Frequent nonhomologous replacement of replicative helicase loaders by viruses in Vibrionaceae.

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.

Identification of promoter activity in gene-less cassettes from Vibrionaceae superintegrons.

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.

LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA.

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.

Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen.

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)10, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.

Dynamics of efflux pumps in antimicrobial resistance, persistence, and community living of Vibrionaceae.

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.

Grimontia kaedaensis sp. nov., isolated from the estuary of Kaeda river in Miyazaki, Japan.

Strain 020920NT was isolated from the estuary of the Kaeda river in the Miyazaki prefecture in Japan. Phylogenetic analysis based on the 16S rRNA gene showed the strain's close evolutionary relationship with bacteria from the genus Grimontia, in the family Vibrionaceae. Phenotypic and chemotaxonomic features of the strain were investigated. Whole genome sequencing revealed that the strain 020920NT genome consists of two chromosomes and a plasmid, for a total of 5.52 Mbp. Calculations of whole genome average nucleotide identity and phylogenetic analysis based on the whole genome sequence showed that the strain represents a new species in the genus Grimontia, for which we propose the name Grimontia kaedaensis sp. nov. with the type strain 020920NT (=LMG 32507T=JCM 34978T).

Elevated estuary water temperature drives fish gut dysbiosis and increased loads of pathogenic vibrionaceae.

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.

The pan-genome of Splendidus clade species in the family Vibrionaceae: Insights into evolution, adaptation, and pathogenicity.

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.

Outbreak of seafood-related food poisoning from undetectable Vibrio parahaemolyticus-like pathogen, Chiang Mai Province, Thailand, December 2020.

OBJECTIVE: On 1 December 2020, the Department of Disease Control of Thailand was notified of a cluster of food poisoning cases among participants at a church festival in Mae Ai district, Chiang Mai province. We conducted an outbreak investigation to confirm diagnosis, describe the epidemiological characteristics of the outbreak, identify possible sources of the outbreak and provide appropriate control measures. METHODS: We reviewed medical records of the food poisoning cases from the health care centres. Active case finding was conducted among participants who had consumed food and water at the festival. An environmental survey was done in the village where the festival was held. A case-control study was conducted to identify the source of the outbreak. Samples for laboratory analysis included rectal swabs and fresh stool specimens from the cases and food handlers, surface swabs of cooking equipment, food, water and ice samples. RESULTS: Among 436 participants surveyed, 368 (84.4%) cases of food poisoning were identified. The most common clinical manifestation was abdominal pain (89.7%), followed by watery diarrhoea (45.7%), nausea (43.5%), vomiting (38.9%), fever (18.5%) and bloody diarrhoea (4.6%). None died in this outbreak. The case-control study showed that mixed spicy seafood salad served in the festival was significantly associated with the disease by both univariable and multivariable analyses. However, the causative agent could not be identified. The environmental investigation suggested this seafood might have been undercooked. CONCLUSION: Clinical manifestations of the cases, incubation period and the suspected seafood salad suggested seafood-related food poisoning. Grimontia hollisae, the organism causing illness similar to Vibrio parahaemolyticus and commonly undetectable in the laboratory with routine testing, might be the pathogen that caused this outbreak. G. hollisae should be in differential diagnosis and identified in seafood-associated outbreaks.

Distribution of Vibrionaceae in farmed Asian sea bass, Lates calcarifer in Thailand and their high prevalence of antimicrobial resistance.

This study describes the etiological agent of Vibriosis along with its distribution and antimicrobial resistance profiles among farmed Asian sea bass (Lates calcarifer) in Thailand. The study isolated 283 Vibrionaceae from 15 Asian sea bass farms located around the provinces of the Andaman Sea and Gulf of Thailand coasts to uncover the distribution and antimicrobial resistance profiles. Bacterial identification based on a combination of the biochemical characteristics, Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis, and the species-specific PCR demonstrated the predominant Vibrionaceae were Vibrio harveyi (n = 56), Photobacterium damselae (n = 35), and V. vulnificus (n = 31), respectively. According to a laboratory challenge experiment, among the six isolates, only V. harveyi was found to cause clinical signs of muscle necrosis and scale loss in Asian sea bass. Antibiotics resistance test results exhibited high resistance to antibiotics such as metronidazole (100%), streptomycin (97%), clindamycin (96%), colistin sulphate (70%) and amoxicillin (59%). Remarkably, 100% of Vibrionaceae isolates are susceptible to florfenicol. The 28 of 29 resistance profiles were multidrug resistances (MDR), with V. vulnificus having the highest MAR value (0.66). The findings of this study advise that a surveillance program, as well as preventive and control measures, be developed for Vibrionaceae to reduce production loss, pathogen proliferation, and antibiotic abuse, whereas AMR data indicate substantial health problems for aquatic animals and humans.

Identification of Key Functions Required for Production and Utilization of the Siderophore Piscibactin Encoded by the High-Pathogenicity Island irp-HPI in Vibrionaceae.

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.

Macroalga-associated bacterial endophyte bioactive secondary metabolites twinning: Cystoseira myrica and its associated Catenococcus thiocycli QCm as a model.

