Salinivibrio kushneri sp. nov., a moderately halophilic bacterium isolated from salterns

1 Salinivibrio kushneri sp. nov., a moderately halophilic bacterium 1 isolated from salterns 2 3 Clara López-Hermoso†, Rafael R. de la Haba†, Cristina Sánchez-Porro and 4 Antonio Ventosa* 5 6 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of 7 Sevilla, 41012 Sevilla, Spain 8 9 †These authors contributed equally to this work. 10 11 * Corresponding author at: Department of Microbiology and Parasitology, Faculty of 12 Pharmacy, University of Sevilla, 41012 Sevilla, Spain. 13 E-mail address: [email protected] (A. Ventosa). 14 15 Running title: Salinivibrio kushneri sp. nov. 16 17 Abbreviations: MLSA, Multilocus Sequence Analysis; ANI, Average Nucleotide 18 Identity, DDH, DNA-DNA hybridization; GGDC, Genome-to-Genome Distance 19 Calculator. 20 21 22 23 24 25 2 Abstract 26 Ten Gram-strain-negative, facultatively anaerobic, moderately halophilic bacterial 27 strains, designated AL184T, IB560, IB563, IC202, IC317, MA421, ML277, ML318, 28 ML328A and ML331, were isolated from water ponds of five salterns located in Spain. 29 The cells were motile, curved rods and oxidase and catalase positive. All of them grew 30 optimally at 37 ºC, at pH 7.2-7.4 and in the presence of 7.5 % (w/v) NaCl. Based on 31 phylogenetic analyses of the 16S rRNA, the isolates were most closely related to 32 Salinivibrio sharmensis DSM 18182T (99.6-98.2 % 16S rRNA gene sequence 33 similarity) and Salinivibrio costicola subsp. costicola DSM 11403T (99.0-98.1 %). 34 According to the MLSA analyses based on four (gyrB, recA, rpoA and rpoD) and eight 35 (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) concatenated gene sequences, the 36 most closely relatives were S. siamensis JCM 14472T (96.8-95.4 % and 94.9-94.7 %, 37 respectively) and S. sharmensis DSM 18182T (94.0-92.6 % and 92.9-92.7 %, 38 respectively). In silico DNA-DNA hybridization (GGDC) and Average Nucleotide 39 Identity (ANI) showed values of 80-100 % and 97.7-100 %, respectively with the 40 related species demonstrating that the ten isolates constituted a single novel species of 41 the genus Salinivibrio. Its pangenome and core genome consist of 6,041 and 1,230 42 genes, respectively. The phylogeny based on the concatenated orthologous core genes 43 revealed that the ten strains form a coherent phylogroup well separated from the rest of 44 the species of the genus Salinivibrio. The major cellular fatty acids of strain AL184T 45 were C16:0 and C18:1. The DNA G+C content range was 51.9-52.5 mol% (Tm) and 50.2- 46 50.9 mol% (genome). Based on the phylogenetic-phylogenomic, phenotypic and 47 chemotaxonomic data, the ten isolates represent a novel species of the genus 48 Salinivibrio, for which the name Salinivibrio kushneri sp. nov. is proposed. The type 49 strain is AL184T (= CECT 9177T = LMG 29817T). 50 3 51 Keywords: Vibrionaceae, Salinivibrio, moderately halophilic bacteria, new species, 52 salterns, genomic analysis. 53 54 Introduction 55 In 1938 Smith [44] described the species Vibrio costicolus isolated from rib 56 bones of bacon. This species was one of the few moderately halophilic bacteria included 57 on the Approved Lists of Bacterial Names [43], correctly named as V. costicola. Early 58 studies on water of ponds from marine salterns showed that halophilic vibrios were 59 easily isolated [50] and lately they were characterized taxonomically as members of V. 60 costicola [11,14], permitting an amended description of this species. This bacterium has 61 been used as a model microorganism for many physiological studies [19,22,33,49], due 62 to the fact that it is able to grow over a wide range of salt concentrations. In 1996 63 Mellado et al. [31] proposed a reclassification of V. costicola into a new genus, named 64 as Salinivibrio costicola, on the basis of the 16S rRNA gene sequence comparison, 65 showing that it constitutes a separate phylogenetic clade with respect to the genus 66 Vibrio, as well as on the phenotypic differences with the most closely related taxa. This 67 genus belongs to the family Vibrionaceae within the class Gammaproteobacteria and 68 includes Gram-stain-negative, curved motile rods, facultatively anaerobic, able to grow 69 on a NaCl range from 0.5 to 20 % (w/v), and catalase and oxidase positive. The DNA 70 G+C content ranges from 49.0 to 51.0 mol% [16,31]. Currently, the genus Salinivibrio 71 comprises four species, one of them with three subspecies: Salinivibrio costicola subsp. 