000 μg ml-1 stock solution. Freshly prepared working solutions were made by diluting the stock solution with methanol/water (50/50 v/v) to achieve 15 μg ml-1 solutions. All mass spectrometry analyses were done on a LCQ DECA XP Plus (Thermo Finnigan, San Jose, CA, USA), Ion Trap mass instrument equipped with an ESI source. Samples (15 μg ml-1 solutions) were infused with a syringe at 5μg min-1 flow rate. Source voltage and capillary voltage were 4.80 kV and 23 V in positive ion mode, while 5 kV and -15 V in negative ion mode. Capillary temperature and sheath gas flow (N2) were, set respectively at 350ºC and 30 arbitrary units in both scan modes. Data were acquired in positive and negative MS total ion scan mode (mass scan range: m/z 100 2000) and in positive MS/MS product ion scan mode; the normalized collision energy (nce %) was optimized for each precursor ion selected: m/z 1030, 38%; 1044, 1058, 1072, 1086 and 1100, 39%; 1464, 1478, 1492 and 1506, 35%. 2.5.2. Liquid chromatography–mass spectrometry analysis A Surveyor HPLC on line with a LCQ DECA XP Plus (Thermo Finnigan, San Jose, CA, USA) Ion Trap mass spectrometer equipped with an ESI source was employed. Separations were performed on an analytical Luna 5 μm C18, 150×4.6mm (Phenomenex, Torrance, CA) protected with a C18-Security Guard cartridge, 4 × 3.0 mm (Phenomenex). The injection volume applied was 10 μl. Mobile phase components encompassed: A - 361 Chapter 4 and Leu-Leu-Asp (-341 Da) from the m/z 1072. In addition, the second one enclosed the peptidic moiety inside the C-terminal product and corresponds to the ions peaks at m/z 721, 608 and 477. Ion at m/z 721 correspond to the loss of C15–β-hydroxyl fatty acid chain -Glu (-351 Da) from the precursor ion, following double hydrogen transfer (DHT) mechanism of the ester bond of the cyclic skeleton and cleavage of one C-terminal amino acid residue (Yang et al., 2006). Moreover, ion at m/z 608 match to the successively loss of Leu (-113 Da) and ion at m/z 477 to the loss of Leu-Leu-H2O (-244 Da). LC-ESI-MS/MS spectra analysis from precursor ion [M+Na]+ at 1100 (Rt 23.01 min) (Fig. 2b) revealed, as above mentioned, two product ion series, comprising m/z 1082, 987, 856 and 759 related to the fatty acid chain, and ions at m/z 721, 608 and 477. Within the first series product ions belonging to m/z 1100 differ of 28 Da from those belonging to m/z 1072, confirming the difference of a -CH2–CH2 in the side chain. Furthermore, in the second series all product ions are identical to the previous mentioned, thus, corresponding to the same amino acid sequences. Indeed, these ion signals are in accordance with two pumilacidin-Leul7 homologues, respectively C15-Glu/Leu/Leu/Leul/Asp/Leu/Leu and C17Glu /Leu/Leu/Leul/Asp/Leu/Leu, (Fig. 3a), which can be related with pumilacidin A ([M+Na]+ 1073 m/z) or F ([M+Na]+ 1073 m/z) or G ([M+Na]+ 1073 m/z) and with C ([M+Na]+ 1101 m/z) types, respectively (Burch et al., 2011, Naruse et al., 1990). Interestingly, the LC-ESI-MS/MS analysis from the precursor ions [M+Na]+ at m/z 1058 and 1086 showed three isobaric peaks for each precursor ion, at Rt 15.98, 16.56 and 17.85 and also 20.46, 21.25 and 22.84 min, respectively. From the precursor ion [M+Na]+ at m/z 1058 the product ion spectra of the peak at Rt 15.98 min also demonstrates two product ion series. In the first