Halomonas Avicenniae sp. nov., Isolated from the Salty Leaves of the Black Mangrove Avicennia Germinans in Puerto Rico

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGYon May 2007, available at: https://doi.org/10.1099/ijs.0.64818-0.” Halomonas avicenniae sp. nov., isolated from the salty leaves of the black mangrove Avicennia germinans in Puerto Rico 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Nelís Soto-Ramírez1, Cristina Sánchez-Porro2, Soniris Rosas1, Wildaomaris González1, Mariam Quiñones1, Antonio Ventosa2, and Rafael Montalvo-Rodríguez1 1 Biology Department Box 9012, University of Puerto Rico, Mayagüez, Puerto Rico 00681 2 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain Author for correspondence: R. Montalvo-Rodríguez; email: titom[email protected], phone: (787) 832-4040 X2421, Fax: (787) 834-3673. Running title: Halomonas avicenniae sp. nov. Subject category: New Taxa (Proteobacteria) Footnote: The GenBank accession number for the 16S rRNA gene sequence of strain MW2aT is DQ888315. Summary 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 A Gram-negative, short to oval shape, bacterium (strain MW2aT) was isolated from the surface of leaves of the black mangrove Avicennia germinans and was subjected to a polyphasic taxonomic study. Strain MW2aT is moderately halophilic growing at NaCl concentrations in the range of 0-25% (w/v) with an optimum at 5% (w/v) NaCl. Growth occurs at temperatures from 12-40ºC (optimal 30-35 ºC) and at pH range from 5.0-9.0 (optimal 7.0-8.0). Strain MW2aT is strictly aerobic. Phylogenetic analysis using the 16S rRNA gene also revealed that this strain belongs to the genus Halomonas. The closest relative is Halomonas marisflavi having a 98.6% 16S rDNA sequence similarity. Its DNA G+C content is 61.5 mol% which is in the range for Halomonas species. DNADNA hybridization with H. marisflavi showed a 42% relatedness and lower percentages were obtained with respect to other related Halomonas species. Its major fatty acids are C16:0, C19:0 cyclo ω8c, C18:1 ω7c and C12:0 3OH. Overall, the phenotypic, genotypic and phylogenetic results presented in this study demonstrate that strain MW2aT should be considered a new species within the genus Halomonas. The name Halomonas avicenniae sp. nov. is proposed for this novel species, with strain MW2aT (= CECT 7193T = CCM 7396T) as the type strain. The genus Halomonas belongs to the family Halomonadaceae within the γProteobacteria and contains 35 species that have been isolated mostly from saline or hypersaline environments (Arahal et al., 2002; Bouchotroch et al., 2001; Dobson & Franzmann, 1996; Garcia et al., 2004; Lee et al., 2005; Lim et al., 2004; Martínez- Cánovas et al., 2004; Martínez-Checa et al., 2005; Mata et al., 2002; Mormile et al., 1999; Quillaguaman et al., 2004; Romanenko et al., 2002; Ventosa et al., 1998; Vreeland et al., 1980; Yoon et al., 2001; 2002) or from unusual habitats like dry mural paintings (Heyrman et al., 2002) and deep-sea hydrothermal vents (Kaye et al., 2004). Phylogenetic analysis using the 16S and 23S rRNA sequences and phenotypic studies demonstrated that this genus is very heterogeneous (Arahal et al., 2002; Mata et al., 2002). One of these distinctive groups is represented by a single species, Halomonas marisflavi (Yoon et al., 2001). So far, there are no reports about the occurrence of members of this genus associated to mangroves that thrive on saline habitats. Avicennia germinans is a type of mangrove that has developed mechanisms to tolerate high salt concentrations in unstable substrates, to obtain oxygen in almost anoxic sediments, and to reproduce in harsh areas. One of the mechanisms consists in secreting salt crystals on their leaves when the concentration is in excess of that normally in their vascular system (Lugo & Snedaker, 1975). In this study, we have determined the taxonomic position of strain MW2a 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 T which was isolated from the surface of leaves of the black mangrove Avicennia germinans during a microbial diversity study. As a result of our studies, the name Halomonas avicenniae sp. nov. is proposed for this novel microorganism. Strain MW2aT was isolated from the surface of the Avicennia germinans leaves. For isolation, a total of 40 leaves were collected randomly from four Avicennia germinans trees growing near the solar salterns of Cabo Rojo, Puerto Rico. These leaves were placed in sterile bags (Whirl Pak®) containing 1% phosphate buffer pH 7 with 15% (w/v) NaCl and mixed. This procedure allowed the suspension of the microorganisms present in the salt crystals and the surface of the leaves. Ten milliliters of the mix were then used for serial dilutions. The dilutions were poured into agar plates containing SeghalGibbons medium at 15% NaCl (Seghal & Gibbons, 1960) (SG). Inoculated plates were incubated at 30ºC. After 3 days of incubation, colonies were selected and purified by the quadrant streak plate method. Pure cultures were transferred into SG medium for further analysis. 