72 costicola [11,16,31,44], Salinivibrio costicola subsp. vallismortis [18], Salinivibrio 73 costicola subsp. alcaliphilus [38], Salinivibrio proteolyticus [2], Salinivibrio siamensis 74 4 [5] and Salinivibrio sharmensis [39]. Members of this genus inhabit salted meats, brines 75 and hypersaline environments. 76 The evolutionary history of several vibrios (including the type species of the 77 genus Salinivibrio) was reconstructed in 2007 by means of multilocus sequence analysis 78 (MLSA) of nine genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S 79 rRNA), proposing the Salinivibrio clade [41]. Later in 2013, the vibrio clades were 80 updated by MLSA based on the eight protein-coding housekeeping genes previously 81 mentioned, delineating a super-clade grouping the genera Salinivibrio, Grimontia and 82 Enterovibrio [42]. Recently, López-Hermoso et al. [27] carried out a study in which 70 83 new isolates belonging to the genus Salinivibrio and the type strains of the species and 84 subspecies of this genus were analyzed by 16S rRNA gene sequence comparison and 85 MLSA. The data showed that the 16S rRNA gene was not able to properly differentiate 86 the new isolates or assign them to the previously described species of Salinivibrio but 87 alternative MLSA analyses allowed to clearly distinguish well-defined phylogroups. 88 They validated the MLSA, based on the concatenation of gyrB, recA, rpoA and rpoD 89 housekeeping genes, in order to replace the DNA-DNA hybridization (DDH) assays in 90 the genus Salinivibrio, establishing a cut-off value for species delineation of 97 % 91 concatenated MLSA similarity. Besides, in this study they observed clearly that some of 92 the isolates (which formed a single phylogroup) could constitute a new species within 93 this genus. 94 In this study, we carried out the taxonomic characterization of ten moderately 95 halophilic bacteria from five salterns located in different places in Spain selected from 96 the previous study of López-Hermoso et al. [27], and the data suggest that they 97 constitute a novel species of the genus Salinivibrio. Their characterization was achieved 98 by following a polyphasic approach. The phylogenetic, genotypic and phenotypic 99 5 characteristics of these strains, including 16S rRNA gene sequence analysis, MLSA, 100 DDH, ANI, GGDC and chemotaxonomic features, have been carried out in order to 101 define their taxonomic status. 102 Materials and methods 103 Bacterial strains and growth conditions 104 The strains used for this study were isolated from water samples collected from 105 different ponds of salterns of Spain: strain AL184T was isolated from Santa Pola, 106 Alicante, strains IB560 and IB563 from Isla Bacuta, Huelva, strains IC202 and IC317 107 from Isla Cristina, Huelva, strain MA421 from La Malahá, Granada, and strains 108 ML277, ML318, ML328A and ML331 were isolated from Es Trenc, Mallorca. The 109 salinity of the samples was determined to be between 5 and 21 % salts [27]. The strains 110 were isolated by plating 0.1 ml of the water samples on SW medium with the pH 111 adjusted between 7.2-7.4 with 1 M KOH and incubated at 37 ºC. After isolation, the 112 strains were subsequently purified three times by plating on the same medium. The 113 composition of SW medium is the following: (g l−1): NaCl, 58.5; MgCl2·6H2O, 9.75; 114 MgSO4·7H2O, 15.25; CaCl2, 0.25; KCl, 1.5; NaHCO3, 0.05; NaBr, 0.175; and yeast 115 extract, 5.0, and solidified with 1.8 % (w/v) agar when necessary. The strains were 116 routinely grown on SW medium and were maintained on the same medium in slant 117 tubes, and for long term preservation at -80 ºC in SW broth with 20 % (v/v) glycerol. S. 118 costicola subsp. costicola DSM 11403T, S. costicola subsp. alcaliphilus DSM 16359T, 119 S. costicola subsp. vallismortis DSM 8285T, S. proteolyticus DSM 19052T, S. 120 sharmensis DSM 18182T and S. siamensis JCM 14472T were used as reference strains 121 for comparison purposes in the present study (Supplementary Table S1). 