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Background The number of patients requiring home enteral nutrition (HEN) continues to increase. Many of these patients are interested in using blended food instead of, or in addition to, commercial enteral formula (CEF). Increased risk of food-borne illness is a concern of blenderized tube-feeding (BTF). This project assessed a standard procedure for minimizing bacterial growth of BTF prepared in the home setting. Methods Fifty participants prepared BTF in their kitchens using a standard preparation procedure to minimize bacterial contamination. BTF was assessed for growth of aerobic microorganisms, Escherichia coli, Staphylococcus aureus, and coliforms at baseline, 24-hour, and 48-hour intervals after preparation for a total of 150 colony forming units (CFU) counts performed. Results No sample had zero aerobic microbial counts; yet no substantial increase in microbial counts was observed during the 48 hours. At baseline and 24 hours, 5/50 (10%) had a CFU count of >104 , and at 48 hours, 6/50 (12%) exceeded 104 CFUs. Out of 150 CFU counts, 2 (1.3%) were just over 105 CFU/mL. Samples exceeding 104 CFU/mL were likely contaminated by common endospore-forming bacteria found in soil or by bacteria in milk that was close to its expiration date. Conclusion In this study, 88% of the samples met the US Food Code criteria for safe food consumption; 10.7% met guidelines for marginal safety by other standards; and 1.3% slightly exceeded 105 CFUs. Established safe food-handling procedures can minimize bacterial contamination of BTF and consequently reduce risk of food-borne infection in HEN patients.

Background Many healthcare facilities and providers prohibit blenderized tube feeding (BTF) for patients who request it due to concerns of high microbial load. The current project compared microbial loads of a standard ready-to-feed polymeric commercial formula (CF), a BTF made using baby food (BTF-BF), and a BTF prepared from blending whole food (BTF-WF), following food safety standards expected of U.S. hospitals. Methods Three tube-feeding formulas (CF, BTF-BF, BTF-WF) were prepared in a U.S. hospital and delivered in vitro to an unoccupied patient room. Samples were collected at zero hour, 2 hours, and 4 hours and compared for growth of aerobic microorganisms, Staphylococus aureus, coliforms, and Escherichia coli. The experiment was conducted in triplicate, 1 week apart. Results No S. aureus or coliform/E. coli were detected at any time point following preparation, and total bacterial count was well below acceptable limits. All 3 feeding formulas at zero hour, 2 hours, and 4 hours for each of the 3 sampling dates were acceptable for human consumption. Conclusion Judicious BTF recipe selection and adherence to safe food handling provide a safe feeding substrate equivalent to CF in the hospital setting. Due to increased use and interest in BTF by patients and their caregivers, healthcare facilities may need to reexamine their policies prohibiting BTF use.

This study aimed to select and validate different methodological strategies to quantify the expression of the virulence genes ascC and ascV by qPCR in Aeromonas salmonicida subsp. salmonicida (Aer. salmonicida).Using the geNorm, Normfinder and BestKeeper algorithms, reference genes for the qPCR were selected based on their in vitro expression stabilities in three Aer. salmonicida strains. Gene amplification efficiency was calculated by Real-time PCR Miner and LinReg PCR programmes, which have not been used previously in the analysis of bacterial gene expression. The expression of the ascC and ascV virulence genes in a virulent Aer. salmonicida strain was evaluated by three quantification models, including single (least or most stable) or three most stable reference genes, combined with constant or specific gene amplification efficiency. The most stable reference genes were gyrB, proC and rpoC, while rpoD and fabD were the least stable. Quantification models showed different expression patterns.The optimal strategy to quantify mRNA expression was to use a combination of the three algorithms and the quantification model including the three most stable reference genes. Real-time PCR Miner or LinReg PCR were valuable tools to estimate amplification efficiency.The methods used in this study gave more reliable expression data using qPCR than previously published methods. The quantification and expression dynamics of virulence genes will contribute to a better understanding of how Aer. salmonicida interacts with its host and the environment, and therefore to the prevention of epizootics due to this pathogen.

