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Considered one of the most devastating coral disease outbreaks in history, stony coral tissue loss disease (SCTLD) is currently spreading throughout Florida's coral reefs and the greater Caribbean. SCTLD affects at least two dozen different coral species and has been implicated in extensive losses of coral cover. Here we show Pseudoalteromonas sp. strain McH1-7 has broad-spectrum antibacterial activity against SCTLD-associated bacterial isolates. Chemical analyses indicated McH1-7 produces at least two potential antibacterials, korormicin and tetrabromopyrrole, while genomic analysis identified the genes potentially encoding an L-amino acid oxidase and multiple antibacterial metalloproteases (pseudoalterins). During laboratory trials, McH1-7 arrested or slowed disease progression on 68.2% of diseased Montastraea cavernosa fragments treated (n = 22), and it prevented disease transmission by 100% (n = 12). McH1-7 is the most chemically characterized coral probiotic that is an effective prophylactic and direct treatment for the destructive SCTLD as well as a potential alternative to antibiotic use., (© 2023. The Author(s).)

The bacterium Vibrio coralliilyticus has been implicated in mass mortalities of corals and shellfish larvae. However, using corals for manipulative infection experiments can be logistically difficult compared to other model organisms, so we aimed to establish oyster larvae infections as a proxy model. Therefore, this study assessed the virulence of six wild-type V. coralliilyticus strains, and mutants of one strain with deletions of known virulence factors, between Pacific oyster larvae ( Crassostrea gigas ) and Hawaiian rice coral ( Montipora capitata ) infection systems. The wild-type strains tested displayed variable virulence in each system, but virulence levels between hosts were not necessarily comparable. Strains RE98 and OCN008 maintained a medium to high level of virulence across hosts and appeared to be more generalist pathogens. Strain H1, in contrast, was avirulent towards coral but displayed a medium level of virulence towards oyster larvae. Interestingly, the BAA-450 type strain had a medium level of virulence towards coral and was the least virulent to oyster larvae. A comparison of known virulence factors determined that the flagellum, motility or chemotaxis, all of which play a significant role in coral infections, were not crucial for oyster infections with strain OCN008. A genomic comparison of the newly sequenced strain H1 with the other strains tested identified 16 genes potentially specific to coral pathogens that were absent in H1. This is both the first comparison of various V. coralliilyticus strains across infection systems and the first investigation of a strain that is non-virulent to coral. Our results indicate that the virulence of V. coralliilyticus strains in coral is not necessarily indicative of virulence in oyster larvae, and that the set of genes tested are not required for virulence in both model systems. This study increases our understanding of the virulence between V. coralliilyticus strains and helps assess their potential threat to marine environments and shellfish industries.

A deadly coral disease outbreak has been devastating the Florida Reef Tract since 2014. This disease, stony coral tissue loss disease (SCTLD), affects at least 22 coral species causing the progressive destruction of tissue. The etiological agents responsible for SCTLD are unidentified, but pathogenic bacteria are suspected. Virulence screens of 400 isolates identified four potentially pathogenic strains of Vibrio spp. subsequently identified as V. coralliilyticus . Strains of this species are known coral pathogens; however, cultures were unable to consistently elicit tissue loss, suggesting an opportunistic role. Using an improved immunoassay, the VcpA RapidTest , a toxic zinc-metalloprotease produced by V. coralliilyticus was detected on 22.3% of diseased Montastraea cavernosa ( n = 67) and 23.5% of diseased Orbicella faveolata ( n = 24). VcpA + corals had significantly higher mortality rates and faster disease progression. For VcpA - fragments, 21.6% and 33.3% of M. cavernosa and O. faveolata , respectively, died within 21 d of observation, while 100% of similarly sized VcpA + fragments of both species died during the same period. Further physiological and genomic analysis found no apparent differences between the Atlantic V. coralliilyticus strains cultured here and pathogens from the Indo-Pacific but highlighted the diversity among strains and their immense genetic potential. In all, V. coralliilyticus may be causing coinfections that exacerbate existing SCTLD lesions, which could contribute to the intraspecific differences observed between colonies. This study describes potential coinfections contributing to SCTLD virulence as well as diagnostic tools capable of tracking the pathogen involved, which are important contributions to the management and understanding of SCTLD., (Copyright © 2020 Ushijima, Meyer, Thompson, Pitts, Marusich, Tittl, Weatherup, Reu, Wetzell, Aeby, Häse and Paul.)

Background: Vibriosis has been implicated in major losses of larvae at shellfish hatcheries. However, the species of Vibrio responsible for disease in aquaculture settings and their associated virulence genes are often variable or undefined. Knowledge of the specific nature of these factors is essential to developing a better understanding of the environmental and biological conditions that lead to larvae mortality events in hatcheries. We tested the virulence of 51 Vibrio strains towards Pacific Oyster (Crassostreae gigas) larvae and sequenced draft genomes of 42 hatchery-associated vibrios to determine groups of orthologous genes associated with virulence and to determine the phylogenetic relationships among pathogens and non-pathogens of C. gigas larvae., Results: V. coralliilyticus strains were the most prevalent pathogenic isolates. A phylogenetic logistic regression model identified over 500 protein-coding genes correlated with pathogenicity. Many of these genes had straightforward links to disease mechanisms, including predicted hemolysins, proteases, and multiple Type 3 Secretion System genes, while others appear to have possible indirect roles in pathogenesis and may be more important for general survival in the host environment. Multiple metabolism and nutrient acquisition genes were also identified to correlate with pathogenicity, highlighting specific features that may enable pathogen survival within C. gigas larvae., Conclusions: These findings have important implications on the range of pathogenic Vibrio spp. found in oyster-rearing environments and the genetic determinants of virulence in these populations.

