Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus: acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo-inositol.

Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo-inositol. Myo-inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo-inositol (iol) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo-inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo-inositol. Within the iol clusters were an MFS-type (iolT1) and an ABC-type (iolXYZ) transporter and analyses showed that both transported myo-inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae, IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna.IMPORTANCEHost associated bacteria such as Vibrio coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo-inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo-inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of myo-inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

NhaR, LeuO, and H-NS Are Part of an Expanded Regulatory Network for Ectoine Biosynthesis Expression.

Bacteria accumulate compatible solutes to maintain cellular turgor pressure when exposed to high salinity. In the marine halophile Vibrio parahaemolyticus, the compatible solute ectoine is biosynthesized de novo, which is energetically more costly than uptake; therefore, tight regulation is required. To uncover novel regulators of the ectoine biosynthesis ectABC-asp_ect operon, a DNA affinity pulldown of proteins interacting with the ectABC-asp_ect regulatory region was performed. Mass spectrometry analysis identified, among others, 3 regulators: LeuO, NhaR, and the nucleoid associated protein H-NS. In-frame non-polar deletions were made for each gene and PectA-gfp promoter reporter assays were performed in exponential and stationary phase cells. PectA-gfp expression was significantly repressed in the ΔleuO mutant and significantly induced in the ΔnhaR mutant compared to wild type, suggesting positive and negative regulation, respectively. In the Δhns mutant, PectA-gfp showed increased expression in exponential phase cells, but no change compared to wild type in stationary phase cells. To examine whether H-NS interacts with LeuO or NhaR at the ectoine regulatory region, double deletion mutants were created. In a ΔleuO/Δhns mutant, PectA-gfp showed reduced expression, but significantly more than ΔleuO, suggesting H-NS and LeuO interact to regulate ectoine expression. However, ΔnhaR/Δhns had no additional effect compared to ΔnhaR, suggesting NhaR regulation is independent of H-NS. To examine leuO regulation further, a PleuO-gfp reporter analysis was examined that showed significantly increased expression in the ΔleuO, Δhns, and ΔleuO/Δhns mutants compared to wild type, indicating both are repressors. Growth pattern analysis of the mutants in M9G 6%NaCl showed growth defects compared to wild type, indicating that these regulators play an important physiological role in salinity stress tolerance outside of regulating ectoine biosynthesis gene expression. IMPORTANCE Ectoine is a commercially used compatible solute that acts as a biomolecule stabilizer because of its additional role as a chemical chaperone. A better understanding of how the ectoine biosynthetic pathway is regulated in natural bacterial producers can be used to increase efficient industrial production. The de novo biosynthesis of ectoine is essential for bacteria to survive osmotic stress when exogenous compatible solutes are absent. This study identified LeuO as a positive regulator and NhaR as a negative regulator of ectoine biosynthesis and showed that, similar to enteric species, LeuO is an anti-silencer of H-NS. In addition, defects in growth in high salinity among all the mutants suggest that these regulators play a broader role in the osmotic stress response beyond ectoine biosynthesis regulation.

The Role of Nutrients and Nutritional Signals in the Pathogenesis of Vibrio cholerae.

Vibrio cholerae, the agent of cholera, is a natural inhabitant of aquatic environments. Over the past decades, the importance of specific nutrients and micronutrients in the environmental survival, host colonization, and pathogenesis of this species has become increasingly clear. For instance, V. cholerae has evolved ingenious mechanisms that allow the bacterium to colonize and establish a niche in the intestine of human hosts, where it competes with commensals (gut microbiota) and other pathogenic bacteria for available nutrients. Here, we discuss the carbon and energy sources utilized by V. cholerae and what is known about the role of nutrition in V. cholerae colonization. We examine how nutritional signals affect virulence gene regulation and how interactions with intestinal commensal species can affect intestinal colonization.

The organosulfur compound dimethylsulfoniopropionate (DMSP) is utilized as an osmoprotectant by Vibrio species.