The metalloendopeptidase AsaP1 is one of the major extracellular virulence factors of A. salmonicida subsp. achromogenes, expressed as a 37-kDa pre-pro-peptide and processed to a 19-kDa active peptide. The aim of this study was to construct mutant strains secreting an AsaP1-toxoid instead of AsaP1-wt, to study virulence of these strains and to test the potency of the AsaP1-toxoid bacterin and the recombinant AsaP1-toxoids to induce protective immunity in Arctic char. Two A. salmonicida mutants were constructed that secrete either AsaP1E294A or AsaP1Y309F . The secreted AsaP1Y309F -toxoid had weak caseinolytic activity and was processed to the 19-kDa peptide, whereas the AsaP1E294A -toxoid was found as a 37-kDa pre-pro-peptide suggesting that AsaP1 is auto-catalytically processed. The LD50 of the AsaP1Y309F -toxoid mutant in Arctic char was significantly higher than that of the corresponding wt strain, and LD50 of the AsaP1E294A -toxoid mutant was comparable with that of an AsaP1-deficient strain. Bacterin based on AsaP1Y309F -toxoid mutant provided significant protection, comparable with that induced by a commercial polyvalent furunculosis vaccine. Detoxification of AsaP1 is very hard, expensive and time consuming. Therefore, an AsaP1-toxoid-secreting mutant is more suitable than the respective wt strain for production of fish bacterins aimed to protect against atypical furunculosis.

Proteorhodopsin (PR), a ubiquitous membrane photoprotein in marine environments, acts as a light-driven proton pump and can provide energy for bacterial cellular metabolism. However, knowledge of f ...

Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections-unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.

Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections-unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.

Vibrio anguillarum causes a fatal haemorrhagic septicaemia in marine fish. During initial stages of infection, host surfaces are colonized; however, few virulence factors required for colonization ...

Vibrio anguillarum, part of the normal flora of the aquatic milieu, causes a fatal haemorrhagic septicaemia in marine fish. In this study, a rainbow trout model was used to characterize the colonization of fish skin by V. anguillarum. Within 5 h after infection, the bacterium penetrated the skin mucosal layer, attached to the scales within 12 h, and formed a biofilm by 24-48 h. Two divergently transcribed putative operons, orf1-wbfD-wbfC-wbfB and wza-wzb-wzc, were shown to play a role in skin colonization and virulence. The first operon encodes proteins of unknown function. The wza-wzb-wzc genes encode a secretin, tyrosine kinase and tyrosine phosphatase, respectively, which are similar to proteins in polysaccharide transport complexes. Compared with the wild type, polar mutations in wza, orf1 and wbfD caused a decrease in exopolysaccharide biosynthesis but not lipopolysaccharide biosynthesis. The wza and orf1 mutants did not attach to fish scales; whereas, the wbfD mutant had a wild-type phenotype. Moreover, the wza and orf1 mutants had decreased exoprotease activity, in particular the extracellular metalloprotease EmpA, as well as mucinase activity suggesting that these mutations also affect exoenzyme secretion. Thus, the exopolysaccharide transport system in V. anguillarum is required for attachment to fish skin, possibly preventing mechanical removal of bacteria via natural sloughing of mucus.

Vibrio anguillarum causes a fatal hemorrhagic septicemia in marine fish that leads to great economical losses in aquaculture world-wide. Vibrio anguillarum strain NB10 serotype O1 is a Gram-negative, motile, curved rod-shaped bacterium, isolated from a diseased fish on the Swedish coast of the Gulf of Bothnia, and is slightly halophilic. Strain NB10 is a virulent isolate that readily colonizes fish skin and intestinal tissues. Here, the features of this bacterium are described and the annotation and analysis of its complete genome sequence is presented. The genome is 4,373,835 bp in size, consists of two circular chromosomes and one plasmid, and contains 3,783 protein-coding genes and 129 RNA genes. Electronic supplementary material The online version of this article (doi:10.1186/s40793-015-0060-7) contains supplementary material, which is available to authorized users.