Pseudomonas aeruginosa is a Gram-negative γ-proteobacterium that forms part of the normal human microbiota and it is also an opportunistic pathogen, responsible for 30% of all nosocomial urinary tract infections. P. aeruginosa carries a highly branched respiratory chain that allows the colonization of many environments, such as the urinary tract, catheters and other medical devices. P. aeruginosa respiratory chain contains three different NADH dehydrogenases (complex I, NQR and NDH-2), whose physiologic roles have not been elucidated, and up to five terminal oxidases: three cytochrome c oxidases (COx), a cytochrome bo3 oxidase (CYO) and a cyanide-insensitive cytochrome bd-like oxidase (CIO). In this work, we studied the composition of the respiratory chain of P. aeruginosa cells cultured in Luria Broth (LB) and modified artificial urine media (mAUM), to understand the metabolic adaptations of this microorganism to the growth in urine. Our results show that the COx oxidases play major roles in mAUM, while P. aeruginosa relies on CYO when growing in LB medium. Moreover, our data demonstrate that the proton-pumping NQR complex is the main NADH dehydrogenase in both LB and mAUM. This enzyme is resistant to HQNO, an inhibitory molecule produced by P. aeruginosa, and may provide an advantage against the natural antibacterial agents produced by this organism. This work offers a clear picture of the composition of this pathogen's aerobic respiratory chain and the main roles that NQR and terminal oxidases play in urine, which is essential to understand its physiology and could be used to develop new antibiotics against this notorious multidrug-resistant microorganism., Competing Interests: The authors have declared that no competing interests exist.

Background: Pseudomonas aeruginosa is a bacterial pathogen that can cause grave and sometimes chronic infections in patients with weakened immune systems and cystic fibrosis. It is expected that sodium/proton transporters in the cellular membrane are crucial for the organism's survival and growth under certain conditions, since many cellular processes rely on the maintenance of Na + and H + transmembrane gradients., Results: This study focused on the role of the primary and secondary proton and/or sodium pumps Mrp, Nuo, NhaB, NhaP, and NQR for growth, biofilm formation, and swarming motility in P. aeruginosa. Using mutants with gene deletions, we investigated the impact of each sodium pump's absence on the overall growth, biofilm formation, motility, and weak acid tolerance of the organism. We found that the absence of some, but not all, of the sodium pumps have a deleterious effect on the different phenotypes of P. aeruginosa., Conclusion: The absence of the Mrp sodium/proton antiporter was clearly important in the organism's ability to survive and function in environments of higher pH and sodium concentrations, while the absence of Complex I, which is encoded by the nuo genes, had some consistent impact on the organism's growth regardless of the pH and sodium concentration of the environment.

The type VI secretion system (T6SS) is a nanomachine capable of killing adjacent microbial cells in a contact-dependent manner. Due to limited studies, relatively little is known about the range of marine bacteria that are susceptible to T6SS attack. Here, 15 diverse marine bacterial isolates from the phyla Bacteroidetes and Ɣ-Proteobacteria were challenged against the marine bacterium and human pathogen, Vibrio cholerae, which has a well described T6SS. V. cholerae killed several of the tested Ɣ-Proteobacteria, including members of the orders Vibrionales, Alteromonadales, Oceanospirillales, and Pseudomonadales. In contrast, V. cholerae co-existed with multiple Bacteroidetes and Ɣ-Proteobacteria isolates, but was killed by Vibrio coralliilyticus. Follow-up experiments revealed that five V. coralliilyticus strains, including known coral and shellfish pathogens survived the T6SS challenge and killed V. cholerae. By using predicted protein comparisons and mutagenesis, we conclude that V. coralliilyticus protected itself in the challenge by using its own T6SS to kill V. cholerae. This study provides valuable insight into the resilience and susceptibility of marine bacteria to the V. cholerae T6SS, and provides the first evidence for a functional T6SS in V. coralliilyticus, both of which have implications for human and ocean health., Competing Interests: The authors have declared that no competing interests exist.

The Na + ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF , a flavin attachment gene, apbE , and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δ nqr and Δ ubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe 2+ (Feo), was increased in V. cholerae Δnqr In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. cholerae Δnqr with Fe 2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. cholerae Δnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation. IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics., (Copyright © 2020 American Society for Microbiology.)

As many as 22 of the 45 coral species on the Florida Reef Tract are currently affected by stony coral tissue loss disease (SCTLD). The ongoing disease outbreak was first observed in 2014 in Southeast Florida near Miami and as of early 2019 has been documented from the northernmost reaches of the reef tract in Martin County down to Key West. We examined the microbiota associated with disease lesions and apparently healthy tissue on diseased colonies of Montastraea cavernosa , Orbicella faveolata , Diploria labyrinthiformis , and Dichocoenia stokesii . Analysis of differentially abundant taxa between disease lesions and apparently healthy tissue identified five unique amplicon sequence variants enriched in the diseased tissue in three of the coral species (all except O. faveolata) , namely an unclassified genus of Flavobacteriales and sequences identified as Fusibacter (Clostridiales) , Planktotalea (Rhodobacterales) , Algicola (Alteromonadales) , and Vibrio (Vibrionales) . In addition, several groups of likely opportunistic or saprophytic colonizers such as Epsilonbacteraeota, Patescibacteria, Clostridiales, Bacteroidetes, and Rhodobacterales were also enriched in SCTLD disease lesions. This work represents the first microbiological characterization of SCTLD, as an initial step toward identifying the potential pathogen(s) responsible for SCTLD., (Copyright © 2019 Meyer, Castellanos-Gell, Aeby, Häse, Ushijima and Paul.)

The Vc-NhaP2 antiporter from Vibrio cholerae exchanges H + for K + or Na + but not for the smaller Li + . The molecular basis of this unusual selectivity remains unknown. Phyre 2 and Rosetta software were used to generate a structural model of the Vc-NhaP2. The obtained model suggested that a cluster of residues from different transmembrane segments (TMSs) forms a putative cation-binding pocket in the middle of the membrane: D133 and T132 from TMS V together with D162 and E157 of TMS VI. The model also suggested that L257, G258, and N259 from TMS IX together with T276, D273, Q280, and Y251 from TMS X as well as L289 and L342 from TMS XII form a transmembrane pathway for translocated ions with a built-in filter determining cation selectivity. Alanine-scanning mutagenesis of the identified residues verified the model by showing that structural modifications of the pathway resulted in altered cation selectivity and transport activity. In particular, L257A, G258A, Q280A, and Y251A variants gained Li + /H + antiport capacity that was absent in the nonmutated antiporter. T276A, D273A, and L289A variants exclusively exchanged K + for H + , while a L342A variant mediated Na + /H + exchange only, thus maintaining strict alkali cation selectivity.