Dimethylsulfoniopropionate (DMSP), a key component of the global geochemical sulfur cycle, is a secondary metabolite produced in large quantities by marine phytoplankton and utilized as an osmoprotectant, thermoprotectant and antioxidant. Marine bacteria can use two pathways to degrade and catabolize DMSP, a demethylation pathway and a cleavage pathway that produces the climate active gas dimethylsulfide (DMS). Whether marine bacteria can also accumulate DMSP as an osmoprotectant to maintain the turgor pressure of the cell in response to changes in external osmolarity has received little attention. The marine halophile Vibrio parahaemolyticus, contains at least six osmolyte transporters, four betaine carnitine choline transport (BCCT) carriers BccT1-BccT4 and two ABC-family ProU transporters. In this study, we showed that DMSP is used as an osmoprotectant by V. parahaemolyticus and several other Vibrio species including V. cholerae and V. vulnificus Using a V. parahaemolyticus proU double mutant, we demonstrated that these ABC transporters are not required for DMSP uptake. However, a bccT null mutant lacking all four BCCTs had a growth defect compared to wild type in high salinity media supplemented with DMSP. Using mutants possessing only one functional BCCT in growth pattern assays, we identified two BCCT-family transporters, BccT1 and BccT2, which are carriers of DMSP. The only V. parahaemolyticus BccT homolog that V. cholerae and V. vulnificus possess is BccT3 and functional complementation in Escherichia coli MKH13 showed V. cholerae VcBccT3 could transport DMSP. In V. vulnificus strains, we identified and characterized an additional BCCT family transporter, which we named BccT5 that was also a carrier for DMSP.Importance DMSP is present in the marine environment, produced in large quantities by marine phytoplankton as an osmoprotectant, and is an important component of the global geochemical sulfur cycle. This algal osmolyte has not been previously investigated for its role in marine heterotrophic bacterial osmotic stress response. Vibrionaceae are marine species, many of which are halophiles exemplified by V. parahaemolyticus, a species that possesses at least six transporters for the uptake of osmolytes. Here, we demonstrated that V. parahaemolyticus and other Vibrio species can accumulate DMSP as an osmoprotectant and show that several BCCT family transporters uptake DMSP. These studies suggest that DMSP is a significant bacterial osmoprotectant, which may be important for understanding the fate of DMSP in the environment. DMSP is produced and present in coral mucus and Vibrio species form part of the microbial communities associated with them. The function of DMSP in these interactions is unclear, but could be an important driver for these associations allowing Vibrio proliferation. This work suggests that DMSP likely has an important role in heterotrophic bacteria ecology than previously appreciated.

Investigations of Dimethylglycine, Glycine Betaine, and Ectoine Uptake by a Betaine-Carnitine-Choline Transporter Family Transporter with Diverse Substrate Specificity in Vibrio Species.

Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.IMPORTANCEVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

CosR Is a Global Regulator of the Osmotic Stress Response with Widespread Distribution among Bacteria.

Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2 Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria.IMPORTANCEVibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.

Structural and functional characterization of a modified legionaminic acid involved in glycosylation of a bacterial lipopolysaccharide.

Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host-pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6.

CRISPR-Cas systems are present predominantly on mobile genetic elements in Vibrio species.

