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Asian Seabass (Lates calcarifer) is widely farmed as a sustainable source of protein for countries in the tropical Indo-West Pacific region. However, microbial species of the gut microbiome of healthy Asian Seabass remain largely uncharacterized and uncultured. Here, we analysed the microbial composition along the gastrointestinal tract of a farmed healthy Asian Seabass. We used different cultivation approaches to obtain isolates from the seabass intestinal tract and describe the isolation and characterization of a novel strain, TLL-SE01 T . Analysis of the strain's 16S rRNA gene indicates that the strain belongs to the family Vibrionaceae with Photobacterium damselae as its closest relative, albeit sharing only 94.8% (aligned region 1553 bp) nucleotide identity. Comparative genomic analysis with all validly published Vibrionaceae species with available genomes revealed average nucleotide identity (ANI) and DNA-DNA hybridisation (DDH) values of around 70% and 24% respectively to strain TLL-SE01 T , which are well below proposed thresholds for species delineation (ANI, 95-96%; DDH, 70%). The alignment fraction and ANI genus demarcation boundaries for all genera in the Vibrionaceae family were determined for which strain TLL-SE01 T is well below the calculated values, indicating that it belongs to a novel genus. Single- and core-gene phylogenetic analysis places strain TLL-SE01 T in a monophyletic clade, further supporting its designation to a novel genus. Phenotypic comparison between strain TLL-SE01 T and its close relatives indicated additional differences, such as growth response at different salt concentrations and different metabolic capabilities. Based on genotypic, phylogenetic and phenotypic differences to other Vibrionaceae species, we propose a novel species in a new genus, Parasalinivibrio latis gen. nov. sp. nov. and strain TLL-SE01 T (= BCRC 81435 T  = JCM 36283 T ) as the type strain., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Extensive research on the diversity and functional roles of the microorganisms associated with reef-building corals has been promoted as a consequence of the rapid global decline of coral reefs attributed to climate change. Several studies have highlighted the importance of coral-associated algae (Symbiodinium) and bacteria and their potential roles in promoting coral host fitness and survival. However, the complex coral holobiont extends beyond these components to encompass other entities such as protists, fungi, and viruses. While each constituent has been individually investigated in corals, a comprehensive understanding of their collective roles is imperative for a holistic comprehension of coral health and resilience., Results: The metagenomic analysis of the microbiome of the coral Oculina patagonica has revealed that fungi of the genera Aspergillus, Fusarium, and Rhizofagus together with the prokaryotic genera Streptomyces, Pseudomonas, and Bacillus were abundant members of the coral holobiont. This study also assessed changes in microeukaryotic, prokaryotic, and viral communities under three stress conditions: aquaria confinement, heat stress, and Vibrio infections. In general, stress conditions led to an increase in Rhodobacteraceae, Flavobacteraceae, and Vibrionaceae families, accompanied by a decrease in Streptomycetaceae. Concurrently, there was a significant decline in both the abundance and richness of microeukaryotic species and a reduction in genes associated with antimicrobial compound production by the coral itself, as well as by Symbiodinium and fungi., Conclusion: Our findings suggest that the interplay between microeukaryotic and prokaryotic components of the coral holobiont may be disrupted by stress conditions, such as confinement, increase of seawater temperature, or Vibrio infection, leading to a dysbiosis in the global microbial community that may increase coral susceptibility to diseases. Further, microeukaryotic community seems to exert influence on the prokaryotic community dynamics, possibly through predation or the production of secondary metabolites with anti-bacterial activity. Video Abstract., (© 2024. The Author(s).)

The aim of this study was to investigate the prevalence of Vibrionaceae family in retail seafood products available in the Qidong market during the summer of 2023 and to characterize Vibrio parahaemolyticus isolates, given that this bacterium is the leading cause of seafood-associated food poisoning. We successfully isolated a total of 240 Vibrionaceae strains from a pool of 718 seafood samples. The breakdown of the isolates included 146 Photobacterium damselae, 59 V. parahaemolyticus, 18 V. campbellii, and 11 V. alginolyticus. Among these, P. damselae and V. parahaemolyticus were the predominant species, with respective prevalence rates of 20.3% and 8.2%. Interestingly, all 59 isolates of V. parahaemolyticus were identified as non-pathogenic. They demonstrated proficiency in swimming and swarming motility and were capable of forming biofilms across a range of temperatures. In terms of antibiotic resistance, the V. parahaemolyticus isolates showed high resistance to ampicillin, intermediate resistance to cefuroxime and cefazolin, and were sensitive to the other antibiotics evaluated. The findings of this study may offer valuable insights and theoretical support for enhancing seafood safety measures in Qidong City., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae . Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2 , that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1 / 2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene., Competing Interests: Competing interests statement:The authors declare no competing interest.

Introduction: as cholera, due to toxigenic bacteria Vibrio cholera (serogroups O1 and O139), is a major public health threat in Africa, the aim of this work was to investigate potentially pathogenic Vibrionaceae bacteria firstly from human stool samples, and secondly from various environmental water points of Saint-Louis city in Senegal., Methods: a hospital-based study was conducted between 2013 and 2015. Stool samples were taken and cultured from daily incoming patients or hospitalized for acute diarrhea at Saint-Louis´ regional hospital. For environment, a monthly longitudinal sampling from January to October 2016 was carried out at 10 sites in the city. We used total DNA extracted from APW (alkaline peptone water) broth solutions and on suspect bacterial colonies to run PCR Multiplex targeting specific DNA fragments to detect Vibrio genus and specific species. In case of positivity, a simplex PCR was performed to test for cholera toxins Ctx, and V. parahaemolyticus TRH and TDH., Results: for 43 patients screened, bacterial culture was positive in 6% of cases but no strain of V. cholerae or other Vibrio sp. was isolated. PCR on 90 APW solutions were positive for Vibrio sp.(n = 43), V. cholera(n = 27), V. mimicus(n = 16), V. parahaemolyticus(8), V. alginolyticus(n = 4), and V. vulnificus(n = 2). Unlike for those on suspected colonies which were positive for a majority of V. parahaemolyticus (n = 40) and V. cholerae non-O1 / O139 (n = 35). Six strains of V. parahaemolyticus carried TRH gene, 3 of which expressed simultaneously virulence TRH and TDH genes. For physicochemical parameters, all temperatures varied similarly according to a unimodal seasonality, as well as salinity., Conclusion: despite the presence of natural populations of Vibrionaceae, even toxigenic ones, was noted in water environment, along with favorable habitat conditions that could play a role in transmission of Vibriosis in the Saint Louis population, we did not isolate any of them from patients screened at the hospital., Competing Interests: The authors declare no competing interests., (Copyright: Seynabou Lo et al.)

Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions. ICs generally encode promoterless genes, whose expression relies on the platform-associated Pc promoter, with the cassette array functioning as an operon-like structure regulated by the distance to the Pc. This is relevant in large sedentary chromosomal integrons (SCIs) carrying hundreds of ICs, like those in Vibrio species. We selected 29 gene-less cassettes in four Vibrio SCIs, and explored whether their function could be related to the transcription regulation of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Additionally, we identified the transcription start sites of gene-less ICs through 5'-RACE. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 cassette downstream, increasing trimethoprim resistance >512-fold in V. cholerae and Escherichia coli. Furthermore, one cassette with an antisense promoter can reduce trimethoprim resistance when cloned downstream. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in SCIs and their clinical importance if captured by mobile integrons., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The marine bacteria of the Vibrionaceae family are significant from the point of view of their role in the marine geochemical cycle, as well as symbionts and opportunistic pathogens of aquatic animals and humans. The well-known pathogens of this group, Vibrio cholerae, V. parahaemolyticus, and V. vulnificus, are responsible for significant morbidity and mortality associated with a range of infections from gastroenteritis to bacteremia acquired through the consumption of raw or undercooked seafood and exposure to seawater containing these pathogens. Although generally regarded as susceptible to commonly employed antibiotics, the antimicrobial resistance of Vibrio spp. has been on the rise in the last two decades, which has raised concern about future infections by these bacteria becoming increasingly challenging to treat. Diverse mechanisms of antimicrobial resistance have been discovered in pathogenic vibrios, the most important being the membrane efflux pumps, which contribute to antimicrobial resistance and their virulence, environmental fitness, and persistence through biofilm formation and quorum sensing. In this review, we discuss the evolution of antimicrobial resistance in pathogenic vibrios and some of the well-characterized efflux pumps' contributions to the physiology of antimicrobial resistance, host and environment survival, and their pathogenicity., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Marine water temperatures are increasing globally, with eastern Australian estuaries warming faster than predicted. There is growing evidence that this rapid warming of coastal waters is increasing the abundance and virulence of pathogenic members of the Vibrionaceae, posing a significant health risk to both humans and aquatic organisms. Fish disease, notably outbreaks of emerging pathogens in response to environmental perturbations such as heatwaves, have been recognised in aquaculture settings. Considerably less is known about how rising sea surface temperatures will impact the microbiology of wild fish populations, particularly those within estuarine systems that are more vulnerable to warming. We used a combination of Vibrio-specific quantitative PCR and amplicon sequencing of the 16S rRNA and hsp60 genes to examine seawater and fish (Pelates sexlineatus) gut microbial communities across a quasi-natural experimental system, where thermal pollution from coal-fired power stations creates a temperature gradient of up to 6 °C, compatible with future predicted temperature increases. At the warmest site, fish hindgut microbial communities were in a state of dysbiosis characterised by shifts in beta diversity and a proliferation (71.5% relative abundance) of the potential fish pathogen Photobacterium damselae subsp. damselae. Comparable patterns were not identified in the surrounding seawater, indicating opportunistic proliferation within estuarine fish guts under thermal stress. A subsequent evaluation of predicted future warming-related risk due to pathogenic Vibrionaceae in temperate estuarine fish demonstrated that warming is likely to drive opportunistic pathogen increases in the upper latitudinal range of this estuarine fish, potentially impacting adaptations to future warming. These findings represent a breakthrough in our understanding of the dynamics of emerging pathogens in populations of wild aquatic organisms within environments likely to experience rapid warming under future climate change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Splendidus clade is the largest clade in Vibrionaceae, and its members are often related to mortality of marine animals with huge economic losses. The molecular bases of their pathogenicity and virulence, however, remain largely unknown. In particular, the complete genome sequences of the Splendidus clade species are rarely registered, which is one of the obstacles to predict core and/or unique genes responsible for their adaptation and pathogenicity, and to perform a fine scale meta-transcriptome during bacterial infection to their hosts. In this study, we obtained the complete genomes of all type strains in the Splendidus clade and revealed that (1) different genome sizes (4.4-5.9 Mb) with V. lentus the biggest and most of them had several big plasmids, likely because of the different features on mobilome elements; (2) the Splendidus clade consists of 19 species except V. cortegadensis, and 3 sub-clades (SC) were defined with the 15 most closely related members as SC1; (3) different carbohydrate degradation preferences may be the result of environmental adaptation; and (4) a broad prediction of virulence factors (VFs) revealed core and species unique VF genes., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The Vibrionaceae family groups genetically and metabolically diverse bacteria thriving in all marine environments. Despite often representing a minor fraction of bacterial assemblages, members of this family can exploit a wide variety of nutritional sources, which makes them important players in biogeochemical dynamics. Furthermore, several Vibrionaceae species are well-known pathogens, posing a threat to human and animal health. Here, we applied the phylogenetic placement coupled with a consensus-based approach using 16S rRNA gene amplicon sequencing, aiming to reach a reliable and fine-level Vibrionaceae characterization and identify the dynamics of blooming, ecologically important, and potentially pathogenic species in different sites of the northern Adriatic Sea. Water samples were collected monthly at a Long-Term Ecological Research network site from 2018 to 2021, and in spring and summer of 2019 and 2020 at two sites affected by depurated sewage discharge. The 41 identified Vibrionaceae species represented generally below 1% of the sampled communities; blooms (up to ~ 11%) mainly formed by Vibrio chagasii and Vibrio owensii occurred in summer, linked to increasing temperature and particulate matter concentration. Pathogenic species such as Vibrio anguilllarum, Vibrio tapetis, and Photobacterium damselae were found in low abundance. Depuration plant samples were characterized by a lower abundance and diversity of Vibrionaceae species compared to seawater, highlighting that Vibrionaceae dynamics at sea are unlikely to be related to wastewater inputs. Our work represents a further step to improve the molecular approach based on short reads, toward a shared, updated, and curated phylogeny of the Vibrionaceae family., (© 2022. The Author(s).)

Piscibactin is a widespread siderophore system present in many different bacteria, especially within the Vibrionaceae family. Previous works showed that most functions required for biosynthesis and transport of this siderophore are encoded by the high-pathogenicity island irp -HPI. In the present work, using Vibrio anguillarum as a model, we could identify additional key functions encoded by irp -HPI that are necessary for piscibactin production and transport and that have remained unknown. Allelic exchange mutagenesis, combined with cross-feeding bioassays and LC-MS analysis, were used to demonstrate that Irp4 protein is an essential component for piscibactin synthesis since it is the thioesterase required for nascent piscibactin be released from the NRPS Irp1. We also show that Irp8 is a MFS-type protein essential for piscibactin secretion. In addition, after passage through the outer membrane transporter FrpA, the completion of ferri-piscibactin internalization through the inner membrane would be achieved by the ABC-type transporter FrpBC. The expression of this transporter is coordinated with the expression of FrpA and with the genes encoding biosynthetic functions. Since piscibactin is a major virulence factor of some pathogenic vibrios, the elements of biosynthesis and transport described here could be additional interesting targets for the design of novel antimicrobials against these bacterial pathogens.