Chemotaxis, the directed movement toward or away from a chemical signal, can be essential to bacterial pathogens for locating hosts or avoiding hostile environments. The coral pathogen Vibrio coralliilyticus chemotaxes toward coral mucus; however, chemotaxis has not been experimentally demonstrated to be important for virulence. To further examine this, in-frame mutations were constructed in genes predicted to be important for V. coralliilyticus chemotaxis. Most Vibrio genomes contain multiple homologs of various chemotaxis-related genes, and two paralogs of each for cheB , cheR , and cheA were identified. Based on single mutant analyses, the paralogs cheB 2 , cheR 2 , and cheA 1 were essential for chemotaxis in laboratory assays. As predicted, the Δ cheA 1 and Δ cheR 2 strains had a smooth-swimming pattern, while the Δ cheB 2 strain displayed a zigzag pattern when observed under light microscopy. However, these mutants, unlike the parent strain, were unable to chemotax toward the known attractants coral mucus, dimethylsulfoniopropionate, and N -acetyl-d-glucosamine. The Δ cheB 2 strain and an aflagellate Δ fliG 1 strain were avirulent to coral, while the Δ cheA 1 and Δ cheR 2 strains were hypervirulent (90 to 100% infection within 14 h on average) compared to the wild-type strain (66% infection within 36 h on average). Additionally, the Δ cheA 1 and Δ cheR 2 strains appeared to better colonize coral fragments than the wild-type strain. These results suggest that although chemotaxis may be involved with infection (the Δ cheB 2 strain was avirulent), a smooth-swimming phenotype is important for bacterial colonization and infection. This study provides valuable insight into understanding V. coralliilyticus pathogenesis and how this pathogen may be transmitted between hosts. IMPORTANCE Corals are responsible for creating the immense structures that are essential to reef ecosystems; unfortunately, pathogens like the bacterium Vibrio coralliilyticus can cause fatal infections of reef-building coral species. However, compared to related human pathogens, the mechanisms by which V. coralliilyticus initiates infections and locates new coral hosts are poorly understood. This study investigated the effects of chemotaxis, the directional swimming in response to chemical signals, and bacterial swimming patterns on infection of the coral Montipora capitata Infection experiments with different mutant strains suggested that a smooth-swimming pattern resulted in hypervirulence. These results demonstrate that the role of chemotaxis in coral infection may not be as straightforward as previously hypothesized and provide valuable insight into V. coralliilyticus pathogenesis., (Copyright © 2018 American Society for Microbiology.)

The bacterium Vibrio coralliilyticus can threaten vital reef ecosystems by causing disease in a variety of coral genera, and, for some strains, increases in virulence at elevated water temperatures. In addition, strains of V. coralliilyticus (formally identified as V. tubiashii) have been implicated in mass mortalities of shellfish larvae causing significant economic losses to the shellfish industry. Recently, strain BAA-450, a coral pathogen, was demonstrated to be virulent towards larval Pacific oysters (Crassostrea gigas). However, it is unclear whether other coral-associated V. coralliilyticus strains can cause shellfish mortalities and if infections are influenced by temperature. This study compared dose dependence, temperature impact, and gross pathology of four V. coralliilyticus strains (BAA-450, OCN008, OCN014 and RE98) on larval C. gigas raised at 23°C and 27°C, and evaluated whether select virulence factors are required for shellfish infections as they are for corals. All strains were infectious to larval oysters in a dose-dependent manner with OCN014 being the most pathogenic and BAA-450 being the least. At 27°C, higher larval mortalities (p < 0.05) were observed for all V. coralliilyticus strains, ranging from 38.8-93.7%. Gross pathological changes to the velum and cilia occurred in diseased larvae, but there were no distinguishable differences between oysters exposed to different V. coralliilyticus strains or temperatures. Additionally, in OCN008, the predicted transcriptional regulator ToxR and the outer membrane protein OmpU were important for coral and oyster disease, while mannose sensitive hemagglutinin type IV pili were required only for coral infection. This study demonstrated that multiple coral pathogens can infect oyster larvae in a temperature-dependent manner and identified virulence factors required for infection of both hosts., Competing Interests: The authors have declared that no competing interests exist.

