Identification and characterization of two contiguous operons required for aerobactin transport and biosynthesis in Vibrio mimicus

In response to iron deprivation, Vibrio mimicus produces aerobactin as a major siderophore. Application of the Fur titration assay to a V. mimicus genomic DNA library followed by further cloning of the surrounding regions led to the identification of two adjacent, iron-regulated operons. One contains three genes encoding homologs of the Escherichia coli FhuCDB and the other, five genes encoding homologs of the E. coli IucABCD IutA. Construction of the V. mimicus polar disruptants in the respective operons allowed us to confirm their functions. The genetic arrangement of the aerobactin-mediated iron acquisition system in V. mimicus is unique in that the aerobactin operon (iucABCD iutA ) is contiguous to the operon (matCDB ) encoding components of an ATP-binding cassette transport system for ferric aerobactin. This is the first report demonstrating that aerobactin transport and biosynthesis genes are present in a species outside the family Enterobacteriaceae.     

Isolation and characterization of a visibly luminous variant ofVibrio fischeri strain ES114 from the sepiolid squidEuprymna scolopes

Vibrio fischeri strains isolated from light organs of the sepiolid squid Euprymna scolpes are non-visibly luminous and fast growing in laboratory culture, whereas in the symbiosis they are visibly luminous and slow growing. A spontaneous, visibly luminous, slow-growing variant was isolated from a laboratory culture of the squid-symbiotic V. fischeri strain ES114. Taxonomic and DNA-homology analyses demonstrated that the variant was V. fischeri and was very similar to the original form. However, the variant grew at one-fourth the rate of the original form, produced 30,000-fold more luminescence, induced luminescence at a lower cell density, and produced a higher level of V. fischeri luminescence autoinducer. Regulation of luminescence, nonetheless, was similar in the two forms and typical of V. fischeri with respect to responses to autoinducer, glucose, the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid), and 3′:5′-cyclic AMP. Compared to the original form, cells of the variant were smaller, exhibited from zero to two polar, sheathed flagella instead of a tuft of three to eight flagella, produced a deeper yellow-orange pigment, did not acidify media containing glycerol, and produced a more distinct pellicle. The two forms also differed in the levels of several outer membrane and soluble proteins. These results establish a distinctive physiological, morphological, and biochemical dimorphism in V. fischeri ES114 in which the variant exhibits several traits similar to V. fischeri cells in the symbiotic state. The variant and its conversion from the original form in laboratory culture may provide insight into the properties of V. fischeri cells in the symbiosis and may serve as a model for elucidating the mechanism for their pleiotropic conversion upon colonization of the squid. Received: 10 January 1995 / Accepted: 24 May 1995     

Contribution of pilA to Competitive Colonization of the Squid Euprymna scolopes by Vibrio fischeri

Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and V. fischeri strains must compete to occupy this privileged niche. We cloned a V. fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V. fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain. V. fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V. fischeri isolates apparently possess pilA. The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.     

Salinity and Temperature Effects on Physiological Responses of <Emphasis Type="Italic">Vibrio fischeri</Emphasis> from Diverse Ecological Niches

Vibrio fischeri is a bioluminescent bacterial symbiont of sepiolid squids (Cephalopoda: Sepiolidae) and monocentrid fishes (Actinopterygii: Monocentridae). V. fischeri exhibit competitive dominance within the allopatrically distributed squid genus Euprymna, which have led to the evolution of V. fischeri host specialists. In contrast, the host genus Sepiola contains sympatric species that is thought to have given rise to V. fischeri that have evolved as host generalists. Given that these ecological lifestyles may have a direct effect upon the growth spectrum and survival limits in contrasting environments, optimal growth ranges were obtained for numerous V. fischeri isolates from both free-living and host environments. Upper and lower limits of growth were observed in sodium chloride concentrations ranging from 0.0% to 9.0%. Sepiola symbiotic isolates possessed the least variation in growth throughout the entire salinity gradient, whereas isolates from Euprymna were the least uniform at <2.0% NaCl. V. fischeri fish symbionts (CG101 and MJ101) and all free-living strains were the most dissimilar at >5.0% NaCl. Growth kinetics of symbiotic V. fischeri strains were also measured under a range of salinity and temperature combinations. Symbiotic V. fischeri ES114 and ET101 exhibited a synergistic effect for salinity and temperature, where significant differences in growth rates due to salinity existed only at low temperatures. Thus, abiotic factors such as temperature and salinity have differential effects between free-living and symbiotic strains of V. fischeri, which may alter colonization efficiency prior to infection.     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.     

Cellulolytic activity of luminous bacteria

Summary The luminous bacteriaVibrio harveyi andV. fischeri degrade cellulose. Cellulase activity was high when carboxymethyl cellulose and cellobiose were used as substrates but low with cellulose powder. The role of these microbes in the digestion of food material in fish gut is also discussed.

