Dual functions of a 4-hydroxyphenylpyruvate dioxygenase for Vibrio splendidus survival and infection.

Vibrio splendidus is a well-documented pathogenic bacterium that can trigger different diseases, including skin ulcer syndrome in Apostichopus japonicus. In our previous study, a gene named Vshppd encoding a 4-hydroxyphenylpyruvate dioxygenase homologue was cloned from pathogenic V. splendidus, and validated to be responsible for the haemolysis activities of V. splendidus. In this study, Vshppd was determined to participate in the catabolism of tyrosine and promote pyomelanin production in Escherichia coli BL21 (DE3) harboring Vshppd. The purified melanin pigment displayed obvious antimicrobial activity against E. coli and Micrococcus luteus and protective effect on V. splendidus under ultraviolet irradiation. As an important virulence factor, Vshppd was further determined to be cytotoxic to the coelomocyte of A. japonicus and cell viability decreased to approximately 68%, 77%, 54% and 44% when 50, 60, 80 and 100 µL of purified rVshppd was present, respectively. To better understand the potential effect of Vshppd mediated oxidative stress, we injceted A. japonicus with the rVshppd, which showed significantly stimulatory effects on the expression of oxidative stress related genes catalase (cat), glutathione S-transferase (gst), glutathione peroxidase (gpx), heat shock protein 70 (hsp70) of A. japonicus. At 48 h, the expression level of cytochrome P450 (cyp450) was down-regulated compared with that treated with BSA. It was suggested that Vshppd exhibited cytotoxicity via altering the oxidative stress. Our result indicated that Vshppd was not only involved in the self-protection, but also contributed to the pathogenesis of V. splendidus by modulating the oxidative stress imbalance in A. japonicus.

Er:YAG Laser Irradiation Induces Behavioral Changes in V. harveyi.

OBJECTIVE: Laser technologies have many different applications in medical, agricultural, and industrial fields. Studies have shown several effects of laser energy on different bacterial species, in a wide variety of settings. Recent reports have found that one of the unique features of bacteria is their ability to communicate among themselves (quorum sensing). We sought to investigate whether low-energy laser irradiation affects bacterial behavior, which is regulated by quorum sensing. METHODS: Laser irradiations were performed using Er:YAG laser (2940 nm wavelength) at output powers of 0.5, 1.5, 2.5, and 4 W on wild-type Vibrio harveyi. Bioluminescence, motility, and biofilm forming capability were assessed on the bacteria after irradiation. RESULTS: After irradiation of bacteria, positive dose/output power dependencies were found in the bioluminescence omitted from tested experimental groups. Motility of colonies on semi-solid media was inhibited as irradiation output power was increased. However, after irradiation, biomass analysis of biofilm samples showed negligible differences between the irradiated samples and controls. CONCLUSIONS: Results indicate the impact of low-energy laser irradiation on bacterial behavior such as quorum sensing and motility, without affecting bacterial growth patterns.

Ultraviolet irradiation is an effective alternative to ozonation as a sea water treatment to prevent Kudoa neurophila (Myxozoa: Myxosporea) infection of striped trumpeter, Latris lineata (Forster).

Myxozoan parasites are known pathogens of cultured finfish. Kudoa neurophila n. comb. (Grossel, Dyková, Handlinger & Munday) has historically infected hatchery-produced striped trumpeter, Latris lineata (Forster in Bloch and Schneider), a candidate species for seacage aquaculture in Australia. We examined the efficacy of four water treatment methods to prevent K. neurophila infection in post-larval (paperfish) and juvenile striped trumpeter. Treatments included dose-controlled ultraviolet irradiation [hydro-optic disinfection (HOD)], ozone with conventional UV (ozone), mechanical filtration at 25 µm and then foam fractionation (primary filtration), and 50-µm-filtered sea water (control). In post-larvae (initially 10.3 ± 2.7 g, mean ± SD, 259 days post-hatching, dph), the infection prevalence (PCR test) after 51 days was 93 ± 12% in the control, 100 ± 0% in primary filtration and 0 ± 0% in both ozone and HOD. Likewise, in juveniles (initially 114 ± 18 g, 428 dph), prevalence was 100 ± 0% in the control and primary filtration treatments with no infection detected in ozone and HOD. Concurrently, there was a 50-100% reduction in heterotrophic bacteria and 100% reduction in presumptive Vibrio sp. in sea water HOD and ozone treatments. HOD with a dose of ≥44 mJ cm(-2) UV was as effective as ozonation at >700 mV ORP for 10 min, in preventing K. neurophila infection.

Function and regulation of Vibrio campbellii proteorhodopsin: acquired phototrophy in a classical organoheterotroph.

Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii. Pigmentation analysis, absorption spectroscopy and photoinduction assays using a heterologous over-expression system established the V. campbellii PR as a functional green light absorbing proton pump. In situ analyses comparing PR expression and function in wild type (WT) V. campbellii with an isogenic ΔpR deletion mutant revealed a marked absence of PR membrane localization, pigmentation and light-induced proton pumping in the ΔpR mutant. Comparative photoinduction assays demonstrated the distinct upregulation of pR expression in the presence of light and PR-mediated photophosphorylation in WT cells that resulted in the enhancement of cellular survival during respiratory stress. In addition, we demonstrate that the master regulator of adaptive stress response and stationary phase, RpoS1, positively regulates pR expression and PR holoprotein pigmentation. Taken together, the results demonstrate facultative phototrophy in a classical marine organoheterotrophic Vibrio species and provide a salient example of how this organism has exploited lateral gene transfer to further its adaptation to the photic zone.

Reducing Vibrio load in Artemia nauplii using antimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture.

