Bacteria isolated from an outbreak with moderate mortalities in farmed sole, Solea senegalensis (Kaup), in the south of Spain were identified as Vibrio harveyi and V. parahaemolyticus. Only bacterial strains showing swarming were virulent in sole and caused mortalities in experimentally inoculated fish. However, the signs of the disease were only reproduced with V. harveyi. The intramuscular inoculation of the extracellular products (ECPs) of both species produced mortalities in inoculated fish and the appearance of surface ulcers in the case of V. harveyi. However, the inoculation of sublethal doses of ECPs to fish showed a protective effect against V. harveyi.

An epizootic causing mortality among cultured summer flounder Paralichthys dentatus occurred in summer of 1998 at a land-based facility on Narragansett Bay, Rhode Island, USA. The disease, flounder infectious necrotizing enteritis (FINE), was characterized by reddening around the anal area, distended abdomens filled with opaque serosanguineous fluid, enteritis and necrosis of the posterior intestine. In extreme cases of the disease, the posterior intestine was detached from the anus and was observed coming out the vent. The intestine of individuals that recovered from the disease ended in a blind-sac; the abdomens of these fish were distended, due to food and water inside the intestinal blind-sac. A bacterium was isolated from ascites fluid and kidney of moribund flounder and identified as the causative agent in challenge experiments. The pathogen was identified as Vibrio carchariae by morphological and biochemical characteristics and sequence of the 16S rRNA. The LD50 estimate was 5 x 10(5) colony-forming units injected intraperitoneally into 100 to 200 g summer flounder.

Vibrio harveyi, which now includes Vibrio carchariae as a junior synonym, is a serious pathogen of marine fish and invertebrates, particularly penaeid shrimp. In fish, the diseases include vasculitis, gastro-enteritis and eye lesions. With shrimp, the pathogen is associated with luminous vibriosis and Bolitas negricans. Yet, the pathogenicity mechanisms are imprecisely understood, with likely mechanisms involving the ability to attach and form biofilms, quorum sensing, various extracellular products including proteases and haemolysins, lipopolysaccharide, and interaction with bacteriophage and bacteriocin-like substances.

Vibrio harveyi is an important aquatic pathogen that produces several virulence factors. In this study, the effect of quorum sensing, bacterial cell-to-cell communication, on the production of the virulence factors caseinase, gelatinase, lipase, hemolysin, and phospholipase, was investigated. The activity of virulence factors was studied through enzymatic plate assays using V. harveyi wild type and mutants with constitutively maximal or minimal quorum sensing activity. The results showed that quorum sensing negatively regulates phospholipase activity as higher activity was observed in mutants with minimal quorum sensing activity than in the mutant with maximal quorum sensing activity.Reverse transcriptase real-time PCR with specific primers revealed that the expression level of three phospholipase genes was 2-fold higher [corrected] in the mutant with minimal quorum sensing activity than in the mutant with maximal quorum sensingactivity. As far as we know, this is the first report of quorum sensing regulation of phospholipase. Finally, caseinase and gelatinase activity were positively regulated by quorum sensing, which is consistent with previous reports, and lipase and hemolysin activity were found to be independent of quorum sensing. Hence, the regulation is different for different virulence factors, with some being either positively or negatively regulated, and others being independent of quorum sensing. This might reflect the need to produce the different virulence factors at different stages during infection.Copyright © 2011 Elsevier B.V. All rights reserved.

The growing number of complete microbial genome sequences provides a powerful tool for studying the biology of microorganisms. In combination with assays for function, genomic-based approaches can facilitate efficient and directed research strategies to elucidate mechanisms of bacterial pathogenicity. As genomic information accrues, the challenge remains to construct a picture of the biology that accurately reflects how individual genes collaborate to create the complex world of microbial specialization.