122 Phenotypic characterization 123 6 Cell morphology and motility were examined by phase-contrast microscopy 124 (Olympus CX41) from exponentially growing cultures. The morphology and 125 pigmentation of colonies were observed on SW solid medium after 24 h of incubation at 126 37 ºC. Growth range and optimum were determined on SW medium with different salt 127 concentrations (0, 1, 2, 3, 4, 5, 7.5, 10, 12.5, 15, 17.5, 20, 21, 22, 23, 24 and 25%, w/v) 128 at pH 7.2-7.4. To determine the optimal and range of temperature and pH supporting the 129 growth of the strains, SW broth cultures were incubated at 5-35ºC at intervals of 5 ºC 130 and from 35-50 ºC in increments of 1 ºC and at pH 4-10 at intervals of 0.5 pH units with 131 the addition of the appropriate buffering capacity to each medium [40]. Growth was 132 determined by monitoring the optical density at 600 nm using a spectrophotometer. 133 Catalase activity was determined by bubble production in 3 % (w/v) H2O2 solution. 134 Oxidase activity was examined using 1 % (v/v) tetramethyl-p-phenylenendiamine [21]. 135 Hydrolysis of aesculin, casein, DNA, gelatin, starch and Tween 80, Voges-Proskauer 136 and methyl red tests, production of indole, phenylalanine deaminase, phosphatase, 137 nitrate and nitrite reduction and Simmon’s citrate were determined as described by 138 Cowan and Steel [6] with the addition of 7.5 % total salts to the medium [36,50]. 139 Growth under anaerobic conditions (with H2/CO2) was determined by incubation in an 140 anaerobic jar using Anaerogen (Oxoid) to generate an anaerobic atmosphere, and an 141 anaerobic indicator (Oxoid), on SW solid medium during one week. Acid production 142 from carbohydrates was determined using a phenol red base supplemented with 1 % 143 carbohydrate and SW medium; this medium was a modification of the original 144 described elsewhere [50]. For determination of the range of substrates used as carbon 145 and energy sources or as carbon, nitrogen and energy sources, the classical medium of 146 Koser [20] as modified by Ventosa et al. [50] was used. This medium contained (per 147 liter): 75 g NaCl, 2 g KCl, 0.2 g MgSO4·7 H2O, 1 g KNO3, 1 g (NH4)2HPO4, 0.5 g 148 7 KH2PO4 and 0.05 g yeast extract (BD). Substrates were added as filter-sterilized 149 solutions to give a final concentration of 1 g l-1, except for carbohydrates, which were 150 used at 2 g l-1. 151 Phylogenetic analysis 152 The almost-complete 16S rRNA gene and four protein-coding housekeeping 153 genes were previously sequenced and analyzed by López-Hermoso et al. [27]. The 154 almost-complete 16S rRNA gene sequence of strains AL184T, IB560, IB563, IC202, 155 IC317, MA421, ML277, ML318, ML328A and ML331 had a length of 1435, 1468, 156 1467, 1478, 1435, 1480, 1483, 1479, 1431 and 1445 bp, respectively; whereas 157 housekeeping genes had a length: gyrB (623 bp), recA (701 bp), rpoA (820 bp) and 158 rpoD (734 bp) [27]. The sequences obtained from the 16S rRNA gene and the four 159 housekeeping genes were assembled by using ChromasPro software (Technelysium Pty) 160 and edited to solve ambiguous positions. Multiple sequence alignments were made 161 using CLUSTAL_X 2.1 [23] and corrected by visual inspection using BioEdit [15] 162 taking into account the corresponding amino acid alignments for protein-coding genes. 163 Phylogenetic trees were constructed using MEGA 5 [47] for neighbour-joining and 164 maximum-parsimony methods and PhyML [13] for the maximum-likelihood (ML) [9] 165 method. Neigbour-joining analyses were performed using Jukes-Cantor parameter 166 model [18]. Maximum-parsimony analyses were carried out using a heuristic search 167 option. For ML analysis, the GTR model was selected and the base frequencies, the rate 168 matrix, the proportion of invariable sites and the gamma distribution were estimated via 169 likelihood. Bootstrap analyses were based on 1000 replications [10]. The sequence 170 accession numbers used in this study are shown in Supplementary Table S1. 171 Additionally, an eight gene (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) MLSA 172 was also carried out as previously described [1,41,42], retrieving the housekeeping 173 8 genes from the available genomes [26], using the methodology indicated above for 174 sequence alignment and tree construction. 