Vibrio zhuhaiensis sp. nov., isolated from a Japanese prawn (Marsupenaeus japonicus)

A Gram-negative, oxidase-positive, facultatively anaerobic bacterium, designated strain E20121, was isolated from the digestive tract of a Japanese prawn (Marsupenaeus japonicus) collected from the coastal sea water area of Zhuhai, Guangdong province, China. The new isolate was determined to be closely related to Vibrio ponticus DSM 16217^T, having 97.6 % 16S rRNA gene sequence similarity. Phylogenetic analysis based on recA, pyrH and rpoA also showed low levels of sequence similarities (72.6–96.6 %) with all species of the genus Vibrio. A multigene phylogenetic tree using concatenated sequences of the four genes (16S rRNA, rpoA, recA and pyrH) clearly showed that the new isolate is different from the currently known Vibrio species. DNA–DNA hybridization experiments revealed similarity values below 70 % with the closest related species V. ponticus DSM 16217^T. Several phenotypic traits enabled the differentiation of strain E20121 from the closest phylogenetic neighbours. The DNA G+C content of strain E20121 was determined to be 47.6 mol % and the major fatty acid components identified were C_16:1ω7c and/or C_16:1ω6c (39.8 %), C_18:1ω7c (13.6 %) and C_16:0 (9.6 %). Based on genotypic, phenotypic, chemotaxonomic, phylogenetic and DNA–DNA hybridization analyses, strain E20121 is proposed to represent a novel species of the genus Vibrio for which the name Vibrio zhuhaiensis sp. nov. is proposed. The type strain is E20121^T(=DSM 25602^T = CCTCC AB 2011174^T).     

Optimization of Multilocus Sequence Analysis for Identification of Species in the Genus Vibrio

Multilocus sequence analysis (MLSA) is an important method for identification of taxa that are not well differentiated by 16S rRNA gene sequences alone. In this procedure, concatenated sequences of selected genes are constructed and then analyzed. The effects that the number and the order of genes used in MLSA have on reconstruction of phylogenetic relationships were examined. The recA, rpoA, gapA, 16S rRNA gene, gyrB, and ftsZ sequences from 56 species of the genus Vibrio were used to construct molecular phylogenies, and these were evaluated individually and using various gene combinations. Phylogenies from two-gene sequences employing recA and rpoA in both possible gene orders were different. The addition of the gapA gene sequence, producing all six possible concatenated sequences, reduced the differences in phylogenies to degrees of statistical (bootstrap) support for some nodes. The overall statistical support for the phylogenetic tree, assayed on the basis of a reliability score (calculated from the number of nodes having bootstrap values of ≥80 divided by the total number of nodes) increased with increasing numbers of genes used, up to a maximum of four. No further improvement was observed from addition of the fifth gene sequence (ftsZ), and addition of the sixth gene (gyrB) resulted in lower proportions of strongly supported nodes. Reductions in the numbers of strongly supported nodes were also observed when maximum parsimony was employed for tree construction. Use of a small number of gene sequences in MLSA resulted in accurate identification of Vibrio species.     

Advanced Microbial Taxonomy Combined with Genome-Based-Approaches Reveals that Vibrio astriarenae sp. nov., an Agarolytic Marine Bacterium,Forms a New Clade in Vibrionaceae