Currently, over 190 species in family Vibrionaceae, including not-yet-cultured taxa, have been described and classified into over nine genera, in which the number of species has doubled compared to the previous vibrio evolutionary update (Vibrio Clade 2.0) (Sawabe et al. 2014). In this study, "Vibrio Clade 3.0," the second update of the molecular phylogenetic analysis was performed based on nucleotide sequences of eight housekeeping genes (8-HKGs) retrieved from genome sequences, including 22 newly determined genomes. A total of 51 distinct clades were observed, of which 21 clades are newly described. We further evaluated the delineation powers of the clade classification based on nucleotide sequences of 34 single-copy genes and 11 ribosomal protein genes (11-RPGs) retrieved from core-genome sequences; however, the delineation power of 8-HKGs is still high and that gene set can be reliably used for the classification and identification of Vibrionaceae. Furthermore, the 11-RPGs set proved to be useful in identifying uncultured species among metagenome-assembled genome (MAG) and/or single-cell genome-assembled genome (SAG) pools. This study expands the awareness of the diversity and evolutionary history of the family Vibrionaceae and accelerates the taxonomic applications in classifying as not-yet-cultured taxa among MAGs and SAGs., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations., Competing Interests: Acknowledgements This work was supported by Human Frontier Science Program Grant RGP0041/2017 (J.M.S.-R. and E.A.G.), National Science Foundation Award #2032315 (E.A.G.), National Institutes of Health Award #R01AR069137 (E.A.G.), Department of Defense MURI Award #W911NF-16-1-0372 (E.A.G.), Spanish Ministry of Science and Innovation/FEDER Funds Grants RTI-2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.) and the Science, Engineering and Research Board (SERB, India) Grant MTR/2019/000392 (A.N.N.). We are grateful to the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and the staff at ID23-1 beamline for assistance during data collection. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The bacterial strain 42Xb2  T was isolated from a female adult krill Nyctiphanes simplex infected with the apostome parasitoid ciliate Pseudocollinia brintoni in January 2007 in the Gulf of California. The strain has the morphological, phenotypic, and molecular characteristics of the bacteria of the family Vibrionaceae. The 16S rRNA gene sequence has a similarity of 97.7% with Enterovibrio pacificus SW014  T and 96.1% similarity with Enterovibrio norvegicus LMG 19839  T . A phylogenomic and a multilocus sequence analyses placed this strain close to the genera Enterovibrio, Grimontia, and Salinivibrio, but clearly forming a separate branch from these bacterial genera. Genomic analyses presented further support this result. A novel genus Veronia gen. nov. and a species Veronia nyctiphanis sp. nov. is here described with CAIM 600  T (= DSM 24592  T  = CECT 7578  T ) as the type strain. Morphological, physiological, and genetic evidence presented here support the unification of Enterovibrio pacificus and Veronia nyctiphanis in the new genus Veronia. Enterovibrio pacificus is reclassified as Veronia pacifica. V. pacifica is assigned as the type species of the new genus Veronia.Genome Sequencing Data The GenBank/EMBL/DDBJ accession numbers for the genome sequence of Veronia nyctiphanis CAIM 600  T is PEIB01 and of Enterovibrio pacificus CAIM 1920  T is LYBM01. The 16S rRNA gene sequence of V. nyctiphanis CAIM 600  T is JX129353., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs largely function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that the QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors., Competing Interests: The authors have declared that no competing interests exist.

Background: The genome of Vibrionaceae bacteria, which consists of two circular chromosomes, is replicated in a highly ordered fashion. In fast-growing bacteria, multifork replication results in higher gene copy numbers and increased expression of genes located close to the origin of replication of Chr 1 (ori1). This is believed to be a growth optimization strategy to satisfy the high demand of essential growth factors during fast growth. The relationship between ori1-proximate growth-related genes and gene expression during fast growth has been investigated by many researchers. However, it remains unclear which other gene categories that are present close to ori1 and if expression of all ori1-proximate genes is increased during fast growth, or if expression is selectively elevated for certain gene categories., Results: We calculated the pangenome of all complete genomes from the Vibrionaceae family and mapped the four pangene categories, core, softcore, shell and cloud, to their chromosomal positions. This revealed that core and softcore genes were found heavily biased towards ori1, while shell genes were overrepresented at the opposite part of Chr 1 (i.e., close to ter1). RNA-seq of Aliivibrio salmonicida and Vibrio natriegens showed global gene expression patterns that consistently correlated with chromosomal distance to ori1. Despite a biased gene distribution pattern, all pangene categories contributed to a skewed expression pattern at fast-growing conditions, whereas at slow-growing conditions, softcore, shell and cloud genes were responsible for elevated expression., Conclusion: The pangene categories were non-randomly organized on Chr 1, with an overrepresentation of core and softcore genes around ori1, and overrepresentation of shell and cloud genes around ter1. Furthermore, we mapped our gene distribution data on to the intracellular positioning of chromatin described for V. cholerae, and found that core/softcore and shell/cloud genes appear enriched at two spatially separated intracellular regions. Based on these observations, we hypothesize that there is a link between the genomic location of genes and their cellular placement.

Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Salinivibrio proteolyticus M318, a halophilic bacterium isolated from fermented shrimp paste, is able to produce polyhydroxyalkanoate (PHA) from different carbon sources. In this study, we report the whole-genome sequence of strain M138, which comprises 2 separated chromosomes and 2 plasmids, and the complete genome contains 3,605,935 bp with an average GC content of 49.9%. The genome of strain M318 contains 3341 genes, 98 tRNA genes, and 28 rRNA genes. The 16S rRNA gene sequence and average nucleotide identity analysis associated with morphological and biochemical tests showed that this strain has high homology to the reference strain Salinivibrio proteolyticus DSM 8285. The genes encoding key enzymes for PHA and ectoine synthesis were identified from the bacterial genome. In addition, the TeaABC transporter responsible for ectoine uptake from the environment and the operon doeABXCD responsible for the degradation of ectoine were also detected. Strain M318 was able to produce poly(3-hydroxybutyrate) [P(3HB)] from different carbon sources such as glycerol, maltose, glucose, fructose, and starch. The ability to produce ectoines at different NaCl concentrations was investigated. High ectoine content of 26.2% of cell dry weight was obtained by this strain at 18% NaCl. This report provides genetic information regarding adaptive mechanisms of strain M318 to stress conditions, as well as new knowledge to facilitate the application of this strain as a bacterial cell factory for the production of PHA and ectoine.

Nonulosonic acid (NulO) biosynthesis in bacteria is directed by nab gene clusters that can lead to neuraminic, legionaminic or pseudaminic acids. Analysis of the gene content from a set mainly composed of Aliivibrio salmonicida and Moritella viscosa strains reveals the existence of several unique nab clusters, for which the NulO products were predicted. This prediction method can be used to guide tandem mass spectrometry studies in order to verify the products of previously undescribed nab clusters and identify new members of the NulOs family.