Improving the accuracy of prediction of gene starts is one of a few remaining open problems in computer prediction of prokaryotic genes. Its difficulty is caused by the absence of relatively strong sequence patterns identifying true translation initiation sites. In the current paper we show that the accuracy of gene start prediction can be improved by combining models of protein-coding and non-coding regions and models of regulatory sites near gene start within an iterative Hidden Markov model based algorithm. The new gene prediction method, called GeneMarkS, utilizes a non-supervised training procedure and can be used for a newly sequenced prokaryotic genome with no prior knowledge of any protein or rRNA genes. The GeneMarkS implementation uses an improved version of the gene finding program GeneMark.hmm, heuristic Markov models of coding and non-coding regions and the Gibbs sampling multiple alignment program. GeneMarkS predicted precisely 83.2% of the translation starts of GenBank annotated Bacillus subtilis genes and 94.4% of translation starts in an experimentally validated set of Escherichia coli genes. We have also observed that GeneMarkS detects prokaryotic genes, in terms of identifying open reading frames containing real genes, with an accuracy matching the level of the best currently used gene detection methods. Accurate translation start prediction, in addition to the refinement of protein sequence N-terminal data, provides the benefit of precise positioning of the sequence region situated upstream to a gene start. Therefore, sequence motifs related to transcription and translation regulatory sites can be revealed and analyzed with higher precision. These motifs were shown to possess a significant variability, the functional and evolutionary connections of which are discussed.

The relationship between gene length and synonymous codon usage bias was investigated in Drosophila melanogaster, Escherichia coli and Saccharomyces cerevisiae. Simulation studies indicate that the correlations observed in the three organisms are unlikely to be due to sampling errors or any potential bias in the methods used to measure codon usage bias. The correlation was significantly positive in E.coli genes, whereas negative correlations were obtained for D. melanogaster and S.cerevisiae genes. When only ribosomal protein genes were used, whose expression levels are assumed to be similar, E.coli and S.cerevisiae showed significantly positive correlations. For the two eukaryotes, the distribution of effective number of codons was different in short genes (300-500 bp) compared with longer genes; this was not observed in E.coli. Both positive and negative correlations can be explained by translational selection. Energetically costly longer genes have higher codon usage bias to maximize translational efficiency. Selection may also be acting to reduce the size of highly expressed proteins, and the effect is particularly pronounced in eukaryotes. The different relationships between codon usage bias and gene length observed in prokaryotes and eukaryotes may be the consequence of these different types of selection.

Quinolones are commonly used for treatment of infections by bacteria of the Enterobacteriaceae family. However, the rising resistance to quinolones worldwide poses a major clinical and public health risk. This study aimed to characterise a novel multiple resistance plasmid carrying three plasmid-mediated quinolone resistance genes in Escherichia coli clinical stain RJ749.

An improved high-performance liquid chromatography separation was developed to detect and quantify beta-exotoxin production in Bacillus thuringiensis culture supernatants. Exotoxin production was assigned to a plasmid in five strains, from three subspecies (B. thuringiensis subsp. thuringiensis serotype 1, B. thuringiensis subsp. tolworthi serotype 9, and B. thuringiensis subsp. darmstadiensis serotype 10). A new exotoxin, called type II beta-exotoxin in this report, was discovered in B. thuringiensis subsp. morrisoni serotype 8ab, purified, and partially characterized. This material is more specific than type I beta-exotoxin and is very active against the Colorado potato beetle, Leptinotarsa decemlineata.

The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.

Bacterial conjugation, also referred to as bacterial sex, is a major horizontal gene transfer mechanism through which DNA is transferred from a donor to a recipient bacterium by direct contact. Conjugation is universally conserved among bacteria and occurs in a wide range of environments (soil, plant surfaces, water, sewage, biofilms, and host-associated bacterial communities). Within these habitats, conjugation drives the rapid evolution and adaptation of bacterial strains by mediating the propagation of various metabolic properties, including symbiotic lifestyle, virulence, biofilm formation, resistance to heavy metals, and, most importantly, resistance to antibiotics. These properties make conjugation a fundamentally important process, and it is thus the focus of extensive study. Here, we review the key steps of plasmid transfer by conjugation in Gram-negative bacteria, by following the life cycle of the F factor during its transfer from the donor to the recipient cell. We also discuss our current knowledge of the extent and impact of conjugation within an environmentally and clinically relevant bacterial habitat, bacterial biofilms.