series (m/z 1040, 945, 814) related to the fatty acid chain, product ion belonging to m/z 1058 differed of 14 Da from those belonging to m/z 1072, also confirming the difference of a –CH2 in the side chain. Within the second series (m/z 721, 608, 477) all product ions are equal and consequently the amino acid sequences the same. Additionally, this signal was in accordance with C14Glu/Leu/Leu/Leul/Asp/Leu/Leu, the third homologue of pumilacidin-Leu7 molecules. Analysis of ion spectra of the peak at Rt 16.56 min (Fig. 4a) reveal ions products at m/z 1040, 959, 828, 731, 707, 594 and 463. Ion at m/z 1040 correspond to the loss of water (- 18 Da) from m/z 1058 and the remaining product ions were deriving, as before, from the initial opening of the lactone ring. In addition, the first series contain the fatty acid chain 368 Chapter 4 and the N-terminal product ions at m/z 959, 828 and 731, corresponding to the losses of Val (-99 Da), Val-Leu-H2O (-230 Da) and Val-Leu-Asp (-327 Da) from m/z 1058, respectively. The second series encompass a different peptidic moiety inside the Cterminal product ions at m/z 707, 594 and 463, where ion at m/z 707 correspond to the loss of C15 –β-hydroxyl fatty acid chain-Glu (-351 Da) from the precursor ion, ion at m/z 594 to the successively loss of Leu (-113 Da) and m/z 463 to the loss of Leu-Leu-H2O (- 244 Da). In fact, these signals are in agreement with the C15Glu/Leu/Leu/Leul/Asp/Leu/Val, pumilacidin-Val7 variant (Fig. 3 b). Related to resulting peak at Rt 17.85 min (Fig. 5a) ions product at m/z 1040, 945, 814, 618, 707, 594 and 463 were detected. Ion at m/z 1040 correspond to the loss of water (- 18 Da) from m/z 1058. The resulting series contained the fatty acid chain and the Nterminal product ions at m/z of 945, 814 and 618 corresponding to the losses of Leu (-113 Da), Leu-Leu-H2O (-244 Da) and Leu-Leu-Asp-Val (-440) from m/z of 1058, respectively, in the first series, and in the second, enclose different peptidic moieties inside the Cterminal products at m/z 707, 594, and 463. Moreover, ion at m/z 707 corresponds to the loss of C15–β-hydroxyl fatty acid chain-Glu (-351 Da) from the precursor ion, m/z 594 to the successively loss of Leu (-113 Da) and m/z of 463 to the loss of Leu-Leu-H2O (-244 Da). These signals are in accordance with the C15-Glu/Leu/Leu/Vall/Asp/Leu/Leu homologue belonging to surfactin molecules (Pecci et al., 2010) (Fig. 3c). Precursor ion analysis at m/z 1086 showed three peaks at Rt 20.46, 21.25 and 22.84 min. Rt 20.46 min product ion spectra resulting, as before, into two product ion series. The peptidic series (m/z 721, 608 and 477) is relative to pumilacidin and the aliphatic series (m/z 1068, 973, 842, 745) differ of –CH2 of the previous homologue. These signals are in conformity with the C16-Glu/Leu/Leu/Leul/Asp/Leu/Leu, the fourth homologue of pumilacidin-Leu7. Rt 21.25 min product ion spectra, also show two product ion series (Fig. 4b), comprising the peptidic one, at m/z 707, 594 and 463, which is relative to the variant of Val7 of pumilacidin molecule and the aliphatic one, enclosing m/z 1068, 987, 856 and 759 which differ of -CH2–CH2 of the previous homologue. C17-Glu/Leu/Leu/Leu/Asp/Leu/Val, the second homologue of pumilacidin-Val7 match these masse signals observed. Finally, at Rt 22.84 min the product ion spectra (Fig. 5b) revealed