Vibrio spp. have been a persistent concern for coastal bivalve hatcheries, which are vulnerable to environmental pathogens in the seawater used for rearing larvae, yet the biogeochemical drivers of oyster-pathogenic Vibrio spp. in their planktonic state are poorly understood. Here, we present data tracking oyster-pathogenic Vibrio bacteria in Netarts Bay and Yaquina Bay in Oregon, USA, as well as in adjacent coastal waters and a local shellfish hatchery, through the 2015 upwelling season. Vibrio populations were quantified using a culture-independent approach of high-throughput Vibrio- specific 16S rRNA gene sequencing paired with droplet digital PCR, and abundances were analyzed in the context of local biogeochemistry. The most abundant putative pathogen in our samples was Vibrio coralliilyticus Environmental concentrations of total Vibrio spp. and V. coralliilyticus were highest in Netarts Bay sediment samples and higher in seawater from Netarts Bay than from nearshore coastal waters or Yaquina Bay. In Netarts Bay, the highest V. coralliilyticus concentrations were observed during low tide, and abundances increased throughout the summer. We hypothesize that the warm shallow waters in estuarine mudflats facilitate the local growth of the V. coralliilyticus pathogen. Samples from larval oyster tanks in Whiskey Creek Shellfish Hatchery, which uses seawater pumped directly from Netarts Bay, contained significantly lower total Vibrio species concentrations, but roughly similar V. coralliilyticus concentrations, than did the bay water, resulting in a 30-fold increase in the relative abundance of the V. coralliilyticus pathogen in hatchery tanks. This suggests that the V. coralliilyticus pathogen is able to grow or persist under hatchery conditions. IMPORTANCE It has been argued that oyster-pathogenic Vibrio spp. have contributed to recent mortality events in U.S. shellfish hatcheries (R. A. Elston, H. Hasegawa, K. L. Humphrey, I. K. Polyak, and C. Häse, Dis Aquat Organ 82:119-134, 2008, https://doi.org/10.3354/dao01982); however, these events are often sporadic and unpredictable. The success of hatcheries is critically linked to the chemical and biological composition of inflowing seawater resources; thus, it is pertinent to understand the biogeochemical drivers of oyster-pathogenic Vibrio spp. in their planktonic state. Here, we show that Netarts Bay, the location of a local hatchery, is enriched in oyster-pathogenic V. coralliilyticus compared to coastal seawater, and we hypothesize that conditions in tidal flats promote the local growth of this pathogen. Furthermore, V. coralliilyticus appears to persist in seawater pumped into the local hatchery. These results improve our understanding of the ecology and environmental controls of the V. coralliilyticus pathogen and could be used to improve future aquaculture efforts, as multiple stressors impact hatchery success., (Copyright © 2018 Gradoville et al.)

Metabolomics is an "omics" approach to quantitatively measure a large set of metabolites. In this chapter, we describe an example method for performing liquid chromatography coupled to mass spectrometry (LC-MS)-based untargeted metabolomics on a cell extract from Vibrio cholerae.

The human pathogen Vibrio parahaemolyticus is a leading cause of seafood-borne illness in the USA, and infections with V. parahaemolyticus typically result from eating raw or undercooked oysters. V. parahaemolyticus has been shown to be highly resistant to oyster depuration, suggesting that the bacterium possesses specific mechanisms or factors for colonizing oysters and persisting during depuration. In this study, we characterized eight different V. parahaemolyticus strains for differences in resistance to oyster depuration, biofilm formation, and motility. While each strain exhibited distinct phenotypes in the various assays, we determined that biofilm formation on abiotic surfaces, such as glass or plastic, does not directly correlate with bacterial retention in oysters during depuration. However, we did observe that the motility phenotype of a strain appeared to be a better indicator for persistence in the oyster. Further studies examining the molecular mechanisms underlying the observed colonization differences by these and other V. parahaemolyticus strains may provide beneficial insights into what critical factors are required for proficient colonization of the Pacific oyster.

LacZ (β-galactosidase) is used to monitor the transcription of genes in reporter strains carrying the lacZ gene under the control of a promotor of interest. This protocol for LacZ activity determinations in Vibrio cholerae following detergent lysis results in 2.5-fold increase of LacZ activities compared to lysis with chloroform., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The genome of Vibrio cholerae encodes three cation-proton antiporters of NhaP-type, Vc-NhaP1, 2, and 3. To examine physiological roles of Vc-NhaP antiporters, triple ΔnhaP1ΔnhaP2ΔnhaP3 and single ΔnhaP3 deletion mutants of V. cholerae were constructed and characterized. Vc-NhaP3 was, for the first time, cloned and biochemically characterized. Activity measurements on the inside-out membrane vesicle experimental model defined Vc-NhaP3 as a potassium-specific cation-proton antiporter. While elimination of functional Vc-NhaP3 resulted in only minor growth defect in potassium-rich medium at pH 6.0, the triple Vc-NhaP mutant demonstrated severe growth defects at both low and high [K + ] at pH 6.0 and failed to grow at high [K + ] in mildly alkaline (pH 8.0 and 8.5) media, as well. Expressed from a plasmid, neither of the Vc-NhaP paralogues was able to complement the severe potassium-sensitive phenotype of the triple deletion mutant completely. Vc-NhaP1 provided much better complementation at acidic pH compared to Vc-NhaP2, despite the fact that Vc-NhaP2 showed much higher antiport activity in sub-bacterial vesicles. In mildly alkaline pH only Vc-NhaP2 complemented the potassium-sensitive phenotype of the triple deletion mutant. Taken together, these data suggest that in vivo all three isoforms operate in concert, contributing to K + resistance of V. cholerae. We suggest that the Vc-NhaP paralogue group might play a role in passing gastric acid barrier by ingested V. cholerae cells.

To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mrp mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δmrp mutant that was complemented by using the mrpA'-F operon on an arabinose-inducible expression vector. Deletion of the mrp operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δmrp mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δmrp strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δmrp mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of V. cholerae despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.

Unlabelled: We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na(+)-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 μmol superoxide min(-1) mg(-1) membrane protein) compared to membranes from the mutant lacking Na(+)-NQR (0.18 ± 0.01 μmol min(-1) mg(-1)). Overexpression of plasmid-encoded Na(+)-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 μmol min(-1) mg(-1)). By analyzing a variant of Na(+)-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae The impact of superoxide formation by the Na(+)-NQR on the virulence of V. cholerae is discussed., Importance: In several studies, it was demonstrated that the Na(+)-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na(+)-NQR as the site of superoxide formation in the cytoplasm of V. cholerae Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on the Na(+)-NQR as the sole electrogenic NADH dehydrogenase., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Vibrio tubiashii is reported to be a bacterial pathogen of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) and has been associated with major hatchery crashes, causing shortages in seed oysters for commercial shellfish producers. Another bacterium, Vibrio coralliilyticus, a well-known coral pathogen, has recently been shown to elicit mortality in fish and shellfish. Several strains of V. coralliilyticus, such as ATCC 19105 and Pacific isolates RE22 and RE98, were misidentified as V. tubiashii until recently. We compared the mortalities caused by two V. tubiashii and four V. coralliilyticus strains in Eastern and Pacific oyster larvae. The 50% lethal dose (LD50) of V. coralliilyticus in Eastern oysters (defined here as the dose required to kill 50% of the population in 6 days) ranged from 1.1 × 10(4) to 3.0 × 10(4) CFU/ml seawater; strains RE98 and RE22 were the most virulent. This study shows that V. coralliilyticus causes mortality in Eastern oyster larvae. Results for Pacific oysters were similar, with LD50s between 1.2 × 10(4) and 4.0 × 10(4) CFU/ml. Vibrio tubiashii ATCC 19106 and ATCC 19109 were highly infectious toward Eastern oyster larvae but were essentially nonpathogenic toward healthy Pacific oyster larvae at dosages of ≥1.1 × 10(4) CFU/ml. These data, coupled with the fact that several isolates originally thought to be V. tubiashii are actually V. coralliilyticus, suggest that V. coralliilyticus has been a more significant pathogen for larval bivalve shellfish than V. tubiashii, particularly on the U.S. West Coast, contributing to substantial hatchery-associated morbidity and mortality in recent years., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology.