Claudin proteins comprise a recently described family of tight junction proteins that differentially regulate paracellular permeability. Since other tight junction proteins show alterations in distribution or expression in inflammatory bowel disease (IBD) we assessed expression of claudins (CL) 2, 3 and 4 in IBD. CL 2 was strongly expressed along the inflamed crypt epithelium, whilst absent or barely detectable in normal colon. In contrast, CL 3 and 4 were present throughout normal colonic epithelium and were reduced or redistributed in the diseased surface epithelium. In a T84-cell culture model of the gut barrier, paracellular permeability decreased with time after plating and correlated with a marked decrease in the expression of CL 2. Addition of IFNgamma/TNFalpha led to further decreases in CL 2 and 3, the redistrbution of CL 4 and a marked increase in paracellular permeability. Conversely, IL-13 dramatically increased CL 2, with little effect on CL 3 or 4, but also resulted in increased paracellular permeability. Expression of CL 2 did not correlate with proliferation or junctional reorganisation after calcium ion depletion. Re-expression of CL 2 in response to IL-13 was inhibited by phophatidylinositol 3 kinase inhibitor, LY294002, which also restored the ion permeability to previous levels. CL 2 expression could be stimulated in the absence of IL-13 by activation of phospho-Akt in the phophatidylinositol 3 kinase pathway. These results suggest that INFgamma/TNFalpha and IL-13 have differential effects on CL 2, 3 and 4 in tight junctions, which may lead to increased permeability via different mechanisms.

Vibrio harveyi is a Gram-negative, halophilic bacterium that is an opportunistic pathogen of commercially farmed marine vertebrate species. To understand the pathogenicity of this species, the genome of V. harveyi QT520 was analyzed and compared to that of other strains. The results showed the genome of QT520 has two unique circular chromosomes and three endogenous plasmids, totaling 6,070,846 bp with a 45% GC content, 5,701 predicted ORFs, 134 tRNAs and 37 rRNAs. Common virulence factors, including ACF, IlpA, OmpU, Flagellin, Cya, Hemolysin and MARTX, were detected in the genome, which are likely responsible for the virulence of QT520. The results of genomes comparisons with strains ATCC 33843 (392 (MAV)) and ATCC 43516 showed that greater numbers genes associated with types I, II, III, IV and VI secretion systems were detected in QT520 than in other strains, suggesting that QT520 is a highly virulent strain. In addition, three plasmids were only observed in the complete genome sequence of strain QT520. In plasmid p1 of QT520, specific virulence factors (cyaB, hlyB and rtxA) were identified, suggesting that the pathogenicity of this strain is plasmid-associated. Phylogenetic analysis of 12 complete Vibrio sp. genomes using ANI values, core genes and MLST revealed that QT520 was most closely related to ATCC 33843 (392 (MAV)) and ATCC 43516, suggesting that QT520 belongs to the species V. harveyi. This report is the first to describe the complete genome sequence of a V. harveyi strain isolated from an outbreak in a fish species in China. In addition, to the best of our knowledge, this report is the first to compare the V. harveyi genomes of several strains. The results of this study will expand our understanding of the genome, genetic characteristics, and virulence factors of V. harveyi, setting the stage for studies of pathogenesis, diagnostics, and disease prevention.

Pili of Vibrio parahaemolyticus were purified from a Kanagawa phenomenon-positive strain (Ha7) that belongs to serogroup O2:K3 and is adhesive to rabbit intestine. The organisms treated with the Fab fraction of antipilus antibody failed to adhere to the intestine. Purified pili had the ability to adhere to the intestine, but the pretreatment of the intestine with purified pili did not allow adherence of the organisms to the intestine. These results suggest that pili of this V. parahaemolyticus strain play an important role in colonization.

We found a positive correlation between cell-associated mannose-sensitive hemagglutination and adherence of Vibrio parahaemolyticus to rabbit enterocytes by investigating 35 strains of V. parahaemolyticus for cell-associated hemagglutinin (cHA) and for the ability to adhere to the enterocytes. We purified a mannose-sensitive cHA from a Kanagawa phenomenon-positive clinical strain of V. parahaemolyticus that exhibited a high level of mannose-sensitive hemagglutination and strongly adhered to the enterocytes. The purified cHA is a heat-labile, tetrameric protein consisting of four identical subunits of approximately 26 kDa each. The adherence to rabbit enterocytes was inhibited in a dose-dependent manner by pretreatment of the bacterial cells with D-mannose and with the Fab fraction of immunoglobulin G against the purified cHA. Furthermore, pretreatment of the enterocytes with the purified cHA inhibited the adherence of V. parahaemolyticus. Immunogold electron microscopy revealed that the cHA is located on the bacterial cell surface and is not associated with pili. These results suggest that cHA is involved in the adherence mechanisms of V. parahaemolyticus to the enterocytes and that the receptors for cHA on the enterocyte appear to be a D-mannose-containing compound.