the presence of the peptidic series (m/z 707, 594 and 463) relative to surfactin molecule, and the aliphatic series (m/z 1068, 973, 842 and 745) differing in -CH2–CH2 of the previous homologue, 369 Chapter 4 which are in accordance with the C17-Glu/Leu/Leu/Vall/Asp/Leu/Leu, the second homologue of surfactin molecule. In fact, detection of chemical differences in pumilacidin and surfactin were previously observed to occur in amino acid position 4 or 7 of the peptidic ring. Thus, pumilacidin variants are consistent with Leu4, Leu7 or Val7 surfactins (Morikawa et al., 1992). The relative abundance of the three surfactin-like biosurfactants variants presented in B. safensis isolates analyzed (Bs2, Bs22, Bs18 and BS27) were inferred from sum of the product ions attributed to the relative precursor ions [M+Na]+. Beyond B. safensis Bs18, all the remaining isolates also presented the same relative abundance on its surfactin-like variants produced, being detected an average of 90% in pumilacidin variants and of 10% in surfactin ones (Table 4). These observations prompt us to suggest that the production of pumilacidin-Leu7, or its variant Val7, together with surfactin seems to be B. safensis species-specific and thus, can be related to its adaptability and defense mechanisms into different ecological niches (Branquinho et al., 2014). Related to B. pumilus species analyzed, full-scan mass spectra derived from Bp11, demonstrated that pumilacidin variant Leu7 and variant Val7 were present (Table 4) with a relative abundance average of 94 and 3%, respectively for each one. Characterization of B. pumilus Bp11 extracts through ESI-MS direct infusion analysis operating in positive full scan mode, showed one cluster of peaks with 14 Da difference on its molecular ion species. Four main signals at 1058, 1072, 1086 and 1100 m/z were detected, which correspond to the sodiated molecules [M+Na]+. The positive LC-ESI-MS/MS analysis on the sodiated molecule at m/z of 1058, 1072, 1086 and 1100 performed on B. pumilus extract, and similar to previously observed to B. safensis isolates, revealed that these signals were in according with the four homologues of pumilacidin-Leu7 variants compounds, respectively C-14, C-15, C-16 and C-17 and also C-15 and C-17 pumilacidinVal7 variant. In contrast with B. safensis, B. pumilus isolates seems to be able to produce just one type of surfactin-like compounds, specifically pumilacidin. Structure-activity relationships should be further conducted, in order to explain in what dimension these differences can interfere with the activity of these compounds. 370 Chapter 4 Characterization of B. subtilis Bs28 extracts through ESI-MS direct infusion analysis operating in positive full scan mode, showed two clusters of peaks with 14 or 28 Da of difference in their molecular ion species (Fig. 6), which in accordance with other work performed by our research team (Pecci et al., 2010), correspond with spectral analysis of surfactin and fengycin compounds. Surfactin is represented by three main mass signals at [M+Na]+, namely at m/z of 1030, 1044 and 1058. Results derived from our LC/ESI-MS/MS analysis performed with gradient 1 are in accordance with the presence of three surfactin homologues, e.g. C13Glu/Leu/Leu/Val/Asp/Leu/Leu, C14-Glu/Leu/Leu/Val/Asp/Leu/Leu and C15-Glu/Leu/Leu/Val/Asp/Leu/Leu and