Background: In bacteria, pan-genomes are the result of an evolutionary "tug of war" between selection and horizontal gene transfer (HGT). High rates of HGT increase the genetic pool and the effective population size (N e ), resulting in open pan-genomes. In contrast, selective pressures can lead to local adaptation by purging the variation introduced by HGT and mutation, resulting in closed pan-genomes and clonal lineages. In this study, we explored both hypotheses, elucidating the pan-genome of Vibrionaceae isolates after a perturbation event in the endangered oasis of Cuatro Ciénegas Basin (CCB), Mexico, and looking for signals of adaptation to the environments in their genomes., Results: We obtained 42 genomes of Vibrionaceae distributed in six lineages, two of them did not showed any close reference strain in databases. Five of the lineages showed closed pan-genomes and were associated to either water or sediment environment; their high N e estimates suggest that these lineages are not from a recent origin. The only clade with an open pan-genome was found in both environments and was formed by ten genetic groups with low N e , suggesting a recent origin. The recombination and mutation estimators (r/m) ranged from 0.005 to 2.725, which are similar to oceanic Vibrionaceae estimations. However, we identified 367 gene families with signals of positive selection, most of them found in the core genome; suggesting that despite recombination, natural selection moves the Vibrionaceae CCB lineages to local adaptation, purging the genomes and keeping closed pan-genome patterns. Moreover, we identify 598 SNPs associated with an unstructured environment; some of the genes associated with these SNPs were related to sodium transport., Conclusions: Different lines of evidence suggest that the sampled Vibrionaceae, are part of the rare biosphere usually living under famine conditions. Two of these lineages were reported for the first time. Most Vibrionaceae lineages of CCB are adapted to their micro-habitats rather than to the sampled environments. This pattern of adaptation is concordant with the association of closed pan-genomes and local adaptation.

Microviridins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that have been isolated from a wide variety of cyanobacterial strains. There are similar gene clusters of RiPPs distributed in the genomes of bacteria belonging to the phyla Proteobacteria and Bacteroidetes. A cryptic gene cluster for the production of microviridin-type peptide was found in the genome of the marine γ-Proteobacterium Grimontia marina. Heterologous production of new microviridin-type peptide named grimoviridin was accomplished in Escherichia coli using the biosynthetic gene cluster of G. marina. The structure of grimoviridin was determined by analysis of MS and NMR data. Grimoviridin contained one isopeptide and two ester bonds, which had exactly the same bridging pattern as other microviridin-type peptides. The absolute stereochemistries of constituent amino acids were determined to be all L-forms by modified Marfey's method. Grimoviridin showed potent inhibitory activity against trypsin with an IC 50 value of 238 nM. This is the first report of heterologous production of microviridin-type peptide using a biosynthetic gene cluster from a Proteobacterium. Key points • Heterologous production afforded new microviridin-type peptide named grimoviridin. • This is the first report of microviridin-type peptide from proteobacterial origin. • Grimoviridin showed potent inhibitory activity against trypsin.

Collagenase products are crucial to isolate primary cells in basic research and clinical therapies, where their stability in collagenolytic activity is required. However, currently standard collagenase products from Clostridium histolyticum lack such stability. Previously, we produced a recombinant 74-kDa collagenase from Grimontia hollisae, which spontaneously became truncated to ~60 kDa and possessed no stability. In this study, to generate G. hollisae collagenase useful as a collagenase product, we designed recombinant 62-kDa collagenase consisting only of the catalytic domain, which exhibits high production efficiency. We demonstrated that this recombinant collagenase is stable and active under physiological conditions. Moreover, it possesses higher specific activity against collagen and cleaves a wider variety of collagens than a standard collagenase product from C. histolyticum. Furthermore, it dissociated murine pancreata by digesting the collagens within the pancreata in a dose-dependent manner, and this dissociation facilitated isolation of pancreatic islets with masses and numbers comparable to those isolated using the standard collagenase from C. histolyticum. Implantation of these isolated islets into five diabetic mice led to normalisation of the blood glucose concentrations of all the recipients. These findings suggest that recombinant 62-kDa collagenase from G. hollisae can be used as a collagenase product to isolate primary cells.

We report here the novel species to encompass the isolate A649 T (=CBAS 716 T  = CBRVS P1061 T ) obtained from viscera of the healthy pufferfish Sphoeroides spengleri (Family Tetraodontidae). Genomic taxonomy analysis demonstrates that the novel strain A649 T had < 95% average amino acid identity/average nucleotide identity (AAI/ANI) and < 70% similarity of genome-to-genome distance (GGDH) towards its closest neighbors which places A649 T into a new Enterovibrio species (Enterovibrio baiacu sp nov.). In silico phenotyping disclosed several features that may be used to differentiate related Enterovibrio species. The nearly complete genome assembly of strain A649 T consisted of 5.4 Mbp and 4826 coding genes.

Salinivibrio proteolyticus strain YCSC6 was isolated from a saturated saltpan and demonstrated to have strong insecticidal activity against turbot's pathogenic ciliate-Uronema marinum. In this study, we sequenced its complete genome. Results showed that it consists of two circular chromosomes: 2.49 Mbps and 0.74 Mbps, respectively. It encodes 3429 protein-coding sequences. Biosynthetic gene clusters predicted to synthesize bacteriocins and antimicrobial peptides were discovered, which might be the key factors to lyse and kill U. marinum. The complete genome sequence of strain YCSC6 provides insights into the fundamental genetic potential for elucidating its insecticidal mechanism against U. marinum.

In comparison to protein coding sequences, the impact of mutation and natural selection on the sequence and function of non-coding (ncRNA) genes is not well understood. Many ncRNA genes are narrowly distributed to only a few organisms, and appear to be rapidly evolving. Compared to protein coding sequences, there are many challenges associated with assessment of ncRNAs that are not well addressed by conventional phylogenetic approaches, including: short sequence length, lack of primary sequence conservation, and the importance of secondary structure for biological function. Riboswitches are structured ncRNAs that directly interact with small molecules to regulate gene expression in bacteria. They typically consist of a ligand-binding domain (aptamer) whose folding changes drive changes in gene expression. The glycine riboswitch is among the most well-studied due to the widespread occurrence of a tandem aptamer arrangement (tandem), wherein two homologous aptamers interact with glycine and each other to regulate gene expression. However, a significant proportion of glycine riboswitches are comprised of single aptamers (singleton). Here we use graph clustering to circumvent the limitations of traditional phylogenetic analysis when studying the relationship between the tandem and singleton glycine aptamers. Graph clustering enables a broader range of pairwise comparison measures to be used to assess aptamer similarity. Using this approach, we show that one aptamer of the tandem glycine riboswitch pair is typically much more highly conserved, and that which aptamer is conserved depends on the regulated gene. Furthermore, our analysis also reveals that singleton aptamers are more similar to either the first or second tandem aptamer, again based on the regulated gene. Taken together, our findings suggest that tandem glycine riboswitches degrade into functional singletons, with the regulated gene(s) dictating which glycine-binding aptamer is conserved., Competing Interests: The authors have declared that no competing interests exist.

Oligomerization of protein into specific quaternary structures plays important biological functions, including regulation of gene expression, enzymes activity, and cell-cell interactions. Here, we report the determination of two crystal structures of the Grimontia hollisae (formally described as Vibrio hollisae) thermostable direct hemolysin (Gh-TDH), a pore-forming toxin. The toxin crystalized in the same space group of P2 1 2 1 2, but with two different crystal packing patterns, each revealing three consistent tetrameric oligomerization forms called Oligomer-I, -II, and -III. A central pore with comparable depth of ~50 Å but differing in shape and size was observed in all determined toxin tetrameric oligomers. A common motif of a toxin dimer was found in all determined structures, suggesting a plausible minimum functional unit within the tetrameric structure in cell membrane binding and possible hemolytic activity. Our results show that bacterial toxins may form a single or highly symmetric oligomerization state when exerting their biological functions. The dynamic nature of multiple symmetric oligomers formed upon release of the toxin may open a niche for bacteria survival in harsh living environments.