Advances in genomic microbial taxonomy have opened the way to create a more universal and transparent concept of species but is still in a transitional stage towards becoming a defining robust criteria for describing new microbial species with minimum features obtained using both genome and classical polyphasic taxonomies. Here we performed advanced microbial taxonomies combined with both genome-based and classical approaches for new agarolytic vibrio isolates to describe not only a novel Vibrio species but also a member of a new Vibrio clade. Two novel vibrio strains (Vibrio astriarenae sp. nov. C7^T and C20) showing agarolytic, halophilic and fermentative metabolic activity were isolated from a seawater sample collected in a coral reef in Okinawa. Intraspecific similarities of the isolates were identical in both sequences on the 16S rRNA and pyrH genes, but the closest relatives on the molecular phylogenetic trees on the basis of 16S rRNA and pyrH gene sequences were V. hangzhouensis JCM 15146^T (97.8% similarity) and V. agarivorans CECT 5085^T (97.3% similarity), respectively. Further multilocus sequence analysis (MLSA) on the basis of 8 protein coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) obtained by the genome sequences clearly showed the V. astriarenae strain C7^T and C20 formed a distinct new clade protruded next to V. agarivorans CECT 5085^T. The singleton V. agarivorans has never been included in previous MLSA of Vibrionaceae due to the lack of some gene sequences. Now the gene sequences are completed and analysis of 100 taxa in total provided a clear picture describing the association of V. agarivorans into pre-existing concatenated network tree and concluded its relationship to our vibrio strains. Experimental DNA-DNA hybridization (DDH) data showed that the strains C7^T and C20 were conspecific but were separated from all of the other Vibrio species related on the basis of both 16S rRNA and pyrH gene phylogenies (e.g., V. agarivorans CECT 5085^T, V. hangzhouensis JCM 15146^T V. maritimus LMG 25439^T, and V. variabilis LMG 25438^T). In silico DDH data also supported the genomic relationship. The strains C7^T also had less than 95% average amino acid identity (AAI) and average nucleotide identity (ANI) towards V. maritimus C210, V. variabilis C206, and V. mediterranei AK1^T, V. brasiliensis LMG 20546^T, V. orientalis ATCC 33934^T, and V. sinaloensis DSM 21326. The name Vibrio astriarenae sp. nov. is proposed with C7 as the type strains. Both V. agarivorans CECT 5058^T and V. astriarenae C7^T are members of the newest clade of Vibrionaceae named Agarivorans.     

Genomic diversity of vibrios associated with the Brazilian coral Mussismilia hispida and its sympatric zoanthids (Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi)

Aims: A taxonomic survey of the vibrios associated with the Brazilian endemic coral Mussismilia hispida and the sympatric zoanthids (i.e. Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi). Methods and Results: Mucus of 54 cnidarian specimens collected in three different places at São Sebastião in two consecutive years (i.e. 2005 and 2006) was used for taxonomic characterization of the cnidarian microbiota. Ninety‐eight of the 151 vibrio isolates fell within the vibrio core group according to partial 16S rDNA sequences. We performed the sequencing of recA and pyrH genes of all vibrio isolates. The most abundant taxa belonged to the vibrio core group (Vibrio harveyi, Vibrio rotiferianus, Vibrio campbellii and Vibrio alginolyticus), Vibrio mediterranei (=Vibrio shillonii) and Vibrio chagasii. With the exception of V. chagasii which was found only in the mucus of M. hispida, the other species appeared in different hosts with no evidence for the presence of host‐specific clones or species. Using rep‐PCR analysis, we observed a high genomic heterogeneity within the vibrios. Each vibrio isolate generated a different rep‐PCR fingerprint pattern. There was a complete agreement between the grouping based on rep‐PCR and concatenated sequences of pyrH, recA and 16S rDNA, but the pyrH gene has the highest discriminatory power for vibrio species identification. Conclusion: The vibrio core group is dominant in the mucus of these cnidarians. There is a tremendous diversity of vibrio lineages within the coral mucus. pyrH gene sequences permit a clear‐cut identification of vibrios. Significance and Impact of the Study: The taxonomic resolution provided by pyrH (but not recA) appears to be enough for identifying species of vibrios and for disclosing putative new taxa. The vibrio core group appears to be dominant in the mucus of the Brazilian cnidarians. The overrepresentation of these vibrios may reflect as yet unknown ecological functions in the coral holobiont.     