Vibrio anguillarum produces several interlinked acylated homoserine lactone (AHL) signal molecules which may influence expression of its virulence factors such as exoprotease production and biofilm formation. Using both thin layer chromatography and HPLC-high resolution mass spectrometry (HPLC-HRMS), we demonstrate in this study that the same types of AHLs are produced by many serotypes of V. anguillarum and that altering in vitro growth conditions (salinity, temperature and iron concentration) has little influence on the AHL-profile. Most strains produced N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-hydroxy-C6-HSL) as the dominant molecules. Also, two spots with AHL activity appeared on TLC plates, which could not be identified as AHL structures. Trace amounts of N-(3-hydroxy-octanoyl)-l-homoserine lactone, N-(3-hydroxy-decanoyl)-l-homoserine lactone and N-(3-hydroxy-dodecanoyl)-l-homoserine lactone (3-hydroxy-C8-HSL, 3-hydroxy-C10-HSL and 3-oxo-C12-HSL, respectively) were also detected by HPLC-HRMS analysis from in vitro cultures. Most studies of quorum sensing (QS) systems have been conducted in vitro, the purpose of our study was to determine if the same acylated homoserine lactones were produced in vivo during infection. Extracts from infected fish were purified using several solid phase extraction strategies to allow chromatographic detection and separation by both TLC and HLPC-HRMS. 3-oxo-C10-HSL and 3-hydroxy-C6-HSL were detected in organs from fish dying from vibriosis, however, compared to in vitro culturing where 3-oxo-C10-HSL is the dominant molecule, 3-hydroxy-C6-HSL was prominent in the infected fish tissues. Hence, the balance between the QS systems may be different during infection compared to in vitro cultures. For future studies of QS systems and the possible specific interference with expression of virulence factors, in vitro cultures should be optimised to reflect the in vivo situation.

N-acylhomoserine lactone-dependent quorum sensing was first discovered in two luminescent marine bacteria, Vibrio fischeri and Vibrio harveyi. The LuxI/R system of V. fischeri is the paradigm of Gram-negative quorum-sensing systems; however, it is not found in all vibrios. A more complex quorum-sensing regulation is found in V. harveyi. Three parallel systems transmit signals via phosphorelays that converge onto one regulatory protein LuxO. Components of the three systems are found only in vibrios. Of the five Vibrio strains analysed, the number and types of signal circuits found in each strain are diverse. The signalling systems have different regulatory responses depending on the type of association the Vibrio strains have with an animal host, which may reflect the diverse roles the vibrios have in structuring and maintaining microniches within the aquatic milieu. Further studies are likely to show that the diversity and complexity of the Vibrio quorum-sensing systems coordinate intraspecies behaviour, niche occupation, and possibly evolution.

Zoospores of the eukaryotic green seaweed Ulva respond to bacterial N-acylhomoserine lactone (AHL) quorum sensing signal molecules for the selection of surface sites for permanent attachment. In this study we have investigated the production and destruction of AHLs in biofilms of the AHL-producing marine bacterium, Vibrio anguillarum and their stability in seawater. While wild type V. anguillarum NB10 was a strong attractor of zoospores, inactivation of AHL production in this strain by either expressing the recombinant Bacillus lactonase coding gene aiiA, or by mutating the AHL biosynthetic genes, resulted in the abolition of zoospore attraction. In seawater, with a pH of 8.2, the degradation of AHL molecules was temperature-dependent, indicating that the AHLs produced by marine bacterial biofilms have short half-lives. The Ulva zoospores sensed a range of different AHL molecules and in particular more zoospores settled on surfaces releasing AHLs with longer (>six carbons) N-linked acyl chains. However, this finding is likely to be influenced by the differential diffusion rates of AHLs from the experimental surface matrix. Molecules with longer N-acyl chains, such as N-(3-oxodecanoyl)- L-homoserine lactone, diffused more slowly than those with shorter N-acyl chains such as N-(3-hydroxy-hexanoyl)- L-homoserine lactone. Image analysis using GFP-tagged V. anguillarum biofilms revealed that spores settle directly on bacterial cells and in particular on microcolonies which we show are sites of concentrated AHL production.

Many bacterial cells communicate using diffusible signal molecules to monitor cell population density via a process termed quorum sensing. In marine Vibrio species, the Vibrio harveyi-type LuxR pro ...

Vibrio anguillarum, a fish pathogen, produces a 38 kDa major outer-membrane porin, which may be involved in environmental adaptation. The gene encoding the 38 kDa porin was cloned and deleted. The deduced protein sequence was 75 % identical to that of the major outer-membrane protein (OMP), OmpU, from Vibrio cholerae. LacZ expression from an ompU : : lacZ transcriptional gene fusion was increased 1.5-fold in the presence of bile salts and was decreased 50- to 100-fold in a toxR mutant compared to that in the wild-type, showing that ompU expression is positively regulated by ToxR and induced by bile salts. Similar to a toxR mutant, an ompU mutant showed a slight decrease in motility, an increased sensitivity to bile salts and a thicker biofilm with better surface area coverage compared to that of the wild-type. When ompU was expressed under a ToxR-independent promoter in the toxR mutant, the phenotypes for bile resistance and biofilm formation, but not motility were complemented to that of the wild-type. In rainbow trout, the ompU mutant showed wild-type virulence via immersion into infected seawater and intraperitoneal injection. The ompU mutant produced two colony morphologies: opaque, which did not grow at 0.2 % bile, and translucent, which grew at 2 % bile. The translucent ompU mutant strain produced a second major OMP that was induced by bile. All ompU mutants showed variations in the amount and length of smooth LPS. In V. anguillarum, OmpU is not required for virulence, possibly due to a second OMP also critical for resistance to bile; however, outside of the fish host, OmpU limits the progression of biofilm formation.