Vibrionaceae is a widespread family of aquatic bacteria that includes emerging pathogens and symbionts. Many Vibrionaceae harbor a type VI secretion system (T6SS), which is a secretion apparatus used to deliver toxins, termed effectors, into neighboring cells. T6SSs mediate both antibacterial and anti-eukaryotic activities. Notably, antibacterial effectors are encoded together with a gene that encodes a cognate immunity protein so as to antagonize the toxicity of the effector. The MIX (Marker for type sIX effectors) domain has been previously defined as a marker of T6SS effectors carrying polymorphic C-terminal toxins. Here, we set out to identify the Vibrionaceae MIX-effector repertoire and to analyze the various toxin domains they carry. We used a computational approach to search for the MIX-effectors in the Vibrionaceae genomes, and grouped them into clusters based on the C-terminal toxin domains. We classified MIX-effectors as either antibacterial or anti-eukaryotic, based on the presence or absence of adjacent putative immunity genes, respectively. Antibacterial MIX-effectors carrying pore-forming, phospholipase, nuclease, peptidoglycan hydrolase, and protease activities were found. Furthermore, we uncovered novel virulence MIX-effectors. These are encoded by "professional MIXologist" strains that employ a cocktail of antibacterial and anti-eukaryotic MIX-effectors. Our findings suggest that certain Vibrionaceae adapted their antibacterial T6SS to mediate interactions with eukaryotic hosts or predators.

Salinivibrio genus is commonly found in salted seafood products. In this study, chitinase produced by Salinivibrio sp. BAO-1801 isolated from salted fermented shrimp was purified and subsequently characterized. The molecular weight of BAO-1801 chitinase was approximately 94.2 kDa by SDS-PAGE analysis. It was classified as a chitinase C based on homology analysis of its N-terminal amino acid residues. This strain BAO-1801 chitinase was then used for synthesis of (GlcNAc)2. Degradation of colloidal chitin and N-acetyl chitooligosaccharides by BAO-1801 chitinase was then analyzed and (GlcNAc)2 was identified as the main product by thin layer chromatography and high-performance liquid chromatography. Effects of temperature and pH on activity and stability of BAO-1801 chitinase were also investigated. Furthermore, this enzyme inhibited fungal growth in a dose-dependent manner. Taken together, these results suggest that this Salinivibrio or its chitinase can be used for the enzymatic degradation of chitin to produce chitobiose in industrial process., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A gram-negative, rod-shaped, motile, oxidase- and catalase-positive, non-pigmented marine bacterium, designated strain OS-11M-2 T , was isolated from a coral sample collected from the Osakura coastal area in Micronesia. Phylogenetic analysis based on 16S ribosomal RNA (rRNA) gene sequences indicated that strain OS-11M-2 T is a member of the family Vibrionaceae, its closest neighbors being Photobacterium damselae subsp. piscicida NCIMB 2058 T (94.9%), Photobacterium damselae subsp. damselae CIP 102761 T (94.75%), Grimontia marina IMCC5001 T (94.5%), Enterovibrio coralii LMG 22228 T (94.5%), and Grimontia celer 96-237 T (94.5%). The major cellular fatty acids were summed feature 3 (21.4%), summed feature 8 (18.5%), iso-C 16:0 (13.8%), and C 16:0 (11.9%). The major respiratory quinone of the bacterium was ubiquinone-8 (Q-8) and its major polar lipid phosphatidylethanolamine. Six amino lipids, two phospholipids, and one polar lipid, all unidentified, were detected. The DNA G+C content was 49.7 mol%. The 16S rRNA gene sequence of OS-11M-2 T was registered in GenBank under accession number MF359550. On the basis of phenotypic, genotypic, and phylogenetic analyses, strain OS-11M-2 T represents a novel genus of the family Vibrionaceae, for which we propose the name Corallibacterium pacifica gen. nov., sp. nov., with the type strain of the type species being OS-11M-2 T (= KCCM 43265 T ). The digital protologue database (DPD) taxon number for strain OS-11M-2T is GA00041.

Bacteria belonging to the genus Pseudovibrio have been frequently found in association with a wide variety of marine eukaryotic invertebrate hosts, indicative of their versatile and symbiotic lifestyle. A recent comparison of the sponge-associated Pseudovibrio genomes has shed light on the mechanisms influencing a successful symbiotic association with sponges. In contrast, the genomic architecture of Pseudovibrio bacteria associated with other marine hosts has received less attention. Here, we performed genus-wide comparative analyses of 18 Pseudovibrio isolated from sponges, coral, tunicates, flatworm, and seawater. The analyses revealed a certain degree of commonality among the majority of sponge- and coral-associated bacteria. Isolates from other marine invertebrate host, tunicates, exhibited a genetic repertoire for cold adaptation and specific metabolic abilities including mucin degradation in the Antarctic tunicate-associated bacterium Pseudovibrio sp. Tun.PHSC04&#95;5.I4. Reductive genome evolution was simultaneously detected in the flatworm-associated bacteria and the sponge-associated bacterium P. axinellae AD2, through the loss of major secretion systems (type III/VI) and virulence/symbioses factors such as proteins involved in adhesion and attachment to the host. Our study also unraveled the presence of a CRISPR-Cas system in P. stylochi UST20140214-052 a flatworm-associated bacterium possibly suggesting the role of CRISPR-based adaptive immune system against the invading virus particles. Detection of mobile elements and genomic islands (GIs) in all bacterial members highlighted the role of horizontal gene transfer for the acquisition of novel genetic features, likely enhancing the bacterial ecological fitness. These findings are insightful to understand the role of genome diversity in Pseudovibrio as an evolutionary strategy to increase their colonizing success across a wide range of marine eukaryotic hosts., Competing Interests: The authors have declared that no competing interests exist.

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 51.9-52.5mol% (T m ) and 50.2-50.9mol% (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 )., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.

Objectives: The eps locus in Vibrio diabolicus is involved in the production of the biotechnologically valuable HE800 EPS. In this study, the distribution and diversity of similar eps gene clusters across Vibrionaceae and its variability in relation to phylogenetic relationship were investigated. The aim was to provide a better knowledge of the eps gene cluster importance and to facilitate discovery of new EPS with potent interesting bioactivities., Results: Seventy percent of the 103 genome sequences examined display such an eps locus with a high level of synteny. However, genetic divergence was found inside some monophyletic clades or even between some strains of the same species. It includes gene insertions, truncations, and deletions. Comparative analysis also reveals some variations in glycosyltransferase and export systems genes. Phylogenetic analysis of the Vibrionaceae eps gene clusters within Vibrionaceae suggests a vertical transfer by speciation but also pinpoints rearrangement events independent of the speciation.