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Resumen Las bacterias luminosasVibrio harveyi yV. fischeri degradan celulosa. La actividad celulásica fue alta cuando se utilizó Carboxy-metil-celulosa o celobiosa como sustrato y baja cuando se utilizó polvo de celulosa. Se discute el papel de estos microorganismos en la digestión intestinal de los alimentos en peces.

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Résumé Les bactéries lumineusesVibrio harveyi etV. fischeri dégradent la cellulose. L'activité cellulolytique était élevée lorsque la carboxméthylcellulose et la cellobiose servaient de substrats, mais elle était faible avec la poudre de cellulose. On discute également de rôle de ces microorganismes dans la digestion du matériau alimentaire.

     

Enteric luminous microflora of the pond-cultured milk fishChanos chanos (Forskal)

Qualitative and quantitative investigations were made on the luminous bacteria associated with the gut of pond cultured milk fishChanos chanos. Significant differences in luminous bacterial numbers were found between gut and pond water and between gut and pond sediment, but not between pond water and sediment. No significant variation in luminous bacterial population among the gut regions was observed. The quantity of ingesta in the fish gut does not appear to influence the biomass of luminous bacteria.Vibrio harveyi andV. fischeri were the 2 most commonly encountered species, and of the 2 luminous species,V. harveyi was predominant.     

Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.     

Chemical and structural characterization of hydroxamate siderophore produced by marine <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

In the present study, 22 different bacteria were isolated from open ocean water from the Gulf of Mannar, India. Of the 22 isolates, 4 were identified as Vibrio spp. (VM1, VM2, VM3 and VM4) and found to produce siderophores (iron-binding chelators) under iron-limited conditions. Different media were found to have an influence on siderophore production. Maximum siderophore production was observed with VM1 isolate in MM9 salts medium at 48 h of incubation. The isolate was confirmed as Vibrio harveyi based on 16S rRNA gene sequencing and phylogenetic analysis. Fourier-transform infrared (FTIR) and ¹H nuclear magnetic resonance (NMR) spectra revealed the hydroxamate nature of the siderophore produced. Further characterization of the siderophore revealed it to be of dihydroxamate nature, forming hexadentate ligands with Fe(III) ions. A narrow shift in ultraviolet (UV)–Vis spectrum was observed on photolysis due to ligand oxidation. Growth-promotion bioassay with Aeromonas hydrophila, Staphylococcus aureus and E. coli confirmed the iron-scavenging property of the siderophore produced by Vibrio harveyi.     

The Effect of Clp Proteins on DnaK-Dependent Refolding of Bacterial Luciferases

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK–DnaJ–GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.     

Inhibition of <Emphasis Type="Italic">Vibrio</Emphasis> biofilm formation by a marine actinomycete strain A66

China remains by far the largest aquaculture producer in the world. However, biofilms formed by pathogenic Vibrio strains pose serious problems to marine aquaculture. To provide a strategy for biofilm prevention, control, and eradication, extracts from 88 marine actinomycetes were screened. Thirty-five inhibited the biofilm formation of Vibrio harveyi, Vibrio vulnificus, and Vibrio anguillarum at a concentration of 2.5% (v/v). Thirty-three of the actinomycete extracts dispersed the mature biofilm. Six extracts inhibited the quorum-sensing system of V. harveyi by attenuating the signal molecules N-acylated homoserine lactones’ activity. Strain A66, which was identified as Steptomyces albus, both attenuated the biofilms and inhibited their quorum-sensing system. It is suggested that strain A66 is a promising candidate to be used in future marine aquaculture.     

Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria

It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.Non-Standard Abbreviation AB medium autoinducer bioassay medium     

Photolyase Confers Resistance to UV Light but Does Not Contribute to the Symbiotic Benefit of Bioluminescence in Vibrio fischeri ES114

Recent reports suggest that the selective advantage of bioluminescence for bacteria is mediated by light-dependent stimulation of photolyase to repair DNA lesions. Despite evidence for this model, photolyase mutants have not been characterized in a naturally bioluminescent bacterium, nor has this hypothesis been tested in bioluminescent bacteria under natural conditions. We have now characterized the photolyase encoded by phr in the bioluminescent bacterium Vibrio fischeri ES114. Consistent with Phr possessing photolyase activity, phr conferred light-dependent resistance to UV light. However, upon comparing ES114 to a phr mutant and a dark ΔluxCDABEG mutant, we found that bioluminescence did not detectably affect photolyase-mediated resistance to UV light. Addition of the light-stimulating autoinducer N-3-oxo-hexanoyl homoserine lactone appeared to increase UV resistance, but this was independent of photolyase or bioluminescence. Moreover, although bioluminescence confers an advantage for V. fischeri during colonization of its natural host, Euprymna scolopes, the phr mutant colonized this host to the same level as the wild type. Taken together, our results indicate that at least in V. fischeri strain ES114, the benefits of bioluminescence during symbiotic colonization are not mediated by photolyase, and although some UV resistance mechanism may be coregulated with bioluminescence, we found no evidence that light production benefits cells by stimulating photolyase in this strain.     