During the warmer summer months, oysters are conditioned to spawn, resulting in massive physiological efforts for gamete production. Moreover, the higher temperatures during the summer typically result in increased bacteria populations in oysters. We hypothesized that these animals are under multiple stresses that lead to possible immune system impairments during the summer months that can possibly lead to death. Here we show that in the summer and the fall animals exposed to a short heat stress respond similarly, resulting in a general trend of more bacteria being found in heat shocked animals than their non-heat shocked counterparts. We also show that naturally occurring bacterial populations are effected by a heat shock. In addition, oysters artificially contaminated with Vibrio parahaemolyticus were also affected by a heat shock. Heat shocked animals contained higher concentrations of V. parahaemolyticus in their tissues and hemolymph than control animals and this was consistent for animals examined during summer and fall. Finally, oyster hemocyte interactions with V. parahaemolyticus differed based on the time of the year. Overall, these findings demonstrate that seasonal changes and/or a short heat shock is sufficient to impact bacterial retention, particularly V. parahaemolyticus, in oysters and this line of research might lead to important considerations for animal harvesting procedures., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Vibrio tubiashii has been linked to disease outbreaks in molluscan species, including oysters, geoducks, and clams, and shellfish hatcheries in the Pacific Northwest have been plagued by intermittent vibriosis outbreaks since 2006. Like V. tubiashii, Vibrio coralliilyticus has recently been described as an oyster pathogen in addition to its role in coral disease. Here, we describe an autolysis phenotype in V. tubiashii and its close relative V. coralliilyticus and characterize the effects of environmental conditions on this phenotype. We also explored whether the survivors of autolysis were resistant to the phenotype and if material from the autolysed culture would either regrow or have a population of viable cells. Ultimately, this work contributes to the larger understanding of bacterial population dynamics as it relates to aquaculture pathogens.

Two virulence factors produced by Vibrio cholerae, cholera toxin (CT) and toxin-corregulated pilus (TCP), are indispensable for cholera infection. ToxT is the central regulatory protein involved in activation of CT and TCP expression. We previously reported that lack of a respiration-linked sodium-translocating NADH-ubiquinone oxidoreductase (Na(+)-NQR) significantly increases toxT transcription. In this study, we further characterized this link and found that Na(+)-NQR affects toxT expression only at the early-log growth phase, whereas lack of Na(+)-NQR decreases CT production after the mid-log growth phase. Such decreased CT production was independent of toxT and ctxB transcription. Supplementing a respiratory substrate, l-lactate, into the growth media restored CT production in the nqrA-F mutant, suggesting that decreased CT production in the Na(+)-NQR mutant is dependent on electron transport chain (ETC) activity. This notion was supported by the observations that two chemical inhibitors, a Na(+)-NQR specific inhibitor 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO) and a succinate dehydrogenase (SDH) inhibitor, thenoyltrifluoroacetone (TTFA), strongly inhibited CT production in both classical and El Tor biotype strains of V. cholerae. Accordingly, we propose the main respiratory enzyme of V. cholerae, as a potential drug target to treat cholera because human mitochondria do not contain Na(+)-NQR orthologs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Na(+)/H(+) antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na(+)/H(+) antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na(+)/H(+) antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na(+) levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na(+)/H(+) antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.

Vibrio tubiashii has been linked to disease outbreaks in molluscan species, including oysters, geoducks, and clams. In particular, oyster hatcheries in the Pacific Northwest have been plagued by intermittent vibriosis since 2006. Accurate detection of vibrios, including V. tubiashii, is critical to the hatcheries in order to allow for rapid remediation efforts. The current methods for detection of Vibrio spp. are not ideal for use at the hatchery. Plating samples require time and is not sensitive to extracelluar pathogenic products, such as the secreted zinc-metalloprotease, VtpA. Other sensitive methods to detect bacteria, such as qPCR, require a high level of laboratory skills and expensive supplies that are prohibitive for use at hatchery sites. Thus, hatcheries would benefit from a sensitive, simple method to detect V. tubiashii and its secreted toxin. Here, we describe the development of two inexpensive and highly specific tests for the shellfish-toxic zinc-metalloprotease secreted by V. tubiashii: enzyme-linked immunoassays (ELISA) and a lateral flow immunoassay (dipstick assay). Both technologies rely on a set of monoclonal antibodies used in a sandwich format, with the capture antibody recognizing a different epitope than the detection antibody on the mature VtpA protein. Both assays are quantitative and give colorimetric readouts. The sandwich ELISA was sensitive when VtpA was diluted into PBS, but was markedly less sensitive in conditions that correlate with the environment of hatchery-derived samples, such as in the presence of seawater, algae, or oyster larvae. In contrast, the dipstick assay remained very sensitive in the presence of these contaminants, is less work-intensive, and much more rapid, making this format the preferred assay method for detecting VtpA on site in a hatchery or environmental setting., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We previously found that inhibition of the TCA cycle, either through mutations or chemical inhibition, increased toxT transcription in Vibrio cholerae. In this study, we found that the addition of malonate, an inhibitor of succinate dehydrogenase (SDH), decreased toxT transcription in V. cholerae, an observation inconsistent with the previous pattern observed. Unlike another SDH inhibitor, 2-thenoyltrifluoroacetone (TTFA), which increased toxT transcription and slightly inhibited V. cholerae growth, malonate inhibited toxT transcription in both the wild-type strain and TCA cycle mutants, suggesting malonate-mediated inhibition of virulence gene expression is independent to TCA cycle activity. Addition of malonate also inhibited ctxB and tcpA expressions but did not affect aphA, aphB, tcpP and toxR expressions. Malonate inhibited cholera toxin (CT) production in both V. cholerae classical biotype strains O395N1 and CA401, and El Tor biotype strain, N16961. Consistent with previous reports, we confirmed that these strains of V. cholerae did not utilize malonate as a primary carbon source. However, we found that the addition of malonate to the growth medium stimulated V. cholerae growth. All together, these results suggest that metabolizing malonate as a nutrient source negatively affects virulence gene expression in V. cholerae.