The abilities of bacterial pathogens to adapt to the environment within the host are essential to their virulence. Microorganisms have adapted to the iron limitation present in mammalian hosts by evolving diverse mechanisms for the assimilation of iron sufficient for growth. In addition, many bacterial pathogens have used the low concentration of iron present in the host as an important signal to enhance the expression of a wide variety of bacterial toxins and other virulence determinants. The molecular basis of coordinate regulation by iron has been most thoroughly studied in Escherichia coli. In this organism, coordinate regulation of gene expression by iron depends on the regulatory gene, fur. Regulation of gene expression by iron in a number of pathogenic organisms is coordinated by proteins homologous to the Fur protein of E. coli. Additional regulatory proteins may be superimposed on the Fur repressor to provide the fine-tuning necessary for the precise regulation of individual virulence genes in response to iron and other environmental signals. Studies of the mechanisms of regulation of iron acquisition systems and virulence determinants by iron should lead to a better understanding of the adaptive response of bacteria to the low-iron environment of the host and its importance in virulence.

Summary: ART is a set of simulation tools that generate synthetic next-generation sequencing reads. This functionality is essential for testing and benchmarking tools for next-generation sequencing data analysis including read alignment, de novo assembly and genetic variation discovery. ART generates simulated sequencing reads by emulating the sequencing process with built-in, technology-specific read error models and base quality value profiles parameterized empirically in large sequencing datasets. We currently support all three major commercial next-generation sequencing platforms: Roche's 454, Illumina's Solexa and Applied Biosystems' SOLiD. ART also allows the flexibility to use customized read error model parameters and quality profiles.

Vibrio harveyi is a Gram-negative bacterium found in tropical and temperate marine environments as a free-living organism or in association with aquatic animals. We report the first sequenced genome of a Vibrio harveyi strain, CAIM 1792, the etiologic agent of the "bright red" syndrome of the Pacific white shrimp Litopenaeus vannamei.

Gram-negative bacteria assemble many proteins into the inner and outer membranes and export a large number of proteins to the periplasm or to the extracellular medium. During the billions of years bacteria have been around, they have evolved a number of different pathways with sophisticated machines to accurately and efficiently move proteins from one location to another. In this review, we first introduce specific proteins that are representative substrates of the protein transport pathways and describe their function. Then, their specific routes from synthesis to their destinations are described mentioning the signal peptide that may initiate their export and discuss what is known about the folding state of the substrates during transport. The membrane translocation device involved, the energy source required for transport, and whether a chaperone is needed will be discussed.© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.

The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.

Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes cholera-like diarrhea in humans and animals. ETEC secretes a heat-labile enterotoxin (LT), which resembles cholera toxin, but the actual mechanism of LT secretion is presently unknown. We have identified a previously unrecognized type II protein secretion pathway in the prototypic human ETEC strain, H10407 (serotype O78:H11). The genes for this pathway are absent from E. coli K-12, although examination of the K-12 genome suggests that it probably once possessed them. The secretory pathway bears significant homology at the amino acid level to the type II protein secretory pathway required by Vibrio cholerae for the secretion of cholera toxin. With this in mind, we determined whether the homologous pathway of E. coli H10407 played a role in the secretion of LT. To this end, we inactivated the pathway by inserting a kanamycin-resistance gene into one of the genes (gspD) of the type II secretion pathway by homologous recombination. LT secretion by E. coli H10407 and the gspD mutant was assayed by enzyme immunoassay, and its biological activity was assessed by using Y-1 adrenal cells. This investigation showed that the protein secretory pathway is functional and necessary for the secretion of LT by ETEC. Our findings have revealed the mechanism for the secretion of LT by ETEC, which previously was unknown, and provide further evidence of close biological similarities of the LT and cholera toxin.

The secretion of enterotoxin by Vibrio cholerae is punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, we show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with [35S]-methionine and then monitoring the turnover of radiolabeled subunits as they assembled within the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of approximately 1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of approximately 13 min, we conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be approximately 20 and approximately 260 micrograms/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. Our findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations.

Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear.