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 The morphology of cells was examined by Nomarsky technique and scanning electron microscopy (SEM) at logarithmic phase of growth under optimal conditions. Electron microscopy procedures were performed as previously described (Díaz-Muñoz & Montalvo-Rodríguez, 2005). Gram stain was performed using both heat-fixed smears and smears fixed in 5% acetic acid (Dussault, 1955). Macroscopic properties were determined using the classical characterization of colony appearance. Optimal conditions for growth were determined by growing MW2aT in SG broth supplemented with 0, 5, 10, 15, 20, 25 and 30% (w/v) NaCl at temperatures of 20, 25, 30, 35, and 40°C, respectively. The pH range for the isolate was tested in SG medium with 5% NaCl adjusted to the following pH values: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 with the addition of the appropriate buffering capacity to each medium as previously described (Montalvo-Rodríguez et al., 2000). The cells were cultivated with constant agitation (150 rpm) and growth was monitored by measuring absorbance at 600 nm. Strain MW2aT is a Gram-negative, nonspore-forming short rod or oval. Cells are 22.6 µm long and 1-2 µm wide at logarithmic phase of growth in SG medium with 5% NaCl at 30ºC. Cells are motile. On SG medium with 5% NaCl colonies were orange, smooth, circular/slightly irregular, convex with an entire margin. Strain MW2aT grows at NaCl concentrations in the range of 0-25% (w/v) in SG medium. It does not grow above 25% (w/v) of NaCl. Growth is observed at temperatures from 12-40ºC and pH range from 5.0-9.0. The optimal growth conditions occur in SG medium at 5% (w/v) NaCl at 30-35ºC and a pH of 7.0-8.0. This combination of conditions produced the shortest generation time (1.42 hours). 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 All biochemical tests were carried out at 5% NaCl and 30°C, unless it is stated otherwise. Catalase activity was determined by adding a 1% (w/v) H2O2 solution to colonies on SG agar medium. Oxidase test was performed using the Dry Slide (Difco) biochemical test (Montalvo-Rodríguez et al; 1998). Hydrolysis of starch, Tween 80, and aesculin were determined as described by Cowan & Steel (1965) with the addition of Basal Salts (5% NaCl, 2% MgSO4 · 7H2O, and 0.2% KCl [w/v]) to the medium. Hydrolysis of gelatin and production of urease were determined according to Cowan & Steel (1965). Citrate utilization was determined on Simmon’s Citrate medium supplemented with Basal Salts. Acid production from carbohydrates was determined using phenol red base supplemented with 0.7% of the carbohydrate and Basal Salts. Motility was determined using SIM medium according to Simmons (1926) supplemented with Basal Salts. Production of H2S was determined using the API 20E system as previously described (Yoon et al., 2001). Growth under anaerobic conditions was determined by incubating strain MW2aT in an anaerobic chamber in SG medium with 5% NaCl. Tests for sugar fermentation and enzymes (qualitative) were carried out using API 20 NE and API ID32E (bioMérieux) inoculated according to the manufacter’s instructions using the inoculated fluid at 5% NaCl and incubated at 30ºC. Nutritional features were determined using the Koser medium (1923) as modified by Ventosa et al. (1982), which contains the Basal Salts described above and (%, w/v): KNO 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 3 , 0.1; (NH4)2HPO4, 0.1; and KH2PO4, 0.05. The filter-sterilized substrate was added to this medium at a final concentration of 0.1% (w/v), with the exception of carbohydrates, which were used at a final concentration of 0.2% (w/v). Strain MW2aT was able to use L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, maltose, D-mannose, glycerol as carbon and energy sources. Antibiotic susceptibility was determined according to the conventional Kirby-Bauer method (Bauer et al., 1966). The phenotypic characteristics