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., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The present study has been conducted towards isolation of moderately halophilic bacteria capable of transforming caffeine into theobromine. A total of 45 caffeine-degrading moderate halophiles were enriched from hypersaline lakes and examined for the biotransformation of caffeine to theobromine by thin-layer chromatography (TLC) and high-performance liquid chromatography analyses. Strain GL6, giving the highest yield of theobromine, was isolated from the Hoz Soltan Lake, 20 % w/v salinity, central Iran, and identified as Salinivibrio costicola based on morphological and biochemical features as well as its 16S rRNA gene sequence analysis (GeneBank Accession No. KT378066) and DNA-DNA relatedness. The biotransformation of caffeine with strain GL6 leads to the formation of two metabolites, identified as theobromine and paraxanthine, but the yield of paraxanthine was much lower. Further study on the production of theobromine from caffeine under resting cell experiment was carried out subsequently. The optimal yield of theobromine (56 %) was obtained after a 32-h incubation using 5 mM of caffeine and 15 g l -1 (wet weight) of biomass in 0.1 M saline phosphate buffer (pH 7.0 and 10 % w/v NaCl) under agitation 180 rpm at 30 °C. The biotransformed theobromine was purified by preparative TLC and subjected to FTIR and mass spectroscopy for chemical identification. This is the first evidence for biotransformation of caffeine into theobromine by strains of the genus Salinivibrio.

Chitinolytic microorganisms secrete a range of chitin modifying enzymes, which can be exploited for production of chitin derived products or as fungal or pest control agents. Here, we explored the potential of 11 marine bacteria ( Pseudoalteromonadaceae , Vibrionaceae ) for chitin degradation using in silico and phenotypic assays. Of 10 chitinolytic strains, three strains, Photobacterium galatheae S2753, Pseudoalteromonas piscicida S2040 and S2724, produced large clearing zones on chitin plates. All strains were antifungal, but against different fungal targets. One strain, Pseudoalteromonas piscicida S2040, had a pronounced antifungal activity against all seven fungal strains. There was no correlation between the number of chitin modifying enzymes as found by genome mining and the chitin degrading activity as measured by size of clearing zones on chitin agar. Based on in silico and in vitro analyses, we cloned and expressed two ChiA-like chitinases from the two most potent candidates to exemplify the industrial potential., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

A novel Gram-stain-negative bacterium, named strain NSCS20N07DT, was isolated from surface seawater of the South China Sea. Cells of this strain contained poly-β-hydroxybutyrate granules. Growth was observed at 15-35 °C with optimum of 30 °C, at a salinity range of 1-6 % (w/v) NaCl with optimum of 3 % and at pH 5-8 with optimum of pH 5. The full-length 16S rRNA gene sequence of strain NSCS20N07DT showed highest similarity to Photobacterium iliopiscarium ATCC 51760T of 96.0 %, followed by Photobacterium kishitanii pjapo.1.1T, Photobacterium phosphoreum ATCC 11040T and Photobacterium aquimaris LC2-065T of 96.0, 95.8 and 95.7 %, respectively. Phylogenetic analysis showed that strain NSCS20N07DT formed a separate clade distinct from species of the genus Photobacterium and other genera within the family Vibrionaceae, indicating that strain NSCS20N07DT represented a novel genus affiliated with this family. The genome size of strain NSCS20N07DT was 2.5 Mb, which was much smaller than those of related species in the family Vibrionaceae. The predominant fatty acids were C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c, C18 : 0 and summed feature 2 (C14 : 0 3-OH/iso-C16 : 1 I). The respiratory quinone was Q-8. The polar lipids were identified as phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and four unidentified lipids. The DNA G+C content was 30.7 mol%. Combined, these results suggest that strain NSCS20N07DT represents a novel species of a new genus, for which the name Paraphotobacterium marinum gen. nov., sp. nov. is proposed. The type strain of Paraphotobacterium marinum is NSCS20N07DT (=KCTC 52126T=MCCC 1A01886T=CIP 111031T).

The luminous bacterial symbionts of anomalopid flashlight fish are thought to be obligately dependent on their hosts for growth and share several aspects of genome evolution with unrelated obligate symbionts, including genome reduction. However, in contrast to most obligate bacteria, anomalopid symbionts have an active environmental phase that may be important for symbiont transmission. Here we investigated patterns of evolution between anomalopid symbionts compared with patterns in free-living relatives and unrelated obligate symbionts to determine if trends common to obligate symbionts are also found in anomalopid symbionts. Two symbionts, "Candidatus Photodesmus katoptron" and "Candidatus Photodesmus blepharus," have genomes that are highly similar in gene content and order, suggesting genome stasis similar to ancient obligate symbionts present in insect lineages. This genome stasis exists in spite of the symbiont's inferred ability to recombine, which is frequently lacking in obligate symbionts with stable genomes. Additionally, we used genome comparisons and tests of selection to infer which genes may be particularly important for the symbiont's ecology compared with relatives. In keeping with obligate dependence, substitution patterns suggest that most symbiont genes are experiencing relaxed purifying selection compared with relatives. However, genes involved in motility and carbon storage, which are likely to be used outside the host, appear to be under increased purifying selection. Two chemoreceptor chemotaxis genes are retained by both species and show high conservation with amino acid sensing genes, suggesting that the bacteria may actively seek out hosts using chemotaxis toward amino acids, which the symbionts are not able to synthesize., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Strain 96-237T, a Gram-reaction-negative, curved- to spiral-shaped motile bacterium, isolated from coastal marine water, was found to be related to species of the genus Grimontia by 16S rRNA gene sequence comparison, sharing 98.3 % similarity to Grimontia marina CECT 8713Tand 98.8 % to 'Grimontiaindica' AK16. Phenotypic analysis revealed that strain 96-237T is slightly halophilic, mesophilic and facultatively anaerobic, fermenting d-glucose, d-ribose, d-mannose, d-mannitol, maltose and sucrose. It was positive for oxidase and indole production and negative for arginine dihydrolase and lysine and ornithine decarboxylases. Its major fatty acids were C16 : 1ω7c/C16 : 1ω6c (SF3), C18 : 1ω7c and C16 : 0. Its DNA G+C content was 48.4 mol%. The strain was different at the species level from all other species of the genusGrimontia, with average nucleotide identity indices of 79.6 % to Grimontia. hollisae CECT 8713T, 87.8 % to G. marina CECT 5069T and 89.1 % to 'G. indica' AK16 genomes. Thus, the strain represents a novel species for which we propose the name Grimontia celer and 96-237T (=CECT 9029T =KCTC 42960T =LMG 29238T) as the type strain.