none of the pumilacidin variants detected for B. safensis or B. pumilus were here achieved. Moreover, fengycin was characterized by four mass signals at [M+H]+, namely at m/z of 1478 and 1464 for fengycin A and m/z of 1492 and 1506 for fengicyn B (Pecci et al., 2010). Our spectral results are in agreement with the previously description, revealing the presence of C16 and C17 homologues of fengycin A and C16 and C17 homologues of fengycin B. The relative abundance achieved in B. subtilis Bsp28 was of 90% for surfactin and of 10% for fengycin. Since biosurfactants production depends of the type and amounts of carbon source and of other nutrients present in the screening culture medium (Soberón-Chávez, 2011), it will consequently influence the composition content of the mixture and the amount of compounds produced. In fact, it is possible that other type of biosurfactants could be produced by these Bacillus spp., nevertheless the cultural optimization and screening conditions were not the aim of the present study. Therefore, a more comprehensive screening can be further performed in order to obtain new potential biosurfactants with putative possible different applications. LB broth has been reported as the culture medium more extensively used for Bacillus biosurfactant production (Rivardo et al., 2009), and therefore it was accepted and applied for our screening evaluation. The distribution of distinct combinations of these molecules within Bacillus pumilus group species recovered from different origins and geographic locations, suggested that biosurfactant production may represent an important survival tool of each species in a specific environment. Moreover, the differences observed in the length of the fatty acid chains and in the amino acid composition of the biosurfactants and its species-specificity, can suggest considerable consequences on its activity and consequently on its possible applications. 371 Chapter 4 4. Conclusion The specificity of biosurfactants molecules produced by species belonging to B. pumilus group supports the speciation recently recognized within this taxonomic group (Branquinho et al., 2014, Liu et al., 2013) and might conferred a distinct niche adaptability. Contrasting with B. subtilis, pumilacidin seems to be the more predominant variant in B. pumilus group species. Further studies exploring the properties of the different biosurfactants characterized could uncover interesting effects broadening potential applications of these compounds. Moreover, our data highlights the failure in the prediction of biosurfactant production based merely on the detection of lipopeptides synthetase genes by a PCR approach. 372 Chapter 4 Table 1. Primers sequences and amplification conditions for screening the main families of lipopeptides biosynthetic genes in Bacillus spp. Lipopetides Gene Primer name Primer sequences (5´to 3´) Annealing temperature (ºC) PCR product size expected References Surfactin/Lichenysin family Surfactin srf/lcha As1-F CGCGGMTACCGVATYGAGC 43 419/422/425/431 Tapi et al., 2010 Ts2-R ATBCCTTTBTWDGAATGTCCGCC sfp sfp-f ATGAAGATTTACGGAATTTA 46 675 Hsieh et al., 2004 sfp-r TTATAAAAGCTCTTCGTACG Lichenysin lchAA LicA-f GTGCCTGATGTAACGAATG 60 735 Nieminen et al., 2007 LicA-r CACTTCCTGCCATATACC lchAB LicB2-f TGATCAGCCGGCCGTTGTCT 60 904 Nieminen et al., 2007 LicB2-r