Vibrio anguillarum , which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system ( vanRI ) and to produce N -(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N -acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum . In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N -hexanoylhomoserine lactone (C6-HSL) and N -(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM , the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N -(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi . However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM ). Downstream of vanM , we identified a homologue of luxN ( vanN ) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi . A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI . However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).

To investigate the involvement of RpoN in flagellum production and pathogenicity of Vibrio (Listonella) anguillarum, the rpoN gene was cloned and sequenced. The deduced product of the rpoN gene displayed strong homology to the alternative σ54 factor (RpoN) of numerous species of bacteria. In addition, partial sequencing of rpoN-linked ORFs revealed a marked resemblance to similarly located ORFs in other bacterial species. A polar insertion or an in-frame deletion in the coding region of rpoN abolished expression of the flagellin subunits and resulted in loss of motility. Introduction of the rpoN gene of V. anguillarum or Pseudomonas putida into the rpoN mutants restored flagellation and motility. The rpoN mutants were proficient in the expression of other proposed virulence determinants of V. anguillarum, such as ability to grow under low available iron conditions, and expression of the LPS O-antigen and of haemolytic and proteolytic extracellular products. The infectivity of the rpoN mutants with respect to the wild-type strain was unaffected following intraperitoneal injection of fish but was reduced significantly when fish were immersed in bacteria-containing water. Thus, RpoN does not appear to regulate any factors required for virulence subsequent to penetration of the fish epithelium, but is important in the infection of fish by water-borne V. anguillarum.

Vibrio anguillarum causes a fatal haemorrhagic septicaemia in marine fish. During initial stages of infection, host surfaces are colonized; however, few virulence factors required for colonization of the host are identified. In this study, in vivo bioluminescent imaging was used to analyse directly the colonization of the whole rainbow trout animal by V. anguillarum. The wild type rapidly colonized both the skin and the intestines by 24 h; however, the bacterial numbers on the skin were significantly higher than in the intestines indicating that skin colonization may be important for disease to occur. Mutants defective for the anguibactin iron uptake system, exopolysaccharide transport, or Hfq, an RNA chaperone, were attenuated for virulence, did not colonize the skin, and penetrated skin mucus less efficiently than the wild type. These mutants, however, did colonize the intestines and were as resistant to 2% bile salts as is the wild type. Moreover, exopolysaccharide mutants were significantly more sensitive to lysozyme and antimicrobial peptides, while the Hfq and anguibactin mutants were sensitive to lysozyme compared with the wild type. Vibrio anguillarum encodes several mechanisms to protect against antimicrobial components of skin mucus enabling an amazingly abundant growth on the skin enhancing its disease opportunities.

Vibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the (1)H, (13)C, and (15)N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum.

Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additionalflagellin proteins, FlaB, -C, and -D. This study identifies the genes encoding FlaB, -C, and -D and a possiblefifthflagellin gene that may encode FlaE. Theflagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci. The genetic organization of these two loci is conserved between both organisms. For each gene, in-frame deletions of the entire gene, the 5*end, and the 3*end were made. Mutant analysis showed that each mutation, except those inflaE, caused a loss offlagellin from thefilament. However, no obvious structural loss in thefilament, as determined by electron microscopy, and only slight decreases in motility were seen. Virulence analysis indicated that all but two of the mutations gave a wild-type phenotype. The 5*-end deletions offlaDandflaE decreased virulence significantly (>10 4 -fold) of infections via both the intraperitoneal and immersion routes. These results indicate that, like FlaA, FlaD and FlaE may also be involved in virulence. For several bacteria, theflagellum has been suggested to be involved in virulence either as a motility organelle or as an organelle that carries an adhesive component. In Campylobacter jejunistudies, theflagellum has been suggested to aid the bacterium either in the adherence to (20) or in the internalization (7) within cultured epithelial cells. Additional studieswithC.jejuni(32)haveshownthatmotilityandnotflagellin A is required for the invasion of intestinal cells; however, flagellin A can serve as a secondary adhesin for the adherence to intestinal cultured cells. For Pseudomonas aeruginosa ,a study utilizing isogenic motility mutants in the burned mouse model showed that motility contributes to the invasiveness of this organism (5). ForVibrio cholerae, earlier studies with motility mutants suggested that either motility (8) or an adhesin (2,11),proposedtobeassociatedwiththeflagellum,isneeded for the colonization of intestinal tissues, whereas a later study (27) indicated that motility but not the flagellar structure is essential for the colonization of rabbits. The polar flagellum of Vibrio anguillarum, the causative agent of vibriosis in marine fish, is a complex structure. The filament is believed to be composed of four flagellin proteins (23) and is covered by a sheath (25). Recently, studies have beeninitiatedtoanalyzetherolethattheflagellumplaysinthe virulence of V. anguillarum. Chemotactic motility has been shown to be required for the invasion of rainbow trout when they are immersed in infected seawater; however, chemotactic motility is not essential for virulence after thefish integument is crossed (26). Moreover, flagellin A has been suggested to have two possible roles in virulence (23). A mutant carrying a deletion of theflaAgene was shown to be partially motile and to be defective in its ability to invade rainbow trout immersed in infected seawater; however, virulence was not decreased if the mutant was introduced into the fish by intraperitoneal injection. Thus,flagellin A is needed for the full motility which is essential for efficient invasion of rainbow trout. A second mutantthatcontainedanin-framedeletionatthe39endofthe

A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.

The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non-motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon-insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum. This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR, was located upstream of one transposon insertion and an in-frame deletion was constructed in the coding region of this gene. The resulting non-chemotactic mutant exhibited wild-type pathogenicity when injected intra-peritoneally into fish but showed a decrease in virulence similar to that seen for the non-motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum.

Proteorhodopsin (PR), a ubiquitous membrane photoprotein in marine environments, acts as a light-driven proton pump and can provide energy for bacterial cellular metabolism. However, knowledge of factors that regulate PR gene expression in different bacteria remains strongly limited. Here, experiments with Vibrio sp. AND4 showed that PR phototrophy promoted survival only in cells from stationary phase and not in actively growing cells. PR gene expression was tightly regulated, with very low values in exponential phase, a pronounced peak at the exponential/stationary phase intersection, and a marked decline in stationary phase. Thus, PR gene expression at the entry into stationary phase preceded, and could therefore largely explain, the stationary phase light-induced survival response in AND4. Further experiments revealed nutrient limitation, not light exposure, regulated this differential PR expression. Screening of available marine vibrios showed that the PR gene, and thus the potential for PR phototrophy, is found in at least three different clusters in the genus Vibrio. In an ecological context, our findings suggest that some PR-containing bacteria adapted to the exploitation of nutrient-rich micro-environments rely on a phase of relatively slowly declining resources to mount a cellular response preparing them for adverse conditions dispersed in the water column.

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.

The most recently discovered secretion pathway in Gram-negative bacteria, the type VI secretion system (T6SS), is present in many species and is considered important for the survival of non-O1 non-O139 Vibrio cholerae in aquatic environments. Until now, it was not known whether there is a functionally active T6SS in wild-type V. cholerae O1 strains, the cause of cholera disease in humans. Here, we demonstrate the presence of a functionally active T6SS in wild-type V. cholerae O1 strains, as evidenced by the secretion of the T6SS substrate Hcp, which required several gene products encoded within the putative vas gene cluster. Our analyses showed that the T6SS of wild-type V. cholerae O1 strain A1552 was functionally activated when the bacteria were grown under high-osmolarity conditions. The T6SS was also active when the bacteria were grown under low temperature (23°C), suggesting that the system may be important for the survival of the bacterium in the environment. A test of the interbacterial virulence of V. cholerae strain A1552 against an Escherichia coli K-12 strain showed that it was strongly enhanced under high osmolarity and that it depended on the hcp genes. Interestingly, we found that the newly recognized osmoregulatory protein OscR plays a role in the regulation of T6SS gene expression and secretion of Hcp from V. cholerae O1 strains.

Vibrio anguillarumutilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like otherVibriospecies,V. anguillarumcontains the geneqrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ54and the σ54-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilizedvanTmRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all fourqrrgenes peaked at high cell density, and exogenously addedN-acylhomoserine lactone molecules induced expression of theqrrgenes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilizedvanTmRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.