A Gram-stain-negative, elliptical and facultatively anaerobic strain, designated SW014T, motile by means of a single polar flagellum and positive for poly-β-hydroxybutyrate accumulation, was isolated from surface seawater of the South Pacific Gyre, during the Integrated Ocean Drilling Program Expedition 329. The strain was able to grow at 10-37 °C (optimum 28 °C). Growth was observed at NaCl concentrations (w/v) of 1-7 % (optimum 3-4 %). The pH range for growth was 7.0-9.0 (optimum pH 8.0). Phylogenetic analysis based on 16S rRNA gene sequences and multilocus sequence analysis indicated that strain SW014T belongs to the genus Enterovibrio within the family Vibrionaceae and is related most closely to Enterovibrio coralii LMG 22228T with 96.3, 83.7, 95.0, 77.1, 84.1 and 85.8 % sequence similarity based on 16S rRNA, recA, rpoA, rpoD, pyrH and ftsZ genes, respectively. The predominant cellular fatty acids were C16 : 1ω7c and/or C16 : 1ω6c, C16 : 0, and C18 : 1ω7c and/or C18 : 1ω6c. The respiratory quinone was ubiquinone-8 (Q-8). The polar lipids of strain SW014T comprised phosphatidylethanolamine, glycolipid, two unidentified aminolipids, two unidentified phospholipids and two unidentified polar lipids. The DNA G+C content was 44.8 mol%. Combining phylogenetic analysis, phenotypic characteristics and chemotaxonomic studies, strain SW014T represents a novel species of the genus Enterovibrio, for which the name Enterovibrio pacificus sp. nov. is proposed. The type strain is SW014T ( = KCTC 42425T = MCCC 1K00500T). Emended descriptions of Enterovibrio coralii and of the genus Enterovibrio are also provided.

Microbial communities can show astonishing ecological and phylogenetic diversity. What is the role of pervasive horizontal gene transfer (HGT) in shaping this diversity in the presence of clonally expanding "killer strains"? Does HGT of antibiotic production and resistance genes erase phylogenetic structure? To answer these questions, we study a spatial eco-evolutionary model of prokaryotes, inspired by recent findings on antagonistic interactions in Vibrionaceae populations. We find toxin genes evolve to be highly mobile, whereas resistance genes minimize mobility. This differential gene mobility is a requirement to maintain a diverse and dynamic ecosystem. The resistance gene repertoire acts as a core genome that corresponds to the phylogeny of cells, whereas toxin genes do not follow this phylogeny and have a patchy distribution. We also show that interstrain HGT makes the emergent phylogenetic structure robust to selective sweeps. Finally, in this evolved ecosystem we observe antagonistic interactions between, rather than within, spatially structure subpopulations, as has been previously observed for prokaryotes in soils and oceans. In contrast to ascribing the diversification and evolution of microbial communities to clonal dynamics, we show that multilevel evolution can elegantly explain the observed phylogenetic structure and ecosystem diversity., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Sponges harbor a complex consortium of microbial communities living in symbiotic relationship benefiting each other through the integration of metabolites. The mechanisms influencing a successful microbial association with a sponge partner are yet to be fully understood. Here, we sequenced the genome of Pseudovibrio sp. POLY-S9 strain isolated from the intertidal marine sponge Polymastia penicillus sampled from the Atlantic coast of Portugal to identify the genomic features favoring the symbiotic relationship. The draft genome revealed an exceptionally large genome size of 6.6 Mbp compared with the previously reported genomes of the genus Pseudovibrio isolated from a coral and a sponge larva. Our genomic study detected the presence of several biosynthetic gene clusters-polyketide synthase, nonribosomal peptide synthetase and siderophore-affirming the potential ability of the genus Pseudovibrio to produce a wide variety of metabolic compounds. Moreover, we identified a repertoire of genes encoding adaptive symbioses factors (eukaryotic-like proteins), such as the ankyrin repeats, tetratrico peptide repeats, and Sel1 repeats that improve the attachment to the eukaryotic hosts and the avoidance of the host's immune response : The genome also harbored a large number of mobile elements (∼5%) and gene transfer agents, which explains the massive genome expansion and suggests a possible mechanism of horizontal gene transfer. In conclusion, the genome of POLY-S9 exhibited an increase in size, number of mobile DNA, multiple metabolite gene clusters, and secretion systems, likely to influence the genome diversification and the evolvability., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Unlabelled: Although plasmids and other episomes are recognized as key players in horizontal gene transfer among microbes, their diversity and dynamics among ecologically structured host populations in the wild remain poorly understood. Here, we show that natural populations of marine Vibrionaceae bacteria host large numbers of families of episomes, consisting of plasmids and a surprisingly high fraction of plasmid-like temperate phages. Episomes are unevenly distributed among host populations, and contrary to the notion that high-density communities in biofilms act as hot spots of gene transfer, we identified a strong bias for episomes to occur in free-living as opposed to particle-attached cells. Mapping of episomal families onto host phylogeny shows that, with the exception of all phage and a few plasmid families, most are of recent evolutionary origin and appear to have spread rapidly by horizontal transfer. Such high eco-evolutionary turnover is particularly surprising for plasmids that are, based on previously suggested categorization, putatively nontransmissible, indicating that this type of plasmid is indeed frequently transferred by currently unknown mechanisms. Finally, analysis of recent gene transfer among plasmids reveals a network of extensive exchange connecting nearly all episomes. Genes functioning in plasmid transfer and maintenance are frequently exchanged, suggesting that plasmids can be rapidly transformed from one category to another. The broad distribution of episomes among distantly related hosts and the observed promiscuous recombination patterns show how episomes can offer their hosts rapid assembly and dissemination of novel functions., Importance: Plasmids and other episomes are an integral part of bacterial biology in all environments, yet their study is heavily biased toward their role as vectors for antibiotic resistance genes. This study presents a comprehensive analysis of all episomes within several coexisting bacterial populations of Vibrionaceae from the coastal ocean and represents the largest-yet genomic survey of episomes from a single bacterial family. The host population framework allows analysis of the eco-evolutionary dynamics at unprecedented resolution, yielding several unexpected results. These include (i) discovery of novel, nonintegrative temperate phages, (ii) revision of a class of episomes, previously termed "nontransmissible," as highly transmissible, and (iii) surprisingly high evolutionary turnover of episomes, manifest as frequent birth, spread, and loss., (Copyright © 2015 Xue et al.)