BACKGROUND: Bacteria are prey for many viruses that hijack the bacterial cell in order to propagate, which can result in bacterial cell lysis and death. Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. Species within the bacterial family Vibrionaceae are marine organisms that encounter large numbers of phages. Our goal was to determine the significance of CRISPR-Cas systems as a mechanism of defense in this group by investigating their prevalence, phylogenetic distribution, and genome context. RESULTS: Herein, we describe all the CRISPR-Cas system types and their distribution within the family Vibrionaceae. In Vibrio cholerae genomes, we identified multiple variant type I-F systems, which were also present in 41 additional species. In a large number of Vibrio species, we identified a mini type I-F system comprised of tniQcas5cas7cas6f, which was always associated with Tn7-like transposons. The Tn7-like elements, in addition to the CRISPR-Cas system, also contained additional cargo genes such as restriction modification systems and type three secretion systems. A putative hybrid CRISPR-Cas system was identified containing type III-B genes followed by a type I-F cas6f and a type I-F CRISPR that was associated with a prophage in V. cholerae and V. metoecus strains. Our analysis identified CRISPR-Cas types I-C, I-E, I-F, II-B, III-A, III-B, III-D, and the rare type IV systems as well as cas loci architectural variants among 70 species. All systems described contained a CRISPR array that ranged in size from 3 to 179 spacers. The systems identified were present predominantly within mobile genetic elements (MGEs) such as genomic islands, plasmids, and transposon-like elements. Phylogenetic analysis of Cas proteins indicated that the CRISPR-Cas systems were acquired by horizontal gene transfer. CONCLUSIONS: Our data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within Vibrionaceae.

Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine.

To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus, a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae, was found to repress ect expression in V. parahaemolyticus In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR, while AphA is an indirect activator of cosR Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness.IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR, which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.

CRISPR-Cas and Contact-Dependent Secretion Systems Present on Excisable Pathogenicity Islands with Conserved Recombination Modules.

Pathogenicity islands (PAIs) are mobile integrated genetic elements that contain a diverse range of virulence factors. PAIs integrate into the host chromosome at a tRNA locus that contains their specific bacterial attachment site, attB, via integrase-mediated site-specific recombination generating attL and attR sites. We identified conserved recombination modules (integrases and att sites) previously described in choleragenic Vibrio cholerae PAIs but with novel cargo genes. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins) and a type VI secretion system (T6SS) gene cluster were identified at the Vibrio pathogenicity island 1 (VPI-1) insertion site in 19 V. cholerae strains and contained the same recombination module. Two divergent type I-F CRISPR-Cas systems were identified, which differed in Cas protein homology and content. The CRISPR repeat sequence was identical among all V. cholerae strains, but the CRISPR spacer sequences and the number of spacers varied. In silico analysis suggests that the CRISPR-Cas systems were active against phages and plasmids. A type III secretion system (T3SS) was present in 12 V. cholerae strains on a 68-kb island inserted at the same tRNA-serine insertion site as VPI-2 and contained the same recombination module. Bioinformatics analysis showed that two divergent T3SSs exist among the strains examined. Both the CRISPR and T3SS islands excised site specifically from the bacterial chromosome as complete units, and the cognate integrases were essential for this excision. These data demonstrated that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes, signifying a novel method of acquisition for both CRISPR-Cas systems and T3SSs.IMPORTANCE This work demonstrated the presence of CRISPR-Cas systems and T3SSs on PAIs. Our work showed that similar recombination modules can associate with different cargo genes and catalyze their incorporation into bacterial chromosomes, which could convert a strain into a pathogen with very different disease pathologies. Each island had the ability to excise from the chromosome as distinct, complete units for possible transfer. Evolutionary analysis of these regions indicates that they were acquired by horizontal transfer and that PAIs are the units of transfer. Similar to the case for phage evolution, PAIs have a modular structure where different functional regions are acquired by identical recombination modules.

Quorum Sensing Regulators Are Required for Metabolic Fitness in Vibrio parahaemolyticus.

Quorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO is active and regulates the two QS master regulators AphA, which is induced, and OpaR, which is repressed. At high cell density the opposite occurs: LuxO is inactive, and therefore OpaR is induced while AphA is repressed. In Vibrio parahaemolyticus, a significant enteric pathogen of humans, the roles of these regulators in pathogenesis are less known. We examined deletion mutants of luxO, opaR, and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to levels in the wild type. The opaR mutant did not show any defect in vivo Colonization was restored to wild-type levels in a luxO opaR double mutant and was also increased in an opaR aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. Transcriptome sequencing (RNA-Seq) analysis of the wild type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.

Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens.

Pseudoalteromonas piscicida is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmented P. piscicida were isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies of P. piscicida on lawns of the naturally occurring marine pathogens Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Photobacterium damselae, and Shewanella algae Inhibition also occurred on lawns of Staphylococcus aureus but not on Escherichia coli O157:H7 or Salmonella enterica serovar Typhimurium. Inhibition was not pH associated, but it may have been related to the secretion of a cysteine protease with strong activity, as detected with a synthetic fluorogenic substrate. This diffusible enzyme was secreted from all three P. piscicida strains. Direct overlay of the Pseudoalteromonas colonies with synthetic fluorogenic substrates demonstrated the activity of two aminopeptidase Bs, a trypsin-like serine protease, and enzymes reactive against substrates for cathepsin G-like and caspase 1-like proteases. In seawater cultures, scanning electron microscopy revealed numerous vesicles tethered to the outer surface of P. piscicida and a novel mechanism of direct transfer of these vesicles to V. parahaemolyticus Vesicles digested holes in V. parahaemolyticus cells, while the P. piscicida congregated around the vibrios in a predatory fashion. This transfer of vesicles and vesicle-associated digestion of holes were not observed in other bacteria, suggesting that vesicle binding may be mediated by host-specific receptors. In conclusion, we show two mechanisms by which P. piscicida inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.IMPORTANCEPseudoalteromonas species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains of P. piscicida and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by which P. piscicida kills V. parahaemolyticus It involves the direct transfer of apparently lytic vesicles from the surface of the Pseudoalteromonas strains to the surface of Vibrio cells, with subsequent digestion of holes in the Vibrio cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls. Pseudoalteromonas piscicida has potential applications in aquaculture and food safety, in control of the formation of biofilms in the environment, and in food processing. These findings may facilitate the probiotic use of P. piscicida to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.

Pathogenicity Island Cross Talk Mediated by Recombination Directionality Factors Facilitates Excision from the Chromosome.

UNLABELLED: Pathogenicity islands (PAIs) are mobile integrated genetic elements (MIGEs) that contain a diverse range of virulence factors and are essential in the evolution of pathogenic bacteria. PAIs are widespread among bacteria and integrate into the host genome, commonly at a tRNA locus, via integrase-mediated site-specific recombination. The excision of PAIs is the first step in the horizontal transfer of these elements and is not well understood. In this study, we examined the role of recombination directionality factors (RDFs) and their relationship with integrases in the excision of two PAIs essential for Vibrio cholerae host colonization: Vibrio pathogenicity island 1 (VPI-1) and VPI-2. VPI-1 does not contain an RDF, which allowed us to answer the question of whether RDFs are an absolute requirement for excision. We found that an RDF was required for efficient excision of VPI-2 but not VPI-1 and that RDFs can induce excision of both islands. Expression data revealed that the RDFs act as transcriptional repressors to both VPI-1- and VPI-2-encoded integrases. We demonstrated that the RDFs Vibrio excision factor A (VefA) and VefB bind at the attachment sites (overlapping the int promoter region) of VPI-1 and VPI-2, thus supporting this mode of integrase repression. In addition, V. cholerae RDFs are promiscuous due to their dual functions of promoting excision of both VPI-1 and VPI-2 and acting as negative transcriptional regulators of the integrases. This is the first demonstration of cross talk between PAIs mediated via RDFs which reveals the complex interactions that occur between separately acquired MIGEs. IMPORTANCE: Deciphering the mechanisms of pathogenicity island excision is necessary for understanding the evolution and spread of these elements to their nonpathogenic counterparts. Such mechanistic insight would assist in predicting the mobility of uncharacterized genetic elements. This study identified extensive RDF-mediated cross talk between two nonhomologous VPIs and demonstrated the dual functionality of RDF proteins: (i) inducing PAI excision and (ii) acting as transcriptional regulators. Findings from this study may be implicated in determining the mobilome contribution of other bacteria with multiple MIGEs.

Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.

Sialic acids are found on all vertebrate cell surfaces and are part of a larger class of molecules known as nonulosonic acids. Many bacterial pathogens synthesize related nine-carbon backbone sugars; however, the role(s) of these non-sialic acid molecules in host-pathogen interactions is poorly understood. Vibrio vulnificus is the leading cause of seafood-related death in the United States due to its ability to quickly access the host bloodstream, which it can accomplish through gastrointestinal or wound infection. However, little is known about how this organism persists systemically. Here we demonstrate that sialic acid-like molecules are present on the lipopolysaccharide of V. vulnificus, are required for full motility and biofilm formation, and also contribute to the organism's natural resistance to polymyxin B. Further experiments in a murine model of intravenous V. vulnificus infection demonstrated that expression of nonulosonic acids had a striking benefit for bacterial survival during bloodstream infection and dissemination to other tissues in vivo. In fact, levels of bacterial persistence in the blood corresponded to the overall levels of these molecules expressed by V. vulnificus isolates. Taken together, these results suggest that molecules similar to sialic acids evolved to facilitate the aquatic lifestyle of V. vulnificus but that their emergence also resulted in a gain of function with life-threatening potential in the human host.

Deciphering the role of multiple betaine-carnitine-choline transporters in the Halophile Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a halophile that is the predominant cause of bacterial seafood-related gastroenteritis worldwide. To survive in the marine environment, V. parahaemolyticus must have adaptive strategies to cope with salinity changes. Six putative compatible solute (CS) transport systems were previously predicted from the genome sequence of V. parahaemolyticus RIMD2210633. In this study, we determined the role of the four putative betaine-carnitine-choline transporter (BCCT) homologues VP1456, VP1723, VP1905, and VPA0356 in the NaCl stress response. Expression analysis of the four BCCTs subjected to NaCl upshock showed that VP1456, VP1905, and VPA0356, but not VP1723, were induced. We constructed in-frame single-deletion mutant strains for all four BCCTs, all of which behaved similarly to the wild-type strain, demonstrating a redundancy of the systems. Growth analysis of a quadruple mutant and four BCCT triple mutants demonstrated the requirement for at least one BCCT for efficient CS uptake. We complemented Escherichia coli MHK13, a CS synthesis- and transporter-negative strain, with each BCCT and examined CS uptake by growth analysis and (1)H nuclear magnetic resonance (NMR) spectroscopy analyses. These data demonstrated that VP1456 had the most diverse substrate transport ability, taking up glycine betaine (GB), proline, choline, and ectoine. VP1456 was the sole ectoine transporter. In addition, the data demonstrated that VP1723 can transport GB, proline, and choline, whereas VP1905 and VPA0356 transported only GB. Overall, the data showed that the BCCTs are functional and that there is redundancy among them.

Alternative sigma factor RpoE is important for Vibrio parahaemolyticus cell envelope stress response and intestinal colonization.