GGCGAATTGTCCGATCATGTCC lchAC LicC-f GCCTATCTGCCGATTGAC 57 1195 Nieminen et al., 2007 LicC-r TATATGCATCCGGCACCA Fengycin/Plipastation family Fengycin fena Af2-F GAATAYMTCGGMCGTMTKGA 45 443/452 Tapi et al., 2010 Tf1-R GCTTTWADKGAATSBCCGCC fenA FENA1F GACAGTGCTGCCTGATGAAA 62 964 Athukorala et al., 2009 FENA1R GTCGGTGCATGAAATGTACG fenB FENB2F CAAGATATGCTGGACGCTGA 62 964 Athukorala et al., 2009 FENB2R ACACGACATTGCGATTGGTA fenD FEND1F TTTGGCAGCAGGAGAAGTTT 62 964 Athukorala et al., 2009 FEND1R GCTGTCCGTTCTGCTTTTTC Pliplastatin ppsa Ap1-F AGMCAGCKSGCMASATCMCC 58 893/959/929 Tapi et al., 2010 Atp1-R GCKATWWTGAARRCCGGCGG Iturin family Iturin lpa-14 lpa-14f ATGAAAATTTACGGAGTATA 50 675 Hsieh et al., 2008 lpa-14r TTATAACAGCTCTTCATACG ituD ituD-f ATGAACAATCTTGCCTTT TTA 50 1203 Hsieh et al., 2008 ituD-r TTATTTTAAAATCCGCAATT a degenerated primers 373 Chapter 4 Table2. Surface tension and emulsification properties of Bacillus spp. studied. Bacillus species Bacterial isolates Origin/Product Year/Location Surface tension E24 (%) Qualitative (mm) Quantitative (mN/m) Cell-free supernatant Cell-free supernatant Purified Biosurfactant (500ug/mL) Cell-free supernatant Average SD Average SD Average SD 24 h 8 days 3 months B. pumilus Bp ATCC14884 3.00 0.00 58.29 0.40 3.90 2.60 2.60 Bp7 Health´s product contaminants (n=3) 2005/Portugal1 17.67 1.44 38.69 0.21 4.29 4.29 2.86 Bp11* 2005/Portugal1 18.67 1.04 35.24 0.39 31.4 0.27 1.30 1.30 1.30 Bp15 2005/Portugal1 17.83 0.76 36.38 0.17 6.67 5.33 5.33 B. altitudinis Ba6 Health´s product ´ contaminants (n=5) 2005/Portugal1 4.50 0.50 45.92 0.50 2.67 2.67 1.33 Ba8 2005/Portugal1 6.00 0.50 41.64 0.37 1.33 1.33 1.33 Ba9 2005/Portugal1 6.17 0.29 45.41 0.15 1.35 1.35 0.00 Ba12 2005/Portugal1 5.00 0.50 47.19 0.29 2.86 2.86 1.43 Ba14 2005/Portugal1 8.83 0.29 43.66 0.55 0.00 0.00 0.00 Ba26 Food´s contaminants/rice (n=1) 2006/Norway2 6.67 0.29 51.24 0.41 0.00 0.00 0.00 Ba20 Plant Growth Promoters (PGPR) (n=2) 1996/USA4 6.00 0.50 47.66 0.30 1.30 1.30 0.00 Ba21 1997/USA4 5.83 0.29 46.40 0.17 2.67 2.67 0.00 374 Chapter 4 Ba30 Food/beans (n=1) 2003/Africa5 7.17 0.58 43.64 0.36 1.43 1.43 0.00 Bacillus safensis Bs2* Animals Gastropods (n=2) 2005/Portugal1 18.83 0.29 36.18 0.31 29.5 0.13 17.81 17.81 8.22 Bs3 2007/Portugal1 19.33 0.76 35.37 0.25 21.28 21.28 21.28 Bs13 Health´s product contaminants (n=3) 2005/Portugal1 18.10 0.50 33.53 0.34 2.70 2.70 2.70 Bs16 2005/Portugal1 18.33 2.36 34.19 0.26 3.85 3.85 2.56 Bs17 2005/Portugal1 18.67 2.75 35.36 0.12 7.14 5.71 2.86 Bs5 Cosmetic´s contaminants (n=4) 2002/Portugal1 4.00 0.50 49.67 0.32 2.86 2.86 1.43 Bs18* 2002/Portugal1 19.17 0.29 34.32 0.20 33.4 0.29 19.12 19.12 16.18 Bs19 2002/Portugal1 17.00 1.80 33.60 0.22 20.00 20.00 13.33 Bs27* 2002/Portugal1 24.17 2.47 30.50 0.18 31.5 0.33 10.39 10.39 2.60 Bs24 Food´s contaminants/salame (n=3) 2004/Italy3 3.50 0.50 46.51 0.42 0.00 0.00 0.00 Bs25 2004/Italy3 4.00 0.50 44.40 0.34 6.49 6.49 2.60 Bs33 2004/Italy3 6.00 0.50 42.59 0.28 1.33 1.33 1.33 Bs22* Plant Growth Promoters (PGPR) (n=2) 1997/USA4 20.10 0.50 32.27 0.13 18.67 16.00 6.67 Bs23 1997/USA4 6.17 0.29 44.30 0.21 12.86 12.00 6.67 Bs31 Food/beans (n=1) 2003/Africa5 7.00 0.50 40.28 0.33 11.54 11.54 2.56 Bacillus subtilis Bsb28* Health´s product contaminants (n=1) 2005/Portugal1 19.67 1.15 30.53 0.31 30.4 0.27 40.00 40.00 40.00 * isolates characterized by