Mutational analysis provides direct evidence for the link between proteorhodopsin light-harvesting and enhanced survival of marine bacteria., Proteorhodopsins are globally abundant photoproteins found in bacteria in the photic zone of the ocean. Although their function as proton pumps with energy-yielding potential has been demonstrated, the ecological role of proteorhodopsins remains largely unexplored. Here, we report the presence and function of proteorhodopsin in a member of the widespread genus Vibrio, uncovered through whole-genome analysis. Phylogenetic analysis suggests that the Vibrio strain AND4 obtained proteorhodopsin through lateral gene transfer, which could have modified the ecology of this marine bacterium. We demonstrate an increased long-term survival of AND4 when starved in seawater exposed to light rather than held in darkness. Furthermore, mutational analysis provides the first direct evidence, to our knowledge, linking the proteorhodopsin gene and its biological function in marine bacteria. Thus, proteorhodopsin phototrophy confers a fitness advantage to marine bacteria, representing a novel mechanism for bacterioplankton to endure frequent periods of resource deprivation at the ocean's surface., Author Summary It is estimated that marine microscopic algae—phytoplankton—are responsible for half of the Earth's photosynthesis. As much as half of the surface ocean bacteria have proteorhodopsins, which are membrane proteins that allow harvesting of energy from sunlight, implying a potentially significant role of non–chlorophyll-based phototrophy in oceanic carbon cycling and energy flux. Functional evidence for specific roles for proteorhodopsins in native marine bacteria and the marine environment remains surprisingly scarce. One reason for this is the lack of marine bacteria (containing proteorhodopsin genes) that can be maintained in laboratory culture and that are tractable to genetic manipulation. In this study, we show that a proteorhodopsin-containing member of the widespread marine genus Vibrio displays light-enhanced survival during starvation in seawater. Furthermore, growth recovery experiments showed that bacteria starving in the light could more rapidly respond to improved growth conditions than those incubated in the dark. We generated a proteorhodopsin deficient Vibrio strain and used it to confirm that light-dependent survival of starvation was mediated by the proteorhodopsin. Proteorhodopsin phototrophy thus provides a physiological mechanism that allows surface ocean bacteria to manage an environment where resource availability fluctuates markedly.

Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.

To understand further the role of the flagellum of Vibrio anguillarum in virulence, invasive and adhesive properties of isogenic motility mutants were analyzed by using a chinook salmon embryo cell line. Adhesion was unaffected but invasion of the cell line was significantly decreased in nonmotile or partially motile mutants, and the chemotactic mutant was hyperinvasive. These results suggest that active motility aids invasion by V. anguillarum , both in vivo and in vitro.

BACKGROUND: The type VI secretion system (T6SS) has emerged as a protein secretion system important to several gram-negative bacterial species. One of the common components of the system is Hcp, initially described as a hemolysin co-regulated protein in a serotype O17 strain of Vibrio cholerae. Homologs to V. cholerae hcp genes have been found in all characterized type VI secretion systems and they are present also in the serotype O1 strains of V. cholerae that are the cause of cholera diseases but seemed to have non-functional T6SS. METHODOLOGY/PRINCIPAL FINDINGS: The serotype O1 V. cholerae strain A1552 was shown to express detectable levels of Hcp as determined by immunoblot analyses using polyclonal anti-Hcp antiserum. We found that the expression of Hcp was growth phase dependent. The levels of Hcp in quorum sensing deficient mutants of V. cholerae were compared with the levels in wild type V. cholerae O1 strain A1552. The expression of Hcp was positively and negatively regulated by the quorum sensing regulators HapR and LuxO, respectively. In addition, we observed that expression of Hcp was dependent on the cAMP-CRP global transcriptional regulatory complex and required the RpoN sigma factor. CONCLUSION/SIGNIFICANCE: Our results show that serotype O1 strains of V. cholerae do express Hcp which is regarded as one of the important T6SS components and is one of the secreted substrates in non-O1 non-O139 V. cholerae isolates. We found that expression of Hcp was strictly regulated by the quorum sensing system in the V. cholerae O1 strain. In addition, the expression of Hcp required the alternative sigma factor RpoN and the cAMP-CRP global regulatory complex. Interestingly, the environmental isolates of V. cholerae O1 strains that showed higher levels of the HapR quorum sensing regulator in comparison with our laboratory standard serotype O1 strain A1552 where also expressing higher levels of Hcp.