175 DNA G+C content and DNA–DNA hybridization 176 The G+C content of the genomic DNA of the ten strains was determined from 177 the midpoint value (Tm) of the thermal denaturation profile [29] by using the equation of 178 Owen and Hill [34], obtained with a Perkin-Elmer UV-Vis Lambda 20 179 spectrophotometer at 260 nm equipped with a PTP-1 peltier system programmed with 180 an increasing temperature of 1 ºC min-1. DNA–DNA hybridization studies were 181 performed using the competition procedure of the membrane method [17], as described 182 in detail by Arahal et al. [3,4]. The hybridization temperature was 52.2 ºC, and the 183 percentage of hybridization was calculated according to Johnson [17]. The experiments 184 were carried out in triplicate and the percentages shown are the mean values. Besides, 185 the genomic G+C content calculation based on the draft genome sequences was 186 performed with the tool enveomics [28]. 187 Chemotaxonomic analysis 188 The fatty acid composition was determined for cells of strain AL184T, selected 189 as representative of this bacterial group, grown for 24 h at 35 ºC in the medium 1 190 described by Romano et al. [39] containing (g l-1): yeast extract, 10.0; NaCl, 100.0; 191 Na3-citrate, 3.0; Na2CO3 3.0, KCl, 2.0; MgSO4·7H2O, 1.0; MnCl2·4H2O, 0.00036; 192 FeSO4, 0.05. The resulting pH was 9.0. The fatty acid profile was obtained with an 193 Agilent 6850 gas chromatograph using the database TSBA6 (MIDI, 2008) [32]. These 194 analyses were carried out at the Spanish Type Culture Collection (CECT), Valencia, 195 Spain. 196 Genome sequencing, assembly and annotation 197 9 Draft genomes of the ten isolates and the type strains of the species and 198 subspecies of Salinivibrio were previously sequenced by López-Hermoso et al. [26]. We 199 also used the graft genome sequence of Salinivibrio costicola subsp. costicola LMG 200 11651T as described by Gorriti et al. [12]. The accession numbers of the draft genome 201 sequences used in this study are shown in Supplementary Table S1. 202 Calculation of in silico DDH and ANI values 203 In silico DDH was calculated by the Genome-to-Genome Distance Calculator 204 (GGDC 2.0) using the BLAST+ method [30]. Results were based on recommended 205 formula 2 (identities/HSP length), which is independent of genome length and is thus 206 robust against the use of incomplete draft genomes. Calculation of the average 207 nucleotide identity (ANI) from the draft genome sequences used previously for in silico 208 DDH was performed with JSpecies [37], whereas OrthoANI percentages were 209 calculated as described by Lee et al. [24]. 210 Core genome phylogenetic reconstruction 211 All predicted protein-coding genes and amino acids annotated from each 212 available genome were compared using an all-versus-all BLAST search [28]. This 213 analysis identified shared reciprocal best matches (defined as > 70 % nucleotide identity 214 or > 40 % amino acid identity) in all pairwise genome comparisons (core orthologues) 215 of the ten Salinivibrio strains and the related taxa of the genus Salinivibrio, as well as 216 the reference strain Vibrio cholerae N16961. The core orthologous genes were 217 individually aligned using MUSCLE [8]. The resulting nucleotide alignments were 218 concatenated to create a core-genome alignment, and the phylogenomic tree was 219 reconstructed by neighbour-joining method with MEGA 5 [47]. Additionally, the core 220 16 mol% (genome) and 51.9-52.5 mol% (Tm). The estimated genome size is 3.3-3.6 Mb. 366 Isolated from marine salterns. 367 The type strain is AL184T (= CECT 9177T = LMG 29817T), isolated from water 368 pond of a solar saltern in Santa Pola, Alicante, Spain. The DNA G+C content of the 369 type strain is 50.2 mol% (genome) and 52.5 mol % (Tm). Strains IB560, IB563, IC202, 370 IC317, MA421, ML277, ML318, ML328A and ML331 are additional strains of the 371 species. The Digital Protologue taxonumber is TA00236. 372 373 374 Acknowledgements 375 C. L-H. was a recipient of a fellowship from the Spanish Ministerio de 376 Educacion, Cultura y Deporte. We thank Dr. Aharon Oren for helpful nomenclature 377 advice and the students Laura Barroso and Cristina Galisteo for theirs teamwork in the 378 laboratory. This study was supported by the Spanish Ministry of Economy and 379 Competitiveness (MINECO) through project CGL2013-46941-P, that included FEDER 380 funds. 381 382 Appendix A. Supplementry data 383 Supplementary data associated with this article can be found, in the online 384 version at XXXXXX. 