Vibrio parahaemolyticus is a halophile that inhabits brackish waters and a wide range of hosts, including crustaceans, fish, mollusks, and humans. In humans, it is the leading cause of bacterial seafood-borne gastroenteritis. The focus of this work was to determine the role of alternative sigma factors in the stress response of V. parahaemolyticus RIMD2210633, an O3:K6 pandemic isolate. Bioinformatics identified five putative extracytoplasmic function (ECF) family of alternative sigma factors: VP0055, VP2210, VP2358, VP2578, and VPA1690. ECF factors typically respond to cell wall/cell envelope stress, iron levels, and the oxidation state of the cell. We have demonstrated here that one such sigma factor, VP2578, a homologue of RpoE from Escherichia coli, is important for survival under a number of cell envelope stress conditions and in gastrointestinal colonization of a streptomycin-treated adult mouse. In this study, we determined that an rpoE deletion mutant strain BHM2578 compared to the wild type (WT) was significantly more sensitive to polymyxin B, ethanol, and high-temperature stresses. We demonstrated that in in vivo competition assays between the rpoE mutant and the WT marked with the ß-galactosidase gene lacZ (WBWlacZ), the mutant strain was defective in colonization compared to the WT. In contrast, deletion of the rpoS stress response regulator did not affect in vivo survival. In addition, we examined the role of the outer membrane protein, OmpU, which in V. cholerae is proposed to be the sole activator of RpoE. We found that an ompU deletion mutant was sensitive to bile salt stress but resistant to polymyxin B stress, indicating OmpU is not essential for the cell envelope stress responses or RpoE function. Overall, these data demonstrate that RpoE is a key cell envelope stress response regulator and, similar to E. coli, RpoE may have several factors that stimulate its function.

Loss of sigma factor RpoN increases intestinal colonization of Vibrio parahaemolyticus in an adult mouse model.

Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation in rpoN (VP2670) in V. parahaemolyticus RIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effects in vivo using a streptomycin-treated mouse model of colonization. We confirmed that deletion of rpoN rendered V. parahaemolyticus nonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. In in vivo competition assays between the rpoN mutant and a wild-type RIMD2210633 strain marked with the ß-galactosidase gene lacZ (WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that the rpoN mutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as the rpoN mutant, which suggested that lack of motility is not the sole cause of the fitness effect. In an in vitro growth competition assay in mouse intestinal mucus, the rpoN mutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ΔrpoN mutant than in the wild type. These data suggest that in V. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization.

Sequence and expression divergence of an ancient duplication of the chaperonin groESEL operon in Vibrio species.

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.

Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus : acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo -inositol.

Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo -inositol. Myo -inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo -inositol ( iol ) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo -inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo -inositol. Within the iol clusters were an MFS-type ( iolT1) and an ABC-type ( iolXYZ) transporter and analyses showed that both transported myo -inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae , IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna. IMPORTANCE: Host associated bacteria such as V. coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo -inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo -inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of myo -inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

Biosynthesis of the osmoprotectant ectoine, but not glycine betaine, is critical for survival of osmotically stressed Vibrio parahaemolyticus cells.

Vibrio parahaemolyticus is a halophile present in marine and estuarine environments, ecosystems characterized by fluctuations in salinity and temperature. One strategy to thrive in such environments is the synthesis and/or uptake of compatible solutes. The V. parahaemolyticus genome contains biosynthesis systems for both ectoine and glycine betaine, which are known to act as compatible solutes in other species. We showed that V. parahaemolyticus had a 6% NaCl tolerance when grown in M9 minimal medium with 0.4% glucose (M9G) with a >5-h lag phase. By using (1)H nuclear magnetic resonance spectroscopy ((1)H-NMR) analysis, we determined that cells synthesized ectoine and glutamate in a NaCl-dependent manner. The most effective compatible solutes as measured by a reduction in lag-phase growth in M9G with 6% NaCl (M9G 6% NaCl) were in the order glycine betaine > choline > proline = glutamate > ectoine. However, V. parahaemolyticus could use glutamate or proline as the sole carbon source, but not ectoine or glycine betaine, which suggests that these are bona fide compatible solutes. Expression analysis showed that the ectA and betA genes were more highly expressed in log-phase cells, and expression of both genes was induced by NaCl up-shock. Under all conditions examined, the ectA gene was more highly expressed than the betA gene. Analysis of in-frame deletions in betA and ectB and in a double mutant showed that the ectB mutant was defective for growth, and this defect was rescued by the addition of glycine betaine, proline, ectoine, and glutamate, indicating that these compounds are compatible solutes for this species. The presence of both synthesis systems was the predominant distribution pattern among members of the Vibrionaceae family, suggesting this is the ancestral state.