In vibrios, regulation of the Vibrio harveyi-like LuxR transcriptional activators occurs post-transcriptionally via small regulatory RNAs (sRNAs) that destabilize the luxR mRNA at a low cell population, eliminating expression of LuxR. Expression of the sRNAs is modulated by the vibrio quorum-sensing phosphorelay systems. However, vanT mRNA, which encodes a LuxR homologue in Vibrio anguillarum, is abundant at low and high cell density, indicating that VanT expression may be regulated via additional mechanisms. In this study, Western analyses showed that VanT was expressed throughout growth with a peak of expression during late exponential growth. VanO induced partial destabilization of vanT mRNA via activation of at least one Qrr sRNA. Interestingly, the sigma factor RpoS significantly stabilized vanT mRNA and induced VanT expression during late exponential growth. This induction was in part due to RpoS repressing expression of Hfq, an RNA chaperone. RpoS is not part of the quorum-sensing regulatory cascade since RpoS did not regulate expression or activity of VanO, and RpoS was not regulated by VanO or VanT. VanT and RpoS were needed for survival following UV irradiation and for pigment and metalloprotease production, suggesting that RpoS works with the quorum-sensing systems to modulate expression of VanT, which regulates survival and stress responses.

A 60 base-pair region of a simian virus 40 DNA fragment was mutagenized to determine base-pairs that are critical for the fragment to bend. The site-directed mutagenesis saturated this region with all possible single base-pair substitutions. The mobility of each mutated fragment was measured by polyacrylamide electrophoresis at 4°C and at 65°C to assess the degree of bend. Four conclusions can be drawn. First, interruptions within the A tracts and changes in the phasing of the A tracts alter the degree of bend. Second, G tracts phased at a half-helical turn from an A tract are additive to the bend. Third, guanine residues in a nearest-neighbor contact with the A tracts modify the bend. Fourth, some mutations that do not obviously relate to the A tracts also alter the DNA bend and suggest clearly that base steps other than ApA are involved in sequence-directed DNA bends.

Many bacterial cells communicate using diffusible signal molecules to monitor cell population density via a process termed quorum sensing. In marine Vibrio species, the Vibrio harveyi-type LuxR protein is a key player in a quorum-sensing phosphorelay cascade, which controls the expression of virulence, symbiotic and survival genes. Previously, we characterized Vibrio anguillarum homologues of LuxR (VanT) and LuxMN (VanMN) and, in this study, we have identified homologues of LuxPQ (VanPQ) and LuxOU (VanOU). In contrast to other Vibrio species, vanT was expressed at low cell density and showed no significant induction as the cell number increased. In addition, although the loss of VanO increased vanT expression, the loss of VanU, unexpectedly, decreased it. Both VanN and VanQ were required for repression of vanT even in a vanU mutant, suggesting an alternative route for VanNQ signal transduction other than via VanU. VanT negatively regulated its own expression by binding and repressing the vanT promoter and by binding and activating the vanOU promoter. The signal relay results in a cellular response as expression of the metalloprotease, empA, was altered similar to that of vanT in all the mutants. Consequently, the V. anguillarum quorum-sensing phosphorelay systems work differently from those of V. harveyi and may be used to limit rather than induce vanT expression.

Bacteria are capable of complex assemblage behavior through cell-to-cell communication using diffusible chemical signal molecules that accumulate to a threshold concentration that activates target genes ([1][1]). This process, termed “quorum sensing,” has not been shown to cross the prokaryote

ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum , a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae , and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.

Vibrio anguillarum possesses at least two N -acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi . In this study, we have cloned an additional gene of this circuit, vanT , encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum ΔvanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ . Three lacZ fusions ( serA :: lacZ , hpdA-hgdA :: lacZ, and sat-vps73 :: lacZ) were identified which exhibited decreased expression in the ΔvanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an l -tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum ΔvanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