Vibrio parahaemolyticus can resist oyster depuration, suggesting that it possesses specific factors for persistence. We show that type I pili, type IV pili, and both flagellar systems contribute to V. parahaemolyticus persistence in Pacific oysters whereas type III secretion systems and phase variation do not.

ToxT is the central regulatory protein involved in activation of the main virulence genes in Vibrio cholerae. We have identified transposon insertions in central metabolism genes, whose disruption increases toxT transcription. These disrupted genes encode the primary respiration-linked sodium pump (NADH:ubiquinone oxidoreductase or NQR) and certain tricarboxylic acid (TCA) cycle enzymes. Observations made following stimulation of respiration in the nqr mutant or chemical inhibition of NQR activity in the TCA cycle mutants led to the hypothesis that NQR affects toxT transcription via the TCA cycle. That toxT transcription increased when the growth medium was supplemented with citrate, but decreased with oxaloacetate, focused our attention on the TCA cycle substrate acetyl-CoA and its non-TCA cycle metabolism. Indeed, both the nqr and the TCA cycle mutants increased acetate excretion. A similar correlation between acetate excretion and toxT transcription was observed in a tolC mutant and upon amino acid (NRES) supplementation. As acetate and its tendency to decrease pH exerted no strong effect on toxT transcription, and because disruption of the major acetate excretion pathway increased toxT transcription, we propose that toxT transcription is regulated by either acetyl-CoA or some close derivative.

Bacterial surface structures called pili have been studied extensively for their role as possible colonization factors. Most sequenced Vibrio genomes predict a variety of pili genes in these organisms, including several types of type IV pili. In particular, the mannose-sensitive hemagglutinin (MSHA) and the PilA pili, also known as the chitin-regulated pilus (ChiRP), are type IVa pili commonly found in Vibrio genomes and have been shown to play a role in the colonization of Vibrio species in the environment and/or host tissue. Here, we report sequence comparisons of two type IVa pilin subunit genes, mshA and pilA, and their corresponding amino acid sequences, for several strains from the three main human pathogenic Vibrio species, V. cholerae, V. parahaemolyticus, and V. vulnificus. We identified specific groupings of these two genes in V. cholerae, whereas V. parahaemolyticus and V. vulnificus strains had no apparent allelic clusters, and these genes were strikingly divergent. These results were compared with other genes from the MSHA and PilA operons as well as another Vibrio pili from the type IVb group, the toxin co-regulated pilus (TCP) from V. cholerae. Our data suggest that a selective pressure exists to cause these strains to vary their MSHA and PilA pilin subunits. Interestingly, V. cholerae strains possessing TCP have the same allele for both mshA and pilA. In contrast, V. cholerae isolates without TCP have polymorphisms in their mshA and pilA sequences similar to what was observed for both V. parahaemolyticus and V. vulnificus. This data suggests a possible linkage between host interactions and maintaining a highly conserved type IV pili sequence in V. cholerae. Although the mechanism underlying this intriguing diversity has yet to be elucidated, our analyses are an important first step towards gaining insights into the various aspects of Vibrio ecology.

Vibrio cholerae has adapted to a wide range of salinity, pH and osmotic conditions, enabling it to survive passage through the host and persist in the environment. Among the many proteins responsible for bacterial survival under these diverse conditions, we have identified Vc-NhaP1 as a K(+)(Na(+))/H(+) antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-exponential phase, after 4-6 h of culture. Using a pH-sensitive GFP, cytosolic pH was shown to be dependent on K(+) in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless Escherichia coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K(+) or Na(+) ions for H(+) within a broad pH range (7.25-9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K(+)(Na(+))/H(+) antiporter of the CPA1 family that is required for bacterial pH homeostasis and growth in an acidic environment.

A Vibrio cholerae tolC mutant showed increased toxT expression in M9 medium, but not in the presence of four amino acids that induce cholera toxin production, and in LB with high osmolarity but not high pH or temperature. TolC did not affect expression of other regulatory genes in the ToxR regulon.

Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic or organellar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regulation of Na+ levels. Na+/H+ antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na+/H+ antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na+, Li+, K+, or Rb+ as well as for Ca2+ and, possibly, NH4+. Questions about ion selectivity and the physiological impact of each particular NhaP antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K+/H+ antiporter while retaining the ability to exchange H+ for Na+ and bind (but not exchange with H+) Li+ in a competitive manner. These and other findings reviewed in this communication make antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize peculiarities of the cation specificity in the NhaP family, the "size-exclusion principle" and the idea of "ligand shading" are discussed.