\n Vibrio parahaemolyticus\n, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. Recent genome sequencing of the clinical\n V. parahaemolyticus\n strain RIMD2210633 identified two sets of genes for the type III secretion system (TTSS), TTSS1 and TTSS2. Here, we constructed a series of mutant strains from RIMD2210633 to determine whether the two putative TTSS apparatus are functional. The cytotoxic activity of mutant strains having a deletion in one of the TTSS1 genes was significantly decreased compared with that of the parent and TTSS2-related mutant strains. In an enterotoxicity assay with the rabbit ileal loop test, intestinal fluid accumulation was diminished by deletion of the TTSS2-related genes while TTSS1-related mutants caused a level of fluid accumulation similar to that of the parent. VopD, a protein encoded in the proximity of the TTSS1 region and a homologue of the\n Yersinia\n YopD, was secreted in a TTSS1-dependent manner. In contrast, VopP, which is encoded by a pathogenicity island on chromosome 2 and is homologous to the\n Yersinia\n YopP, was secreted via the TTSS2 pathway. These results provide evidence that\n V. parahaemolyticus\n TTSSs function as secretion systems and may have a role in the pathogenicity of the organism. This is the first report of functional TTSSs in\n Vibrio\n species. The presence of TTSS apparatus gene homologues was demonstrated in other vibrios, such as\n Vibrio alginolyticus\n,\n Vibrio harveyi\n, and\n Vibrio tubiashii\n, suggesting that some other vibrios also contain TTSS and that the TTSS has a role in protein secretion in those organisms during interaction with eukaryotic cells.\n

The Type III secretion system (T3SS) is a protein export pathway that is widespread in Gram-negative bacteria and delivers effector proteins directly into eukaryotic cells. At its core lie the injectisome (a sophisticated transmembrane secretion apparatus) and a complex network of specialized chaperones that target secretory proteins to the antechamber of the injectisome. The assembly of the system, and the subsequent secretion of proteins through it, undergo fine-tuned, hierarchical regulation. Here, we present the current understanding of the injectisome assembly process, secretion hierarchy, and the role of chaperones. We discuss these events in light of available structural and biochemical dissection and propose future directions essential to revealing mechanistic insight into this fascinating nanomachine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Type VI Secretion System (T6SS) is a nanomolecular apparatus that allows the delivery of effector molecules through the cell envelope of a donor bacterium to prokaryotic and/or eukaryotic cells, playing a role in the bacterial competition, virulence, and host interaction. T6SS is patchily distributed in bacterial genomes, suggesting an association with horizontal gene transfer (HGT). In fact, T6SS gene loci are eventually found within genomic islands (GIs), and there are some reports in plasmids and integrative and conjugative elements (ICEs). The impact that T6SS may have on bacteria fitness and the lack of evidence on its spread mechanism led us to question whether plasmids could represent a key mechanism in the spread of T6SS in bacteria. Therefore, we performed an in-silico analysis to reveal the association between T6SS and plasmids. T6SS was mined on 30,660 plasmids from NCBI based on the presence of at least six T6SS core proteins. T6SS was identified in 330 plasmids, all belonging to the same type (T6SS), mainly in Proteobacteria (328/330), particularly in Rhizobium and Ralstonia. Interestingly, most genomes carrying T6SS-harboring plasmids did not encode T6SS in their chromosomes, and, in general, chromosomal and plasmid T6SSs did not form separate clades.© 2022. The Author(s).

Outer membrane vesicles (OMVs) can function as nanoscale vectors that mediate bacterial interactions in microbial communities. How bacteria recognize and recruit OMVs inter-specifically remains largely unknown, thus limiting our understanding of the complex physiological and ecological roles of OMVs. Here, we report a ligand-receptor interaction-based OMV recruitment mechanism, consisting of a type VI secretion system (T6SS)-secreted lipopolysaccharide (LPS)-binding effector TeoL and the outer membrane receptors CubA and CstR. We demonstrated that Cupriavidus necator T6SS1 secretes TeoL to preferentially associate with OMVs in the extracellular milieu through interactions with LPS, one of the most abundant components of OMVs. TeoL associated with OMVs can further bind outer membrane receptors CubA and CstR, which tethers OMVs to the recipient cells and allows cargo to be delivered. The LPS-mediated mechanism enables bacterial cells to recruit OMVs derived from different species, and confers advantages to bacterial cells in iron acquisition, interbacterial competition, and horizontal gene transfer (HGT). Moreover, our findings provide multiple new perspectives on T6SS functionality in the context of bacterial competition and HGT, through the recruitment of OMVs.