LC/ESI-MS/MS SD – standard deviation 375 Chapter 4 Table 3. PCR amplification results for main families of lipopeptides in B. pumilus, B. safensis, B. altitudinis and B. subtilis isolates. Species Isolates Surfactin/Lichenysin family Fengycin/Plipastatin family Iturin family srf/lch sfp lchAA lchAB lchAC fen fenA fenB fenD pps lpa-14 ituD B. pumilus Bp ATCC14884 + - - - - - - - - - - - Bp7 + - - - - + - - - - - - Bp11 + - - - - + - - - - - - Bp15 + - - - - + - - - - - - B. altitudinis Ba6 + - - - - - - - - - - - Ba8 + - - - - + - - - - - - Ba9 + - - - - - - - - - - - Ba12 + - - - - - - - - - - - Ba14 + - - - - + - - - - - - Ba26 + - - - - - - - - - - - Ba30 + - - - - + - - - - - - Ba20 + - - - - - - - - - - - B. safensis Bs2 + - - - - - - - - - - - Bs3 + - - - - - - - - - - - Bs5 - - - - - + - - - - - - Bs13 + - - - - - - - - - - - Bs16 + - - - - + - - - - - - 376 Chapter 4 Bs17 + - - - - - - - - - - - Bs18 + - - - - + - - - - - - Bs19 + - - - - + - - - - - - Bs21 + - - - - - - - - - - - Bs22 + - - - - + - - - - - - Bs23 + - - - - + - - - - - - Bs24 + - - - - + - - - - - - Bs25 + - - - - - - - - - - - Bs27 + - - - - - - - - - - - Bs31 + - - - - + - - - - - - Bs33 + - - - - + - - - - - - B. subtilis Bsp28 + + - - - + - - - - - - B. safensis 377 Chapter 4 Figure 6. Full-scan mass spectra of Bsp28 extract through (+) ESI-MS direct infusion analysis. Clusters of surfactin and fengycin are shown in the ranges of m/z 1000–1100 and 1450–1550, respectively. 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1058.5 1044.5 1030.5 1506.6 1478.6 1492.5 1464.5 384 Chapter 4 Acknowledgments Dr. Irène Ouoba, Dr. Joseph W. Kloepper, Dr. Cecilie From and Dr. Maria Morea are gratefully acknowledged for providing isolates Bi30 and Bs31; INR7 (AP18), SE 34 (AP281), SE 49 (AP3) and SE 52 (AP7); FEL 55, UNG22 and MIL46, respectively. This study was funded by Fundação para a Ciência e a Tecnologia (FCT), which belongs to the Ministry of Education and Science from Portugal, through grant no. PEstC/EQB/LA0006/2011 Raquel Branquinho was supported by a fellowship from FCT (SFRH/BD/77518/2011). 385 Chapter 4 5. References 1. Arima, K., Kakinuma, A., Tamura, G. 1968. Surfactin, a crystalline peptide lipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun. 31, 488–494. 2. Athukorala, S.N., Fernando, W.G., Rashid, K.Y. 2009. Identification of antifungal antibiotics of Bacillus species isolated from different microhabitats using polymerase chain reaction and MALDI-TOF mass spectrometry. Can. J. Microbiol. 55, 1021-1032. 3. Banat, I.M., Franzetti, A., Gandolfi, I., Bestetti, G., Martinotti, M., Fracchia, L., Smyth T., Marchant R. 2010. Microbial biosurfactants, production, applications and future potential. Appl. Microbiol. Biotechnol. 87, 427–444. 4. Batool, M., Khalid, M.H., Hassan, M.N., Yusuf, H.F. 2011 Homology modeling of an antifungal metabolite plipastatin synthase from the Bacillus subtilis 168. Bioinformation. 7, 384-7. 5. Branquinho, R., Meirinhos-Soares, L., Carriço, J.A., Pintado, M., Peixe, L. V. 2014 Phylogenetic and clonality analysis of Bacillus pumilus isolates uncovered a highly heterogeneous population of different closely related species and clones. FEMS Microbiol. Ecol. (submitted). 6. Burch, A.Y., Browne, P.J., Dunlap, C.A., Price, N.P., Lindow, S.E. 2011. Comparison of biosurfactant detection methods reveals hydrophobic surfactants and contactregulated production. Environ. Microbiol. 