The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na(+)/H(+) antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.0 from high concentrations of external K(+). Resistance of V. cholerae to Na(+) was found to be independent of Vc-NhaP2. When assayed in inside-out membrane vesicles derived from antiporter-deficient Escherichia coli, Vc-NhaP2 catalyzed the electroneutral K(+)(Rb(+))/H(+) exchange with a pH optimum of approximately 7.75 with an apparent K(m) for K(+) of 1.62 mM. In the absence of K(+), it exhibited Na(+)/H(+) antiport, albeit rather weakly. Interestingly, while Vc-NhaP2 cannot exchange Li(+) for protons, elimination of functional Vc-NhaP2 resulted in a significantly higher Li(+) resistance of V. cholerae cells growing at pH 6.0, suggesting the possibility of Vc-NhaP2-mediated Li(+)/K(+) antiport. The peculiar cation specificity of Vc-NhaP2 and the presence of its two additional paralogues in the same genome make this transporter an attractive model for detailed analysis of the structural determinants of the substrate specificity in alkali cation exchangers.

Vibrio tubiashii, a causative agent of severe shellfish larval disease, produces multiple extracellular proteins, including a metalloprotease (VtpA), as potential virulence factors. We previously reported that VtpA is toxic for Pacific oyster (Crassostrea gigas) larvae. In this study, we show that extracellular protease production by V. tubiashii was much reduced by elevated salt concentrations, as well as by elevated temperatures. In addition, V. tubiashii produced dramatically less protease in minimal salts medium supplemented with glucose or sucrose as the sole carbon source than with succinate. We identified a protein that belongs to the TetR family of transcriptional regulators, VtpR, which showed high homology with V. cholerae HapR. We conclude that VtpR activates VtpA production based on the following: (i) a VtpR-deficient V. tubiashii mutant did not produce extracellular proteases, (ii) the mutant showed reduced expression of a vtpA-lacZ fusion, and (iii) VtpR activated vtpA-lacZ in a V. cholerae heterologous background. Moreover, we show that VtpR activated the expression of an additional metalloprotease gene (vtpB). The deduced VtpB sequence showed high homology with a metalloprotease, VhpA, from V. harveyi. Furthermore, the vtpR mutant strain produced reduced levels of extracellular hemolysin, which is attributed to the lower expression of the V. tubiashii hemolysin genes (vthAB). The VtpR-deficient mutant also had negative effects on bacterial motility and did not demonstrate toxicity to oyster larvae. Together, these findings establish that the V. tubiashii VtpR protein functions as a global regulator controlling an array of potential virulence factors.

Vibrio tubiashii is a re-emerging pathogen of molluscs that secretes a variety of extracellular products (ECPs), including a metalloprotease and a cytolysin/haemolysin. Previously, we reported that the V. tubiashii haemolysin locus consists of two ORFs (vthB and vthA), similar to that of the homologous haemolysin genes (vvhB and vvhA) found in Vibrio vulnificus. Here, we demonstrate that the concomitant expression of both V. tubiashii genes resulted in significantly higher haemolytic activity than the vthA gene alone. In addition, we created a VthAB- mutant strain of V. tubiashii that was virtually devoid of haemolytic activity in liquid media. Interestingly, significant production of an additional haemolysin(s) was observed on blood plates. Moreover, we have previously reported that in V. tubiashii, proteolytic and haemolytic activities are inversely produced during bacterial growth. Here, we study this correlation in more detail and present evidence that the VtpA metalloprotease inhibits haemolytic activity in culture supernatants, based on the following evidence: (i) loss of metalloprotease activity by either mutation or EDTA inhibition resulted in increased haemolytic activity; (ii) overexpression of the vtpA gene resulted in decreased haemolytic activity; (iii) purified VtpA metalloprotease directly diminished haemolytic activity by purified VthA haemolysin. Importantly, we found not only that vthAB gene expression remained high throughout growth but also that there were no dramatic differences in vthAB gene expression between the parent and VtpA- mutant strains. Thus, our results strongly suggest that the V. tubiashii metalloprotease directly targets its haemolysin.

Vibrio tubiashii has recently re-emerged as a pathogen of bivalve larvae, causing a marked increase in the mortality of these species within shellfish rearing facilities. This has resulted in substantial losses of seed production and thus created the need for specific as well as sensitive detection methods for this pathogen. In this project, quantitative PCR (qPCR) primers were developed and optimized based upon analysis of the V. tubiashii vtpA gene sequence, encoding a metalloprotease known to cause larval mortality. Standard curves were developed utilizing dilutions of known quantities of V. tubiashii cells that were compared to colony forming unit (CFU) plate counts. The assay was optimized for detection of vtpA with both lab-grown V. tubiashii samples and filter-captured environmental seawater samples seeded with V. tubiashii. In addition, the primers were confirmed to specifically detect only V. tubiashii when tested against a variety of non-target Vibrio species. Validation of the assay was completed by analyzing samples obtained from a shellfish hatchery. The development of this rapid and sensitive assay for quantitative detection of V. tubiashii will accurately determine levels of this bacterium in a variety of seawater samples, providing a useful tool for oyster hatcheries and a method to assess the presence of this bacterium in the current turbulent ocean environment.

The mrp operon from Vibrio cholerae encoding a putative multisubunit Na(+)/H(+) antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na(+) and Li(+) as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li(+), Na(+), and K(+) ions in pH-dependent manner with maximal activity at pH 9.0-9.5. Exchange was electrogenic (more than one H(+) translocated per cation moved in opposite direction). The apparent K(m) at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li(+), Na(+), and K(+), respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems., (Copyright 2008 S. Karger AG, Basel.)

Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.

The ability to move toward favorable environmental conditions, called chemotaxis, is common among motile bacteria. In particular, aerotaxis has been extensively studied in Escherichia coli and was shown to be dependent on the aer and tsr genes. Three putative aer gene homologs were identified in the Vibrio cholerae genome, designated aer-1 (VC0512), aer-2 (VCA0658), and aer-3 (VCA0988). Deletion analyses indicated that only one of them, aer-2, actively mediates an aerotaxis response, as assayed in succinate soft agar plates as well as a capillary assay. Complementation studies confirmed that Aer-2 is involved in aerotaxis in V. cholerae. In addition, overexpression of aer-2 resulted in a marked increase of the aerotactic response in soft agar plates. No observable phenotypes in V. cholerae mutants deleted in the aer-1 or aer-3 genes were detected under standard aerotaxis testing conditions. Furthermore, the V. cholerae aer-1 and aer-3 genes, even when expressed from a strong independent promoter, did not produce any observable phenotypes. As found in other bacterial species, the results presented in this study indicate the presence of a secondary aerotaxis transducer in V. cholerae.