Genes Similar to the Vibrio parahaemolyticus Virulence-Related Genes tdh,tlh, and vscC2 Occur in Other Vibrionaceae Species Isolated from a Pristine Estuary

Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2α gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections.     

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.     

Multi-gene analysis reveals previously unrecognized phylogenetic diversity in Aliivibrio

The “Vibrio fischeri species group” recently was reclassified as a new genus, Aliivibrio, comprising four species, Aliivibrio fischeri, Aliivibrio logei, Aliivibrio salmonicida, and Aliivibrio wodanis. Only limited phylogenetic analysis of strains within Aliivibrio has been carried out, however, and taxonomic ambiguity is evident within this group, especially for phenotypically unusual strains and certain strains isolated from bioluminescent symbioses. Therefore, to examine in depth the evolutionary relationships within Aliivibrio and redefine the host affiliations of symbiotic species, we examined several previously identified and newly isolated strains using phylogenetic analysis based on multiple independent loci, gapA, gyrB, pyrH, recA, rpoA, the luxABE region, and the 16S rRNA gene. The analysis resolved Aliivibrio as distinct from Vibrio, Photobacterium, and other genera of Vibrionaceae, and resolved A. fischeri, A. salmonicida, A. logei, and A. wodanis as distinct, well-supported clades. However, it also revealed that several previously reported strains are incorrectly identified and that substantial unrecognized diversity exists in this genus. Specifically, strain ATCC 33715 (Y-1) and several other strains having a yellow-shifted luminescence were not members of A. fischeri. Furthermore, no strain previously identified as A. logei grouped with the type strain (ATCC 29985^T), and no bona-fide strain of A. logei was identified as a bioluminescent symbiont. Several additional strains identified previously as A. logei group instead with the type strain of A. wodanis (ATCC BAA-104^T), or are members of a new clade. Two strongly supported clades were evident within A. fischeri, a phylogenetic structure that might reflect differences in the host species or differences in the ecological incidence of strains. The results of this study highlight the importance of basing taxonomic conclusions on examination of type strains.     

Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae,isolated from coral reef seawater off Ishigaki Island,Japan

Two phylogenetically distinct Vibrionaceae strains C4II189^T and C4V358^T isolated from reef seawater off Ishigaki Island, Japan, in 2014 were studied with advanced genome-based taxonomy approaches. All aspects of phylogenetic (16S rRNA phylogeny, MLSA), phenotypic and genetic (ANI, DDH, AAI, and the number of core genes) cohesions between the two identified species were high enough to propose them as members of a new genus within the family Vibrionaceae. Consequently, an eighth genus Thaumasiovibrio gen. nov. is proposed that contains two new species Thaumasiovibrio occultus sp. nov. strain C4II189^T (=DSM 101554^T = JCM 31629^T) (type species) and Thaumasiovibrio subtropicus sp. nov. strain C4V358^T (=DSM 101555^T = JCM 31630^T). Thaumasiovibrio species were phylogenetically distinct from the other Vibrionaceae species based on pyrH gene sequences. The combination of catalase negative, sensitivity to vibriostatic agent O/129, and green colony formation on TCBS for the phylogenetically affiliated strains was the diagnostic features for the current tentative identification of this genus.     

Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain

A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams (Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase α-chain (rpoA), RecA protein (recA), the α-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA–DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9^T(=CECT 7244^T=LMG 24045^T).     

Vibrio taketomensis sp. nov. by genome taxonomy

Two novel strains C4III282^T and C4III291 were isolated from seawater collected a site off the Taketomi coral reef. Phylogenetic analysis based on the 16S rRNA sequences revealed that the two strains belong to the genus Vibrio. MLSA using eight protein-coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) showed that C4III282^T and C4III291 are closely related to the members of the Ponticus clade, namely Vibrio panuliri JCM 19500^T, Vibrio ponticus DSM 16217^T, and “Vibrio rhodolitus” G98. ANI and in silico DDH values with members of the Ponticus clade were 77.6-78.7% and 22.2-23.1, respectively. The name Vibrio taketomensis sp. nov. is proposed with C4III282^T (CAIM 1928^T = DSM 106943^T = JCM 33434^T) as the type strain.     