Marine ecosystems represent the largest biome on the earth. Until now, the relationships between the marine microbial inhabitants and the macroalgal species unclear, and the previous studies are insufficient. So, more research is required to advance our understanding of macroalgal- microbial interactions. In this study, we tried to investigate the relationship between the brown marine macroalga, Cystoseira myrica and its associated bacterial endophyte, Catenococcus thiocycli, as the first study concerning the production of bioactive secondary metabolites from a macroalgal species comparing with its associated endophytic bacteria. Secondary metabolites were extracted from alga and its bacterial endophyte with ethyl acetate and methanol. All extracts contained significant quantities of phenolics, flavonoids, tannins, and saponins. Strikingly, extracts possess antioxidant, anti-inflammatory and antimicrobial activities which were significantly correlated to phenolic and flavonoid contents.

Vibrio Clade 3.0: New Vibrionaceae Evolutionary Units Using Genome-Based Approach.

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.

Bacterial Communities of Ballan Wrasse (Labrus bergylta) Eggs at a Commercial Marine Hatchery.

Ballan wrasse (Labrus bergylta, Ascanius 1767) are cleaner fish cultured in northern Europe to remove sea lice from farmed Atlantic salmon (Salmo salar, Linnaeus 1758). Despite increasing appreciation for the importance of the microbiota on the phenotypes of vertebrates including teleosts, the microbiota of wrasse eggs has yet to be described. Therefore, the aim of this present study was to describe the bacterial component of the microbiota of ballan wrasse eggs shortly after spawning and at 5 days, once the eggs had undergone a routine incubation protocol that included surface disinfection steps in a common holding tank. Triplicate egg samples were collected from each of three spawning tanks and analysis of 16S rRNA gene sequences revealed that 88.6% of reads could be identified to 186 taxonomic families. At Day 0, reads corresponding to members of the Vibrionaceae, Colwelliaceae and Rubritaleaceae families were detected at greatest relative abundances. Bacterial communities of eggs varied more greatly between tanks than between samples deriving from the same tank. At Day 5, there was a consistent reduction in 16S rRNA gene sequence richness across the tanks. Even though the eggs from the different tanks were incubated in a common holding tank, the bacterial communities of the eggs from the different tanks had diverged to become increasingly dissimilar. This suggests that the disinfection and incubation exerted differential effects of the microbiota of the eggs from each tank and that the influence of the tank water on the composition of the egg microbiota was lower than expected. This first comprehensive description of the ballan wrasse egg bacterial community is an initial step to understand the role and function of the microbiota on the phenotype of this fish. In future, mass DNA sequencing methods may be applied in hatcheries to screen for pathogens and as a tool to assess the health status of eggs.

Veronia nyctiphanis gen. nov., sp. nov., Isolated from the Stomach of the Euphausiid Nyctiphanes simplex (Hansen, 1911) in the Gulf of California, and Reclassification of Enterovibrio pacificus as Veronia pacifica comb. nov.

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.

Structure of the 4-O-[1-Carboxyethyl]-d-Mannose-Containing O-Specific Polysaccharide of a Halophilic Bacterium Salinivibrio sp. EG9S8QL.

The moderately halophilic strain Salinivibrio sp. EG9S8QL was isolated among 11 halophilic strains from saline mud (Emisal Salt Company, Lake Qarun, Fayoum, Egypt). The lipopolysaccharide was extracted from dried cells of Salinivibrio sp. EG9S8QL by the phenol-water procedure. The OPS was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. The OPS was found to be composed of linear tetrasaccharide repeating units of the following structure: →2)-ß-Manp4Lac-(1→3)-α-ManpNAc-(1→3)-ß-Rhap-(1→4)-α-GlcpNAc-(1→, where Manp4Lac is 4-O-[1-carboxyethyl]mannose.

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae.

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.

Vibrionaceae core, shell and cloud genes are non-randomly distributed on Chr 1: An hypothesis that links the genomic location of genes with their intracellular placement.

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.

Population genomics of Vibrionaceae isolated from an endangered oasis reveals local adaptation after an environmental perturbation.

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 (Ne), 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 Ne 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 Ne, 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.

A structure-function analysis of interspecies antagonism by the 2-heptyl-4-alkyl-quinolone signal molecule from Pseudomonas aeruginosa.

In recent years, the alkyl-quinolone molecular framework has already provided a rich source of bioactivity for the development of novel anti-infective compounds. Based on the quorum-sensing signalling molecules 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS) from the nosocomial pathogen Pseudomonas aeruginosa, modifications have been developed with markedly enhanced anti-biofilm bioactivity towards important fungal and bacterial pathogens, including Candida albicans and Aspergillus fumigatus. Here we show that antibacterial activity of HHQ against Vibrionaceae is species-specific and it requires an exquisite level of structural fidelity within the alkyl-quinolone molecular framework. Antibacterial activity was demonstrated against the serious human pathogens Vibrio vulnificus and Vibrio cholerae as well as a panel of bioluminescent squid symbiont Allivibrio fischeri isolates. In contrast, Vibrio parahaemolyticus growth and biofilm formation was unaffected in the presence of HHQ and all the structural variants tested. In general, modification to almost all of the molecule except the alkyl-chain end, led to loss of activity. This suggests that the bacteriostatic activity of HHQ requires the concerted action of the entire framework components. The only exception to this pattern was deuteration of HHQ at the C3 position. HHQ modified with a terminal alkene at the quinolone alkyl chain retained bacteriostatic activity and was also found to activate PqsR signalling comparable to the native agonist. The data from this integrated analysis provides novel insights into the structural flexibility underpinning the signalling activity of the complex alkyl-quinolone molecular communication system.