The pathogenicity of Vibrio cholerae is influenced by sodium ions which are actively extruded from the cell by the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). To study the function of the Na(+)-NQR in the respiratory chain of V. cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na(+)-NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na(+)-NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na(+) to 0.4 mM at 14.7 mM Na(+), indicating that the concentration of the coupling cation influences the redox state of the quinone pool in V. cholerae membranes. During respiration, V. cholerae cells produced extracellular superoxide with a specific activity of 10.2 nmol min(-1) mg(-1) in the wild type compared to 3.1 nmol min(-1) mg(-1) in the NQR deletion strain. Raising the Na(+) concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-type V. cholerae strain by at least 70%. Rates of respiratory H(2)O(2) formation by wild-type V. cholerae cells (30.9 nmol min(-1) mg(-1)) were threefold higher than rates observed with the mutant strain lacking the Na(+)-NQR (9.7 nmol min(-1) mg(-1)). Our study shows that environmental Na(+) could stimulate ubisemiquinone formation by the Na(+)-NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones.

Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.

The ability of motile bacteria to swim toward or away from specific environmental stimuli, such as nutrients, oxygen, or light provides cells with a survival advantage, especially under nutrient-limiting conditions. This behavior, called chemotaxis, is mediated by the bacteria changing direction by briefly reversing the direction of rotation of the flagellar motors. A sophisticated signal transduction system, consisting of signal transducer proteins, a histidine kinase, a response regulator, a coupling protein, and enzymes that mediate sensory adaptation, relates the input signal to the flagellar motor. Chemotaxis has been extensively studied in bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium, and depends on the activity of single copies of proteins in a linear pathway. However, growing evidence suggests that chemotaxis in other bacteria is more complex with many bacterial species having multiple paralogues of the various chemotaxis genes found in E. coli and, in most cases, the detailed functions of these potentially redundant genes have not been elucidated. Although the completed genome of Vibrio cholerae, the causative agent of cholera, predicted a multitude of genes with homology to known chemotaxis-related genes, little is known about their relative contribution to chemotaxis or other cellular functions. Furthermore, the role of chemotaxis during the environmental or infectious phases of this organism is not yet fully understood. This review will focus on the complex relationship between chemotaxis and virulence in V. cholerae.

Vibrio cholerae is known to secrete a large number of proteins into the extracellular milieu, including the important virulence factor cholera toxin (CT). However, one of the most abundant proteins found in V. cholerae supernatants is the zinc-metalloprotease HA/protease (HAP). Whereas efficient protein secretion in Escherichia coli requires ATP hydrolysis and the proton motive force (pmf), little is known about the energy requirements for protein secretion in V. cholerae. To analyze some of the energy requirements for protein secretion in V. cholerae, HAP accumulation in culture supernatants following growth in the presence of various ionophores was assayed. Extracellular production of HAP was strongly reduced in the presence of monensin, an artificial Na(+)/H(+) antiporter that collapses the DeltapNa(+) across the membrane without affecting Deltapsi, whereas the protonophore CCCP had no significant effect on the extracellular accumulation of HAP. In contrast, extracellular protease production in Pseudomonas aeruginosa was affected by CCCP, but not monensin. Furthermore, extracellular protease production of V. cholerae, but not P. aeruginosa, was increased in increasing amounts of NaCl in the culture medium. Together these results indicate that the V. cholerae HAP requires an intact sodium motive force (smf) for its efficient translocation across the membranes, whereas extracellular protease production by P. aeruginosa requires only pmf. As the entry of some bacteriophage genomes has been reported to require pmf, the effects of ionophores on the efficiency of tranduction of V. cholerae by the CTXPhi phage were analyzed. CTXPhi transduction was strongly affected by CCCP, but not monensin, suggesting that phage entry requires pmf but not smf. Understanding the energy requirements for these potentially important virulence aspects of pathogens might lead to novel intervention strategies.

The membrane energetics of the intestinal pathogen Vibrio cholerae involves both H(+) and Na(+) as coupling ions. The sequence of the c subunit of V. cholerae F(0)F(1) ATPase suggested that this enzyme is H(+) specific, in contrast to the results of previous studies on the Na(+)-dependent ATP synthesis in closely related Vibrio spp. Measurements of the pH gradient and membrane potential in membrane vesicles isolated from wild-type and DeltaatpE mutant V. cholerae show that the F(1)F(0) ATPase of V. cholerae is an H(+), not Na(+), pump, confirming the bioinformatics assignments that were based on the Na(+)-binding model of S. Rahlfs and V. Müller (FEBS Lett. 404:269-271, 1999). Application of this model to the AtpE sequences from other bacteria and archaea indicates that Na(+)-specific F(1)F(0) ATPases are present in a number of important bacterial pathogens.

Although the antimicrobial effects of silver salts were noticed long ago, the molecular mechanism of the bactericidal action of Ag(+) in low concentrations has not been elucidated. Here, we show that low concentrations of Ag(+) induce a massive proton leakage through the Vibrio cholerae membrane, which results in complete deenergization and, with a high degree of probability, cell death.

The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O(2) (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na(+)()-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na(+)-NQR generates a sodium gradient and a Delta Psi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na(+)-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na(+) across the membrane.

The Vibrio cholerae genome revealed the presence of multiple sets of chemotaxis genes, including three cheA gene homologs. We found that the cheA-2, but not cheA-1 or cheA-3, gene is essential for chemotaxis under standard conditions. Loss of chemotaxis had no effect on virulence factor expression in vitro.