Transoceanic Spreading of Pathogenic Strains of Vibrio parahaemolyticus with Distinctive Genetic Signatures in the recA Gene

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. Consistent multilocus sequence typing for V. parahaemolyticus has shown difficulties in the amplification of the recA gene by PCR associated with a lack of amplification or a larger PCR product than expected. In one strain (090–96, Peru, 1996), the produced PCR product was determined to be composed of two recA fragments derived from different Vibrio species. To better understand this phenomenon, we sequenced the whole genome of this strain. The hybrid recA gene was found to be the result of a fragmentation of the original lineage-specific recA gene resulting from a DNA insertion of approximately 30 kb in length. This insert had a G+C content of 38.8%, lower than that of the average G+C content of V. parahaemolyticus (45.2%), and contained 19 ORFs, including a complete recA gene. This new acquired recA gene deviated 24% in sequence from the original recA and was distantly related to recA genes from bacteria of the Vibrionaceae family. The reconstruction of the original recA gene (recA3) identified the precursor as belonging to ST189, a sequence type reported previously only in Asian countries. The identification of this singular genetic feature in strains from Asia reveals new evidence for genetic connectivity between V. parahaemolyticus populations at both sides of the Pacific Ocean that, in addition to the previously described pandemic clone, supports the existence of a recurrent transoceanic spreading of pathogenic V. parahaemolyticus with the corresponding potential risk of pandemic expansion.     

Photobacterium piscicola sp. nov., isolated from marine fish and spoiled packed cod

Five isolates from marine fish (W3^T, WM, W1S, S2 and S3) and three isolates misclassified as Photobacterium phosphoreum, originating from spoiled modified atmosphere packed stored cod (NCIMB 13482 and NCIMB 13483) and the intestine of skate (NCIMB 192), were subjected to a polyphasic taxonomic study. Phylogenetic analysis of 16S rRNA gene sequences showed that the isolates were members of the genus Photobacterium. Sequence analysis using the gapA, gyrB, pyrH, recA and rpoA loci showed that these isolates formed a distinct branch in the genus Photobacterium, and were most closely related to Photobacterium aquimaris, Photobacterium kishitanii, Photobacterium phosphoreum and Photobacterium iliopiscarium. The luxA gene was present in isolates W3^T, WM, W1S, S2 and S3 but not in NCIMB 13482, NCIMB 13483 and NCIMB 192. AFLP and (GTG)₅-PCR fingerprinting indicated that the eight isolates represented at least five distinct genotypes. DNA–DNA hybridizations revealed 89% relatedness between isolate W3^T and NCIMB 192, and values below 70% with the type strains of the phylogenetically closest species, P. iliopiscarium LMG 19543^T, P. kishitanii LMG 23890^T, P. aquimaris LMG 26951^T and P. phosphoreum LMG4233^T. The strains of this new taxon could also be distinguished from the latter species by phenotypic characteristics. Therefore, we propose to classify this new taxon as Photobacterium piscicola sp. nov., with W3^T (=NCCB 100098^T = LMG 27681^T) as the type strain.     

Vibrio plantisponsor sp. nov., a diazotrophic bacterium isolated from a mangrove associated wild rice (Porteresia coarctata Tateoka)