The Iron-Dependent Regulator Fur Controls Pheromone Signaling Systems and Luminescence in the Squid Symbiont Vibrio fischeri ES114

Bacteria often use pheromones to coordinate group behaviors in specific environments. While high cell density is required for pheromones to achieve stimulatory levels, environmental cues can also influence pheromone accumulation and signaling. For the squid symbiont Vibrio fischeri ES114, bioluminescence requires pheromone-mediated regulation, and this signaling is induced in the host to a greater extent than in culture, even at an equivalent cell density. Our goal is to better understand this environment-specific control over pheromone signaling and bioluminescence. Previous work with V. fischeri MJ1 showed that iron limitation induces luminescence, and we recently found that ES114 encounters a low-iron environment in its host. Here we show that ES114 induces luminescence at lower cell density and achieves brighter luminescence in low-iron media. This iron-dependent effect on luminescence required ferric uptake regulator (Fur), which we propose influences two pheromone signaling master regulators, LitR and LuxR. Genetic and bioinformatic analyses suggested that under low-iron conditions, Fur-mediated repression of litR is relieved, enabling more LitR to perform its established role as an activator of luxR. Interestingly, Fur may similarly control the LitR homolog SmcR of Vibrio vulnificus. These results reveal an intriguing regulatory link between low-iron conditions, which are often encountered in host tissues, and pheromone-dependent master regulators.     

Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.     

Synthesis of reserve polyhydroxyalkanoates by luminescent bacteria

The ability of marine luminescent bacteria to synthesize polyesters of hydroxycarboxylic acids (polyhydroxyalkanoates, PHA) as reserve macromolecules was studied. Twenty strains from the collection of the luminescent bacteria CCIBSO (WDCM839) of the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, assigned to different taxa (Photobacterium leiognathi, Ph. phosphoreum, Vibrio harveyi, and V. fischeri) were analyzed. The most productive strains were identified, and the conditions ensuring high polymer yields in batch culture (40–70% of the cell dry mass weight) were determined. The capacity for synthesizing two-and three-component polymers containing hydroxybutyric acid as the main monomer and hydroxyvaleric and hydroxyhexanoic acids was revealed in Ph. leiognathi and V. harveyi strains. The results allow luminescent microorganisms to be regarded as new producers of multicomponent polyhydroxyalkanoates.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Complementation studies of the DnaK–DnaJ–GrpE chaperone machineries from <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>, both in vivo and in vitro

The marine bacterium Vibrio harveyi is a potential indicator organism for evaluating marine environmental pollution. The DnaK–DnaJ–GrpE chaperone machinery of V. harveyi has been studied as a model of response to stress conditions and compared to the Escherichia coli DnaK system. The genes encoding DnaK, DnaJ and GrpE of V. harveyi were cloned into expression vectors and grpE was sequenced. It was found that V. harveyi possesses a unique organization of the hsp gene cluster (grpE–gltP–dnaK–dnaJ), which is present exclusively in marine Vibrio species. In vivo experiments showed that suppression of the E. coli dnaK mutation by V. harveyi DnaK protein was weak or absent, while suppression of the dnaJ and grpE mutations by V. harveyi DnaJ and GrpE proteins was efficient. These results suggest higher species-specificity of the DnaK chaperone than the GrpE and DnaJ cochaperones. Proteins of the DnaK chaperone machinery of V. harveyi were purified to homogeneity and their efficient cooperation with the E. coli chaperones in the luciferase refolding reaction and in stimulation of DnaK ATPase activity was demonstrated. Compared to the E. coli system, the purified DnaK–DnaJ–GrpE system of V. harveyi exhibited about 20% lower chaperoning activity in the luciferase reactivation assay. ATPase activity of V. harveyi DnaK protein was at least twofold higher than that of the E. coli model DnaK but its stimulation by the cochaperones DnaJ and GrpE was significantly (10 times) weaker. These results indicate that, despite their high structural identity (approximately 80%) and similar mechanisms of action, the DnaK chaperones of closely related V. harveyi and E.coli bacteria differ functionally.     

Isolation of lytic bacteriophage against Vibrio harveyi

Aims: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Methods and Results: Water samples from discharge channels and grow‐out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage‐enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Conclusions: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Significance and Impact of the Study: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.