13, 2681-1691. 7. Chowdhury, S.R., Basak, R.K., Sen, R., Adhikari, B. 2011.Characterization and emulsifying property of a carbohydrate polymer produced by Bacillus pumilus UW-02 isolated from waste water irrigated agricultural soil. Int. J. Biol. Macromol. 48, 705-12. 8. Delcambe, L., Peypoux, F., Besson, F., Guinand, M., Michel, G. 1977 Structure of iturin-like substances. Biochem. Sco. Trans. 5, 1122–1124. 9. Duitman, H.E., Hamoen, L.W., Rembold, M., Venema, G., Seitz, H., Saenger, W., Bernhard, F., Reinhard, R., Schmidt, M., Ullrich, C., Stein, T., Leenders, F., Vater, J. 1999. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase and a fatty acid synthase. PNAS 96, 13294-13299. 10. Fracchia, L., Cavallo, M., Martinotti, M.G., Banat, I.M. 2012. Biosurfactants and Bioemulsifiers Biomedical and Related Applications – Present Status and Future Potentials, in: Ghista, D.N. (Eds.), Biomedical Science, Engineering and Technology, InTech. DOI: 10.5772/23821. 11. Hathout, Y., Ho, Y-P., Ryzhov, V., Demirev, P., Fenselau, C. 2000 Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis. J. Nat. Prod. 63, 1492–1496. 386 Chapter 4 12. Hsieh, F-C., Li, M-C., Lin, T-C., Kao, S-S. 2004. Rapid Detection and characterization of Surfactin-Producing Bacillus subtilis and Closely Related Species Based on PCR. Curr. Microbiol. 49, 186-191. 13. Hsieh, F-C., Lin, T-C., Meng, M., Kao, S-S. 2008. Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A. Cur. Microbiol. 56, 1-5. 14. Iqbal, S., Khalid, Z.M., Malik, K.A. 1995. Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by gamma ray-induced mutant of Pseudomonas aeruginosa. Lett. App. Microbiol. 21, 176-179. 15. Ławniczak, L., Marecik, R., Chrzanowski, L. 2013. Contributions of biosurfactants to natural or induced bioremediation. Appl. Microbiol. Biot. 97, 2327-2339. 16. Liu, X., Ren, B., Chen, M., Wang, H., Kokare, C.R., Zhou, X., Wang, J., Dai, H., Song, F., Liu, M., Wang, J., Wang, S., Zhang, L. 2010. Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3. Appl. Microbiol. Biotechnol. 87, 1881-93. 17. Liu, Y., Lai, Q., Dong, C., Sun, F., Wang, L., Li, G., Shao, Z. 2013. Phylogenetic diversity of the Bacillus pumilus group and the marine ecotype revealed by Multilocus Sequence Analysis. PLoS ONE 8, 1-11. 18. Martinotti, M.G., Allegrone, G., Cavallo, M., Fracchia, L. 2013. Biosurfactants, in: Piemonte, V., De Falco, M., Basile, A. (Eds), Sustainable Development in Chemical Engineering Innovative Technologies, John Wiley & Sons Ltd, Chichester, UK. doi: 10.1002/9781118629703.ch9. 19. Melo, F., Fiore, M., Moraes, L.A., Silva-Stenico, M., Scramin, S., Teixeira, M., Melo, L. 2009 Antifungal compound produced by the cassava endophyte Bacillus pumilus MAIIIM4a. Sci. Agric. 66, 583-592. 20. Mootz, H.D., Finking, R., Marahiel, M.A. 2001. 4′-Phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis. J. Biol. Chem. 276, 3728937298. 21. Morikawa, M., Hirata, Y., Imanaka, T. 2000. A study on the structure-function relationship of lipopeptide biosurfactants. Biochim. Biophys. Acta. 1488, 211–218. 22. Morikawa, M., Ito, M., Imanaka, T. 1992. Isolation of a new surfactin producer Bacillus pumilus A 1, and cloning and nucleotide sequence of the regulator gene, psf 1. J. Ferment. Bioeng. 74, 255-261. 