385 386 References 387 [1] Amin, A.K.M.R., Tanaka, M., Al-Saari, N., Feng, G., Mino, S., Ogura, Y., Hayashi, 388 T., Meirelles, P.M., Thompson, F.L., Gomez-Gil, B., Sawabe, T., Sawabe, T. (2017) 389 Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. 390 17 within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, 391 Japan. Syst. Appl. Microbiol. 40, 290–296. 392 [2] Amoozegar, M.A., Schumann, P., Hajighasemi, M., Fatemi, A.Z., Karbalaei- 393 Heidari, H.R. 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Int. J. Syst. Evol. Microbiol. 67, 1613-1617. 537 538 Legends of figures 539 540 Fig. 1. Neighbour-joining tree based on the 16S rRNA gene sequence comparison, 541 showing the phylogenetic position of strains AL184T, IB560, IB563, IC202, IC317, 542 MA421, ML277, ML318, ML328A and ML331 and their relationship with other 543 species and subspecies of the genus Salinivibrio. The GenBank/EMBL/DDBJ accession 544 number of each sequence is shown in parenthesis. Filled circles indicate nodes that were 545 also obtained in maximum-parsimony and maximum-likelihood trees based on the same 546 sequences. Bootstrap values over 70 % are shown at the nodes. Bar, 0.01 substitutions 547 per nucleotide position. The species Vibrio cholerae CECT 514T was used as outgroup. 548 549 550 Fig. 2. Neighbour-joining tree based on the concatenated gyrB, recA, rpoA and rpoD 551 gene sequence comparison, showing the phylogenetic position of strains AL184T, 552 IB560, IB563, IC202, IC317, MA421, ML277, ML318, ML328A and ML331 and their 553 relationship with other species and subspecies of the genus Salinivibrio. Filled circles 554 indicate nodes that were also obtained in maximum-parsimony and maximum- 555 likelihood trees based on the same sequences. Bootstrap values over 70 % are shown at 556 the nodes. Bar, 0.05 substitutions per nucleotide position. The species Vibrio cholerae 557 N16961 was used as outgroup. 558 559 24 560 Fig. 3. Core phylogenomic tree of the 16 available genomes of the genus Salinivibrio. 561 This tree was constructed based on the neighbour-joining distance calculated from the 562 alignment of 1,230 shared orthologous genes of these genomes. All genomes were 563 sequenced by López-Hermoso et al. [26] and Gorriti et al. [12]. Filled circles indicate 564 nodes that were also obtained in maximum-parsimony and maximum-likelihood trees 565 based on the same sequences. Bootstrap values over 70 % are shown above the branch. 566 Bar, 0.01 substitutions per nucleotide position. The species Vibrio cholerae N16961 567 was used as outgroup. 568 569 570 Fig. 4. Venn diagram showing the core orthologous and unique proteins for the type 571 strains of species of the genus Salinivibrio. 572 25 Table 1. Differential characteristics between Salinivibrio kushneri sp. nov. and related 573 species and subspecies of the genus Salinivibrio. 574 1, Salinivibrio kushneri sp. nov. (10 strains); 2, S. siamensis JCM 14472T; 3, S. sharmensis 575 DSM 18182T; 4, S. costicola subsp. alcaliphilus DSM 16359T; 5, S. costicola subsp. costicola 576 DSM 11403T; 6, S. proteolyticus DSM 19052T; 7, S. costicola subsp. vallismortis DSM 8285T. 577 All strains were curve rods. They were positive for catalase, oxidase, hydrolysis of DNA, 578 Voges-Proskauer and phosphatase, and utilization of ribose as sole carbon and energy source. 579 All strains produced acid from fructose, D-glucose, maltose and D-trehalose. All strains were 580 negative for acid production from lactose, raffinose and D-xylose. All data are from this study 581 unless otherwise indicated. +, positive, -, negative. 582 Characteristic 1 2 3 4 5 6 7 Colony pigmentation Cream Creama Creamb Creampinkc Creamd Creamwhitee Creamwhitef NaCl range (%, w/v) 2- 20 1-22a 6-16b 2-25c 0.5-20d 1-17e 0-12.5f NaCl optimum (%, w/v) 7.5 10a 10b 10c 10d 5e 2.5f Temperature range (ºC) 17-49 10-47a 25-40b 10-40c 5-45d 10-45e 20-50f Temperature optimum (ºC) 37 37a 35b 30c 37d 35e 37f pH range 5-10 5-9a 6-10b 7-10.5c 5-10d 5-9.5e 5-10f pH optimum 7.2-7.4 8a 9b 9c 7.5d 8e 7.3f Anaerobic growth + + + - + + + Nitrate reduction + + + + - + - Nitrite reduction - - - + - + - Hydrolysis of Tween 80 - + - - - - + Acid production from ribose + + + - - + + Utilization of sucrose as sole carbon and energy source + - - + + + + Utilization of alanine as sole carbon, nitrogen + + + + + - -