of strain MW2aT are summarized and compared to the type strains of related Halomonas species in Table 1 and also in the species description. For the characterization and sequencing of the 16S rRNA gene, MW2aT was grown in SG medium at 5% NaCl and incubated at 30°C. Genomic DNA was extracted from cells in lysis buffer followed by phenol/chloroform extraction and ethanol precipitation. This DNA was used as template for subsequent PCR amplification. Reaction conditions and amplification protocols were performed as described elsewhere (Hezayen et al., 2002). The resulting amplicon was purified using the MinElute PCR purification kit according to the manufacturer instructions. Purified PCR products were sent to a DNA sequencing facility (Macrogen in Korea). Distance analysis of the resulting DNA sequence was performed using the PHYLIP program (version 3.63) (Felsenstein, 1993). A multiplesequence alignment was made by using the Clustal W program with 16S rRNA gene sequences of closely related organisms (as determined by the BLAST analysis) (Maidak et al., 1996). The 16S rRNA gene similarity values were calculated by pairwise comparison of the sequences within the alignment. Seqboot was used to generate 100 bootstrapped data sets. Distance matrices were calculated with dnadist. One hundred trees were inferred by using the neighbor software. Any bias introduced by the order of sequence addition was minimized by randomizing the input order. Consense was used to determine the most frequent branching order. The final tree was drawn using TREEVIEW (Page, 1996). 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 In silico analysis using the BLAST program (National Center for Biotechnology) of strain MW2aT using an almost complete 16S rRNA gene sequence (1433 bp) revealed that this strain was related to Halomonas, having the closest sequence similarity to Halomonas marisflavi KCCM 80003T (98.5%) which is the only species representing one of the different groups of this genus. Additional 16S rRNA gene sequence similarities between MW2aT and Halomonas elongata ATCC 33173T, Halomonas salina DSM 5928Tand Halomonas halodurans DSM 5160T were 92.8, 92.8 and 91.7 %, respectively. MW2aT also showed low sequence similarity to Chromohalobacter salexigens DSM 3043T (93.7%). Phylogenetical analysis using the neighbor joining algorithm revealed that strain MW2aT was closely related to Halomonas marisflavi forming a branch in this cluster with a boostrap value of 100% (Fig. 1). The phylogenetic position of strain MW2aT was also confirmed in a tree generated with the maximum-parsimony algorithm. Fatty acids were analysed by GC at the Belgian Co-ordinated Collections of Microorganisms, Laboratory of Microbiology of Gent (BCCM/LMG), Gent, Belgium (Kämpfer & Kroppenstedt, 1996; Miller, 1982). Cells were cultured on SW10 medium (8.1% NaCl, 0.7% MgCl2, 0.96% MgSO4, 0.036% CaCl2, 0.2% KCl, 0.006% NaHCO3, 0.0026% NaBr, 0.5 % yeast extract [Difco]) (Ventosa et al., 1982) for 24 hours at pH 7.0, 30ºC. The predominant fatty acids of strain MW2a 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 T were C16:0, C19:0 cyclo ω8c, C18:1 ω7c and C12:0 3OH. This composition is very similar to those described for other Halomonas species (Table 2). The G+C content of genomic DNA was determined from the mid-point value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962) using the equation of Owen & Hill (1979), as previously described in detail by Ventosa et al. (1999). The DNA G+C content of strain MW2aT is 61.5 mol %, which is in the range for species belonging to the genus Halomonas (Arahal & Ventosa, 2005; Franzmann et al., 1989). DNA-DNA hybridization studies were performed by the competition procedure of the membrane method (Johnson, 1994), described in detail by Mormile et al. (1999). The hybridization temperature was 57.3ºC, which is within the limit of validity for the filter method (De Ley & Titjgat, 1970) and the percentage of hybridization was calculated according to Johnson (1994). The experiments were carried out in triplicate. The percentage of DNA-DNA hybridization between strain MW2aT and Halomonas marisflavi KCCM 80003T was 42%, while with respect to Halomonas elongata ATCC 33173Tand Halomonas salina DSM 5928Twere 23% and 13%, respectively. These levels of DNA-DNA hybridization are low enough to classify strain MW2aT as a genotypically distinct species within the genus Halomonas (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Lugo, A. E. & Snedaker, S. C. (1975). Properties of a mangrove forest in southern Florida. In Actas, International Symposium on Biological Management of Mangroves. pp. 170-212. Edited by Walsh, G.; Snedaker, S.C.; Teas, H.. Gainesville: University of Florida. 