Two Gram negative, facultatively anaerobic, halophilic, motile, slightly curved rod-shaped bacterial strains MSSRF60^T and MSSRF64 were isolated from the roots of a mangrove-associated wild rice collected in the Pichavaram mangroves, India. These strains possess the key functional nitrogenase gene nifH. Phylogenetic analysis based on the 16S rRNA, recA, gapA, mreB, gyrB and pyrH, gene sequences revealed that strains MSSRF60^T and MSSRF64 belong to the genus Vibrio, and had the highest sequence similarity with the type strains of Vibrio diazotrophicus LMG 7893^T (99.7, 94.8, 98.5, 97.9, 94.0 and 90.7%, respectively), Vibrio areninigrae J74^T (98.2, 87.5, 91.5, 88.9, 86.5 and 84.6% respectively) and Vibrio hispanicus LMG 13240^T (97.8, 87.1, 91.7, 89.8, 84.1 and 81.9%, respectively). The fatty acid composition too confirmed the affiliation of strains MSSRF60^T and MSSRF64 to the genus Vibrio. These strains can be differentiated from the most closely related Vibrio species by several phenotypic traits. The DNA G + C content of strain MSSRF60^T was 41.8 mol%. Based on phenotypic, chemotaxonomic, genotypic (multilocus sequence analysis using five genes and genomic fingerprinting using BOX-PCR) and DNA–DNA hybridization analyses, strains MSSRF60^T and MSSRF64 represent a novel species of the genus Vibrio, for which the name Vibrio plantipsonsor sp. nov. is proposed. The type strain is MSSRF60^T (=DSM 21026^T = LMG 24470^T = CAIM 1392^T).     

The fur Gene as a New Phylogenetic Marker for Vibrionaceae Species Identification

Microbial taxonomy is essential in all areas of microbial science. The 16S rRNA gene sequence is one of the main phylogenetic species markers; however, it does not provide discrimination in the family Vibrionaceae, where other molecular techniques allow better interspecies resolution. Although multilocus sequence analysis (MLSA) has been used successfully in the identification of Vibrio species, the technique has several limitations. They include the fact that several locus amplifications and sequencing have to be performed, which still sometimes lead to doubtful identifications. Using an in silico approach based on genomes from 103 Vibrionaceae strains, we demonstrate here the high resolution of the fur gene in the identification of Vibrionaceae species and its usefulness as a phylogenetic marker. The fur gene showed within-species similarity higher than 95%, and the relationships inferred from its use were in agreement with those observed for 16S rRNA analysis and MLSA. Furthermore, we developed a fur PCR sequencing-based method that allowed identification of Vibrio species. The discovery of the phylogenetic power of the fur gene and the development of a PCR method that can be used in amplification and sequencing of the gene are of general interest whether for use alone or together with the previously suggested loci in an MLSA.     

Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the P_BAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.     

Culturable Heterotrophic Bacteria Associated with Healthy and Bleached Scleractinian Madracis decactis and the Fireworm Hermodice carunculata from the Remote St. Peter and St. Paul Archipelago, Brazil

We report on the first characterization of the culturable heterotrophic bacteria of the scleractinian Madracis decactis. In addition, we characterized the culturable bacteria associated with the fireworm Hermodice carunculata, observed predating partially bleached coral colonies. Our study was carried out in the remote St. Peter and St. Paul Archipelago (SPSPA), Mid-Atlantic Ridge, Brazil. We constituted a 403 isolates collection and subsequently characterized it by means of pyrH and 16S rRNA partial sequences. We identified Photobacterium, Bacillus, and Vibrio species as members of the culturable microbiota of healthy M. decactis. V. campbellii, V. harveyi, V. communis, and V. maritimus were the most commonly found Vibrio species in healthy corals, representing more than 60 % of all vibrio isolates. Most of the vibrios isolated from the fireworm’s tissues (n = 143; >90 %) were identified as V. shiloi. However, we did not recover V. shiloi from bleached M. decactis. Instead, we isolated V. communis, a novel Photobacterium species, Bacillus, Kocuria, and Pseudovibrio, suggesting a possible role of other facultative anaerobic bacteria and/or environmental features (such as water quality) in the onset of bleaching in SPSPA’s M. decactis.     