We report on the preparation of a new type of immunotoxin by conjugation of an epidermal growth factor receptor (EGFR)-binding peptide and an R46E mutation of thermostable direct hemolysin from Grimontia hollisae, (Gh-TDH(R) (46E) /EB). The hybrid immunotoxin was purified to homogeneity and showed a single band with slight slower mobility than that of Gh-TDH(R) (46E) . Cytotoxicity assay of Gh-TDH(R) (46E) /EB on EGFR highly, moderately, low, and non-expressed cells, A431, MDA-MB-231, HeLa, and HEK293 cells, respectively, showed apparent cytotoxicity on A431 and MDA-MB-231 cells but not on HeLa or HEK293 cells. In contrast, no cytotoxicity was observed for these cells treated with either Gh-TDH(R) (46E) or EB alone, indicating enhanced cytotoxic efficacy of Gh-TDH(R) (46E) by the EGFR binding moiety. Further antitumor activity assay of Gh-TDH(R) (46E) /EB in a xenograft model of athymic nude mice showed obvious shrinkage of tumor size and degeneration, necrosis, and lesions of tumor tissues compared to the normal tissues. Therefore, the combination of Gh-TDH(R) (46E) with target affinity agents opens new possibilities for pharmacological treatment of cancers and potentiates the anticancer drug's effect., (© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Antibiotic resistance in bacteria spreads quickly, overtaking the pace at which new compounds are discovered and this emphasizes the immediate need to discover new compounds for control of infectious diseases. Terrestrial bacteria have for decades been investigated as a source of bioactive compounds leading to successful applications in pharmaceutical and biotech industries. Marine bacteria have so far not been exploited to the same extent; however, they are believed to harbor a multitude of novel bioactive chemistry. To explore this potential, genomes of 21 marine Alpha- and Gammaproteobacteria collected during the Galathea 3 expedition were sequenced and mined for natural product encoding gene clusters., Results: Independently of genome size, bacteria of all tested genera carried a large number of clusters encoding different potential bioactivities, especially within the Vibrionaceae and Pseudoalteromonadaceae families. A very high potential was identified in pigmented pseudoalteromonads with up to 20 clusters in a single strain, mostly NRPSs and NRPS-PKS hybrids. Furthermore, regulatory elements in bioactivity-related pathways including chitin metabolism, quorum sensing and iron scavenging systems were investigated both in silico and in vitro. Genes with siderophore function were identified in 50% of the strains, however, all but one harboured the ferric-uptake-regulator gene. Genes encoding the syntethase of acylated homoserine lactones were found in Roseobacter-clade bacteria, but not in the Vibrionaceae strains and only in one Pseudoalteromonas strains. The understanding and manipulation of these elements can help in the discovery and production of new compounds never identified under regular laboratory cultivation conditions. High chitinolytic potential was demonstrated and verified for Vibrio and Pseudoalteromonas species that commonly live in close association with eukaryotic organisms in the environment. Chitin regulation by the ChiS histidine-kinase seems to be a general trait of the Vibrionaceae family, however it is absent in the Pseudomonadaceae. Hence, the degree to which chitin influences secondary metabolism in marine bacteria is not known., Conclusions: Utilizing the rapidly developing sequencing technologies and software tools in combination with phenotypic in vitro assays, we demonstrated the high bioactive potential of marine bacteria in an efficient, straightforward manner - an approach that will facilitate natural product discovery in the future.

Bacterial quorum sensing (QS) and the corresponding signals, acyl homoserine lactones (AHLs), were first described for a luminescent Vibrio species. Since then, detailed knowledge has been gained on the functional level of QS; however, the abundance of AHLs in the family of Vibrionaceae in the environment has remained unclear. Three hundred and one Vibrionaceae strains were collected on a global research cruise and the prevalence and profile of AHL signals in this global collection were determined. AHLs were detected in 32 of the 301 strains using Agrobacterium tumefaciens and Chromobacterium violaceum reporter strains. Ethyl acetate extracts of the cultures were analysed by ultra-high performance liquid chromatography-high resolution mass spectrometry (MS) with automated tandem MS confirmation for AHLs. N-(3-hydroxy-hexanoyl) (OH-C6) and N-(3-hydroxy-decanoyl) (OH-C10) homoserine lactones were the most common AHLs found in 17 and 12 strains, respectively. Several strains produced a diversity of different AHLs, including N-heptanoyl (C7) HL. AHL-producing Vibrionaceae were found in polar, temperate and tropical waters. The AHL profiles correlated with strain phylogeny based on gene sequence homology, however not with geographical location. In conclusion, a wide range of AHL signals are produced by a number of clades in the Vibrionaceae family and these results will allow future investigations of inter- and intra-species interactions within this cosmopolitan family of marine bacteria.

Heat-shock proteins are molecular chaperones essential for protein folding, degradation and trafficking. The human pathogen Vibrio vulnificus encodes a copy of the groESEL operon in both chromosomes and these genes share <80 % similarity with each other. Comparative genomic analysis was used to determine whether this duplication is prevalent among Vibrionaceae specifically or Gammaproteobacteria in general. Among the Vibrionaceae complete genome sequences in the database (31 species), seven Vibrio species contained a copy of groESEL in each chromosome, including the human pathogens Vibrio cholerae, Vibrio parahaemolyticus and V. vulnificus. Phylogenetic analysis of GroEL among the Gammaproteobacteria indicated that GroESEL-1 encoded in chromosome I was the ancestral copy and GroESEL-2 in chromosome II arose by an ancient gene duplication event. Interestingly, outside of the Vibrionaceae within the Gammaproteobacteria, groESEL chromosomal duplications were rare among the 296 genomes examined; only five additional species contained two or more copies. Examination of the expression pattern of groEL from V. vulnificus cells grown under different conditions revealed differential expression between the copies. The data demonstrate that groEL-1 was more highly expressed during growth in exponential phase than groEL-2 and a similar pattern was also found in both V. cholerae and V. parahaemolyticus. Overall these data suggest that retention of both copies of groESEL in Vibrio species may confer an evolutionary advantage., (© 2014 The Authors.)

The majority of bacteria engaged in bioluminescent symbiosis are environmentally acquired and facultatively symbiotic. A few enigmatic bioluminescent symbionts have not been successfully cultured, which has led to speculation that they may be obligately dependent on their hosts. Here, we report the draft genome of the uncultured luminous symbiont of an anomalopid flashlight fish, 'Candidatus Photodesmus katoptron'. The genome of the anomalopid symbiont is reduced by 80% compared with close relatives and lacks almost all genes necessary for amino acid synthesis and for metabolism of energy sources other than glucose, supporting obligate dependence on the host for growth. 'Candidatus Photodesmus katoptron' is the first described obligate mutualistic symbiont of a vertebrate. Unlike most other obligate mutualists, the anomalopid symbiont genome has retained complete pathways for chemotaxis and motility as well as most genes involved in cell wall production, consistent with the hypothesis that these bacteria may be transmitted environmentally during an extra-host phase., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Aims: The relationship of Atlantic salmon gastrointestinal (GI) tract bacteria to environmental factors, in particular water temperature within a commercial mariculture system, was investigated., Methods and Results: Salmon GI tract bacterial communities commercially farmed in south-eastern Tasmania were analysed, over a 13-month period across a standard commercial production farm cycle, using 454 16S rRNA-based pyrosequencing. Faecal bacterial communities were highly dynamic but largely similar between randomly selected fish. In postsmolt, the faecal bacteria population was dominated by Gram-positive fermentative bacteria; however, by midsummer, members of the family Vibrionaceae predominated. As fish progressed towards harvest, a range of different bacterial genera became more prominent corresponding to a decline in Vibrionaceae. The sampled fish were fed two different commercial diet series with slightly different protein, lipid and digestible energy level; however, the effect of these differences was minimal., Conclusions: The overall data demonstrated dynamic hind gut communities in salmon that were related to season and fish growth phases but were less influenced by differences in commercial diets used routinely within the farm system studied., Significance and Impact of the Study: This study provides understanding of farmed salmon GI bacterial communities and describes the relative impact of diet, environmental and farm factors., (© 2014 The Society for Applied Microbiology.)