Certain gram-negative pathogens are known to control virulence gene expression through cell-cell communication via small diffusible signal molecules termed autoinducers. This intercellular signal transduction mechanism termed quorum sensing depends on the interaction of an N-acylhomoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators. Vibrio anguillarum is a gram-negative pathogen capable of causing a terminal hemorrhagic septicemia known as vibriosis in fish such as rainbow trout. In this study, we sought to determine whether V. anguillarum employs AHLs to regulate virulence gene expression. Spent V. anguillarum culture supernatants stimulated bioluminescence in a recombinant lux-based Escherichia coli AHL biosensor strain, whereas they both stimulated and inhibited AHL-mediated violacein pigment production in Chromobacterium violaceum. This finding suggested that V. anguillarum may produce multiple AHL signal molecules. Using high-performance liquid chromatography and high-resolution tandem mass spectrometry, we identified the major V. anguillarum AHL as N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), a structure which was unequivocally confirmed by chemical synthesis. The gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AHL synthases. Further sequencing downstream of vanI revealed a second gene (vanR) related to the LuxR family of transcriptional activators. Although deletion of vanI abolished ODHL synthesis, no reduction of either metalloprotease production or virulence in a fish infection model was observed. However, the vanI mutant remained capable of weakly activating both bioluminescence and violacein in the E. coli and C. violaceum biosensors, respectively, indicating the existence of additional layers of AHL-mediated regulatory complexity.

In this report, we describe the cytotoxic activity of the cholera hemagglutinin/protease (HA/protease). A concentrated protein sample from the 37 degrees C overnight culture supernatant of CVD110, a delta ctxA, delta zot, delta Ace and hlyA::(ctxB mer) mutant of El Tor biotype Ogawa serotype strain E7946 caused morphological changes in cultured MDCK-I epithelial cells and altered their arrangement of filamentous actin (F-actin) and Zonula occludens-associated protein ZO-1. The drastic morphological changes can be inhibited by Zincov, a specific bacterial metalloprotease inhibitor. The cytotoxic fractions of the sample after FPLC gelfiltration fractionation showed two visible protein bands with molecular weights of approximately 34- and 32 kDa. Microsequencing of these two proteins revealed that they were the cholera HA/protease.

Previously, the double-transposon (Tn) mutant VAN20 of Vibrio anguillarum (Va) 775.17B was isolated. This mutant lacked a major surface antigen (MSA) suggested to be a lipopolysaccharide (LPS) and showed a 10 5 -fold increase in the 50% lethal dose (LD 50 ) when fish were infected intraperitoneally. In this study, the two Tn insertion sites within the chromosome were identified, a plasmid insertion mutation was made at each locus in a more virulent strain of Va , NB10, and the virulence was analyzed. One mutant displayed a 10 4 -fold increase in LD 50 , whereas the second mutant showed the wild-type (wt) phenotype. However, both mutants still expressed the MSA, suggesting that there may be more than two Tn insertions in VAN20 or that a double mutation is required to prevent production of the MSA. The DNA locus for the virulent phenotype was cloned and sequenced. A potential transcriptional unit consisting of three putative open reading frames (ORFs) was identified. The Tn was located in the second ORF, virC (virulence). The first ORF (34.8 kDa) showed 30% homology to the Escherichia coli and Salmonella typhimurium cysG (cysteine) genes. The virC gene (51.4 kDa) and the third ORF (24 kDa) showed no homology to other proteins in GenBank. Plasmid insertion mutants were made within each of these ORFs and the virulence was assayed. Only the virC mutant showed a loss in virulence, indicating that virC is a novel gene that is essential for the virulence of Va .

Bacteria can communicate with members of their own species and others to coordinate their behavior in response to cell density. This phenomenon, known as quorum sensing, relies on the production and sensing of one or more secreted signal molecules. A recent study identifies a complex quorum sensing network in the human pathogen Vibrio cholerae.

Synthetic DNA fragments were constructed to determine the effect of G tracts, in conjunction with periodically spaced A tracts, on DNA bends. Relative length measurements showed that the G tracts spaced at the half helical turn enhanced the DNA bend. When the G tract was interrupted with a thymine or shortened to one or two guanines, the relative lengths decreased. If the G tract was replaced with either an A tract or a T tract, the bend was cancelled. Replacement with a C tract decreased the relative length to that of a thymine interruption suggesting that bend enhancement due to G tracts requires A tracts on the same strand.

Five fragments of DNA exhibiting sequence directed bends were isolated from the Simian Virus 40 genome using a two-dimensional polyacrylamide gel fractionation. The bend sites were mapped for each fragment using the circular permutation test. All five sites have multiple, short runs of A residues with helical spacing typical of other bent fragments. Base pairs important for the bends were determined for one fragment by utilizing a random, single base pair mutagenesis. Of 28 mutants with decreased or increased bends, 14 had alterations that could be interpreted to affect the spaced runs of A residues, supporting their role in bends as predicted by the ApA wedge model. One major mutation was not explainable by existing models. The remaining minor mutations may only be due to small, local DNA conformational changes in the surrounding B-DNA.