23. Naruse, N., Tenmyo, O., Kobaru, S., Kamei, H., Miyaki, T., Konishi, M., Oki, T. 1990. Pumilacidin, a complex of new antiviral antibiotics. Production, isolation, chemical properties, structure and biological activity. J. Antibiot. 43, 267-280. 387 Chapter 4 24. Nerurkar, A.S. 2010. Structural and molecular characteristics of lichenysin and its relationship with surface activity. Adv. Exp. Med. Biol. 672, 304-315. 25. Nieminen, T., Rintaluoma, N., Andersson, M., Taimisto, A.M., Ali-Vehmas, T., Seppälä, A., Priha, O., Salkinoja-Salonen, M. 2007. Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk. Vet. Microbiol. 124, 329-339. 26. Pacwa-Płociniczak, M., Płaza, G.A., Piotrowska-Seget, Z., Cameotra,S.S. Environmental Applications of Biosurfactants: Recent Advances Int. J. Mol. Sci. 12, 633–654. 27. Pecci, Y., Rivardo, F., Martinotti, M.G., Allegrone, G. 2010. LC/ESI-MS/MS characterization of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain. J. Mass. Spectrom. 45, 772-778. 28. Rivardo, F., Turner, R.J., Allegrone, G., Ceri, H., Martinotti, M.G. 2009. Anti-adhesion activity of two biosurfactants produced by Bacillus spp. prevents biofilm formation of human bacterial pathogens. Appl. Microbiol. Biotechnol. 83, 541-553. 29. Roongsawang, N., Thaniyavarn, J., Thaniyavarn, S., Kameyama, T., Haruki, M., Imanaka, T., Morikawa, M., Kanaya, S. 2002. Isolation and characterization of halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides: bacillomycin L, plipastatin and surfactin. Extremophiles. 6, 499–506. 30. Seydlová, G., Čabala, R., Svobodová, J. 2011. Surfactin - Novel Solutions for Global Issues, in: Olsztynska, S. (Ed.), Biomedical Engineering, Trends, Research and Technologies, InTech. DOI: 10.5772/13015. 31. Soberón-Chávez, G., 2011, Biosurfactants: From Genes to Applications, Springer Berlin Heidelberg, Berlin, Heidelberg. 32. Tapi, A., Chollet-Imbert, M., Scherens, B., Jacques, P. 2010 New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction. Appl. Microbiol. Biotechnol. 85,1521-1531. 33. Vanittanakom, N., Loeffler, W., Koch, U., Jung, G. 1986. Fengycina novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J. Antibiot. 39, 888–901. 34. Wu, C.Y., Chen, C.L., Lee, Y.H., Cheng, Y.C., Wu, Y.C., Shu, H.Y., Götz, F., Liu, S.T. 2007. Nonribosomal synthesis of fengycin on an enzyme complex formed by fengycin synthetases. J. Biol. Chem. 282, 5608-5616. 35. Yang, S.Z., Wei, D.Z., Mu, B.Z. 2006. Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism. J. Biochem. Biophys. Methods. 68, 69–74. 388 Chapter 4 4.2. Linezolid resistant ST2/CC5 Staphylococcus epidermidis – biofilm producer Publication: Emergence of Methicillin and Linezolid Resistant ST2/CC5 Staphylococcus epidermidis, Portugal, 2012 This study, reports the emergency of clinical methicillin and linezolid Staphylococcus epidermidis strains in Portugal, which belong to the worldwide successful clonal lineage ST2/CC5 (former CC2), also including the ability of S. epidermidis from patient 1 adhere to abiotic surfaces as a biofilm form. This isolate was used in the study enclosed in section 3.3. The data described in this manuscript also highlight the need for strict infection control procedures along with revision of therapeutic strategies to preserve linezolid therapeutic effectiveness. 389 [Document text truncated for crawler view.]