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N₂-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of São Sebastião (São Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n=76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n=7), V. alginolyticus (n=8), V. campbellii (n=3), and V. parahaemolyticus (n=1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N₂ might explain why they are so abundant in the mucus of different coral species.     

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

Ten Gram-strain-negative, facultatively anaerobic, moderately halophilic bacterial strains, designated AL184^T, IB560, IB563, IC202, IC317, MA421, ML277, ML318, ML328A and ML331, were isolated from water ponds of five salterns located in Spain. The cells were motile, curved rods and oxidase and catalase positive. All of them grew optimally at 37 °C, at pH 7.2–7.4 and in the presence of 7.5% (w/v) NaCl. Based on phylogenetic analyses of the 16S rRNA, the isolates were most closely related to Salinivibrio sharmensis BAG^T (99.6–98.2% 16S rRNA gene sequence similarity) and Salinivibrio costicola subsp. costicola ATCC 35508^T (99.0–98.1%). According to the MLSA analyses based on four (gyrB, recA, rpoA and rpoD) and eight (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) concatenated gene sequences, the most closely relatives were S. siamensis JCM 14472^T (96.8–95.4% and 94.9–94.7%, respectively) and S. sharmensis DSM 18182^T (94.0–92.6% and 92.9–92.7%, respectively). In silico DNA–DNA hybridization (GGDC) and average nucleotide identity (ANI) showed values of 23.3–44.8% and 80.2–91.8%, respectively with the related species demonstrating that the ten isolates constituted a single novel species of the genus Salinivibrio. Its pangenome and core genome consist of 6041 and 1230 genes, respectively. The phylogeny based on the concatenated orthologous core genes revealed that the ten strains form a coherent phylogroup well separated from the rest of the species of the genus Salinivibrio. The major cellular fatty acids of strain AL184^T were C_16:0 and C_18:1. The DNA G + C content range was 51.9–52.5 mol% (T_m) and 50.2–50.9 mol% (genome). Based on the phylogenetic-phylogenomic, phenotypic and chemotaxonomic data, the ten isolates represent a novel species of the genus Salinivibrio, for which the name Salinivibrio kushneri sp. nov. is proposed. The type strain is AL184^T (= CECT 9177^T = LMG 29817^T).     

Multilocus Sequence Analysis for the Assessment of Phylogenetic Diversity and Biogeography in Hyphomonas Bacteria from Diverse Marine Environments

Hyphomonas, a genus of budding, prosthecate bacteria, are primarily found in the marine environment. Seven type strains, and 35 strains from our collections of Hyphomonas, isolated from the Pacific Ocean, Atlantic Ocean, Arctic Ocean, South China Sea and the Baltic Sea, were investigated in this study using multilocus sequence analysis (MLSA). The phylogenetic structure of these bacteria was evaluated using the 16S rRNA gene, and five housekeeping genes (leuA, clpA, pyrH, gatA and rpoD) as well as their concatenated sequences. Our results showed that each housekeeping gene and the concatenated gene sequence all yield a higher taxonomic resolution than the 16S rRNA gene. The 42 strains assorted into 12 groups. Each group represents an independent species, which was confirmed by virtual DNA-DNA hybridization (DDH) estimated from draft genome sequences. Hyphomonas MLSA interspecies and intraspecies boundaries ranged from 93.3% to 96.3%, similarity calculated using a combined DDH and MLSA approach. Furthermore, six novel species (groups I, II, III, IV, V and XII) of the genus Hyphomonas exist, based on sequence similarities of the MLSA and DDH values. Additionally, we propose that the leuA gene (93.0% sequence similarity across our dataset) alone could be used as a fast and practical means for identifying species within Hyphomonas. Finally, Hyphomonas'' geographic distribution shows that strains from the same area tend to cluster together as discrete species. This study provides a framework for the discrimination and phylogenetic analysis of the genus Hyphomonas for the first time, and will contribute to a more thorough understanding of the biological and ecological roles of this genus.