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 Maidak, B.L., Olsen, G.J., Larsen, N., Overbeek, R., McCaughey, M.J., & Woese, C.R. (1996). The ribosomal database project (RDP). Nucleic Acids Res 24, 82-85. Marmur, J. & Doty, P. (1962). 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Yoon, J.-H., Lee, K.-C., Kho, Y. H., Kang, K. H., Kim, C.-J. & Park, Y.-H. (2002). Halomonas alimentaria sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 52, 123–130. 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 Table 1. Phenotypic characteristics that distinguish MW2aT from other type strains of the genus Halomonas. 429 430 431 432 433 434 435 436 437 438 Strains: 1, MW2aT; 2, Halomonas marisflavi KCCM 80003T; 3, Halomonas elongata DSM 33173T; 4, Halomonas salina ATTC 49509T; 5, Halomonas halodurans DSM 5160T; 6, Halomonas halmophila DSM 5349T; 7, Halomonas marina DSM 4741T; 8, Halomonas eurihalina DSM 5720T. Data from Arahal et al. (2001; 2002), Baumann et al. (1983), Franzmann et al. (1989), Hebert & Vreeland (1987), Mata et al. (2002), Mellado et al. (1995), Yoon et al. (2001) and this study. +, Positive; -, negative, +/-, weak reaction; nd, no data. Characteristic 1 2 3 4 5 6 7 8 Cell morphology rods or ovals rods or ovals short rod short rod rod short rod Rod rod Pigmentation Orange yellow white cream none cream Cream cream Beta-galactosidase + - + + - - + + Motility + + + - + + + - Exopolysaccharide production - - - - - - + + Oxidase - - - + + + - - Facultative anaerobe - + + - - - - - Acid production from: Glucose + + + - + + + - Sucrose + + + - + + - - Maltose +/- + + - - + - - Mannitol + +/- + - + + + - Xylose + + - nd - - - + Fructose + + - - + + + - Lactose - + + - - + - - L-arabinose + + + - - + - - Nitrate reduction - - + + - - - + Simmon's citrate + + + + + - - + Hydrolisis of: Gelatine + + - - - - - + Urea - - + + + - + + Tween 80 - - - - + - + + Aesculin + + - - + - - + DNA G+C content (mol%) 61.5 59.0 60.5 60.7-64.2 63.2 63.0 62.8 59.1-65.7 439 Table 2. Cellular fatty acid composition (%) of strain MW2aT, the closely related Halomonas species, H. marisflavi, and the type species of the genus Halomonas, H. elongata. 440 441 442 443 The three strains were grown on SW-10 medium for 24 hours at pH 7.0 and 30ºC. Fatty acid composition Strain MW2aTH. marisflavi KCCM 80003T H. elongata ATCC 33173T C10:0 1.0 2.4 4.7 C10:0 3OH - - 3.6 C12:0 0.8 - 4.7 C12:0 2OH 3.2 3.2 - C12:0 3OH 11.3 11.0 15.3 C14:0 0.4 0.5 - C16:1 ω7c* 2.6 3.5 4.5 C16:0 37.4 33.4 25.6 C16:0 3OH - - 1.0 C17 cyclo 3.2 2.6 1.6 C17:0 - - 0.5 C18:1 ω7c 16.6 25.7 25.6 C18:0 1.2 2.1 0.6 C19 cyclo ω8c 22.3 15.7 10.6 C19:010 methyl - - 0.5 *Fatty acid C16:1 ω7c is included in summed feature 3 (C16:1 ω7c and/or iso-C 15:0 2OH) 444 445 446 447 448 449 450 451 452 453 454 Legend to figure: Figure 1. Neighbour-joining distance tree using the 16S rRNA gene sequences of MW2aT and closely related species. Bar represents 1 substitution per 100 nucleotides. Bootstrap values higher than 40% are shown. Z. palmae DSM 10491T (D14555) was used as the outgroup. Cobetia marina DSM 4741 T ( AJ306890) Chromohalobacter israelensis DSM6768 T (AF211862) 455 Halomonas halophila DSM 4770 T (M93353) Halomonas maura DSM 13445 T (AJ271864) Chromohalobacter nigrandesensis DSM 14323 T (AJ277205) 52 47 Zymobacter palmae DSM 10491 T (D14555) Strain MW2a T (DQ888315) Halomonas marisflavi KCCM 80003 T (AF251143) Halomonas organivorans CECT 5995 T (AJ 616910) Halomonas halmophila ATCC 19717 T ( A J306889) Halomonas eurihalina ATCC 49336 T (X87218) Halomonas pacifica DSM 4742 T (L42616) Halomonas ve tosae DSM 15911n T (AY647306) Halomonas halodenitrificans ATCC 13511 T (L04942) Halomonas desiderata DSM 9502 T (X92417) Halomonas campisalis ATCC 700597 T (AF054286) Halomonas pantelleriense DSM 9661 T (X93493) Halomonas subglaciescola DSM 4683 T (AJ306892) Halomonas halodurans DSM 5160 T (L42619) Halomonas meridiana DSM 5425 T (M93356) Halomonas magadiensis DSM 15367 T (X92150) Halomonas hydrothermalis DSM 15725 T (AF212218) Halomonas sulfidaeris DSM 15722 T (AF212204) Halomonas neptunia DSM 15720 T (AF212202) Chromohalobacter canadensis ATCC 43984 T (AF211861) Chromohalobacter marismortui ATCC 17056 T (X87219) 100 90 61 56 100 100 89 99 96 74 100 54 100 53 73 55 100 0.01