We propose antimicrobial photodynamic therapy (aPDT) as an alternative strategy to reduce the use of antibiotics in shrimp larviculture systems. The growth of a multiple antibiotic resistant Vibrio harveyi strain was effectively controlled by treating the cells with Rose Bengal and photosensitizing for 30 min using a halogen lamp. This resulted in the death of >50% of the cells within the first 10 min of exposure and the 50% reduction in the cell wall integrity after 30 min could be attributed to the destruction of outer membrane protein of V. harveyi by reactive oxygen intermediates produced during the photosensitization. Further, mesocosm experiments with V. harveyi and Artemia nauplii demonstrated that in 30 min, the aPDT could kill 78.9% and 91.2% of heterotrophic bacterial and Vibrio population respectively. In conclusion, the study demonstrated that aPDT with its rapid action and as yet unreported resistance development possibilities could be a propitious strategy to reduce the use of antibiotics in shrimp larviculture systems and thereby, avoid their hazardous effects on human health and the ecosystem at large.

Prodigiosin from Vibrio sp. DSM 14379; a new UV-protective pigment.

Pigments such as melanin, scytonemin and carotenoids protect microbial cells against the harmful effects of ultraviolet (UV) radiation. The role in UV protection has never been assigned to the prodigiosin pigment. In this work, we demonstrate that prodigiosin provides a significant level of protection against UV stress in Vibrio sp. DSM 14379. In the absence of pigment production, Vibrio sp. was significantly more susceptible to UV stress, and there was no difference in UV survival between the wild-type strain and non-pigmented mutant. The pigment's protective role was more important at higher doses of UV irradiation and correlated with pigment concentration in the cell. Pigmented cells survived high UV exposure (324 J/m(2)) around 1,000-fold more successfully compared to the non-pigmented mutant cells. Resistance to UV stress was conferred to the non-pigmented mutant by addition of exogenous pigment extract to the growth medium. A level of UV protection equivalent to that exhibited by the wild-type strain was attained by the non-pigmented mutant once the prodigiosin concentration had reached comparable levels to those found in the wild-type strain. In co-culture experiments, prodigiosin acted as a UV screen, protecting both the wild-type and non-pigmented mutants. Our results suggest a new ecophysiological role for prodigiosin.

Effects of combined treatment of sodium hypochlorite/ionizing radiation and addition of vitamin B(1) on microbial flora of oyster and short-necked clam.

To determine the synergistic disinfection effect of the combined treatments of sodium hypochlorite (NaClO), irradiation, and vitamin B(1), the bactericidal effects of the treatments on natural microflora of oyster and short-necked clam were investigated. Then, bacteria isolated from the samples were identified by 16S rDNA sequencing. Oyster and short-necked clam were mainly contaminated with Vibrio spp. and Bacillus spp. Total number of aerobic bacteria ranged from 10(2) to 10(4) colony forming units (CFU)/g initially. More than 100 mg/L of NaClO with 1000 mg/L vitamin B(1) and 2 kGy irradiation treatment for oyster and short-necked clam can reduce the total aerobic bacteria to the level of lower than a detection limit (10 CFU/g). Synergistic effects were observed for all combined treatment against natural microflora. The results suggest that a significant synergistic benefit can be achieved by a combination of NaClO-ionizing radiation treatment with the addition of vitamin B(1) to reduce the microbial population contaminated in oyster and short-necked clam.

Osmoadaptation among Vibrio species and unique genomic features and physiological responses of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a moderately halophilic bacterium found in estuarine and marine coastal ecosystems worldwide. Although the ability of V. parahaemolyticus to grow and proliferate in fluctuating saline environments is well known, the underlying molecular mechanisms of osmoadaptation are unknown. We performed an in silico analysis of V. parahaemolyticus strain RIMD2210633 for genes homologous to osmotic stress response genes in other bacteria. We uncovered two putative compatible solute synthesis systems (encoded by ectABC and betABI) and six putative compatible solute transporters (encoded by four bcct loci and two proVWX loci). An ectoine synthesis system clustered with a betaine/carnitine/choline transporter and a ProU transporter (encoded by homologues of proVWX from Escherichia coli), and a betaine synthesis system clustered with a ProU transporter (encoded by homologues of proVXW from Pseudomonas syringae). This is at least double the number present in V. cholerae, V. fischeri, or V. vulnificus. Six additional Vibrio species contain both ectABC and betABI, i.e., V. alginolyticus 12G01, V. angustum, V. harveyi BAA-1116, V. splendidus LGP32, Vibrio sp. strain MED222, and Vibrio sp. strain Ex25. V. harveyi HY01 and V. splendidus 12B01 only encoded the betaine system. In addition, V. alginolyticus had a compendium of systems identical to that found in V. parahaemolyticus. Comparative physiological analysis of RIMD2210633 with V. vulnificus YJ016, V. cholerae N16961, and V. fischeri ES114 grown at different salinities and temperatures demonstrated that V. parahaemolyticus had a growth advantage under all of the conditions examined. We demonstrate, by one-dimensional nuclear magnetic resonance analysis, that V. parahaemolyticus is capable of de novo synthesis of ectoine at high salinity whereas a Delta ectB knockout strain is not. We constructed a single-knockout mutation in proU1, but no growth defect was noted, indicating transporter system redundancy. We complemented E. coli MKH13, a compatible solute transporter-negative strain, with bcct2 and demonstrated uptake of betaine at high salt concentrations.

Physiological alteration of the marine bacterium Vibrio angustum S14 exposed to simulated sunlight during growth.

Growth experiments on the marine bacterium Vibrio angustum S14 were conducted under four light conditions using a solar simulator: visible light (V), V + ultraviolet A (UV-A), V + UV-A + UV-B radiation, and dark. Growth was inhibited mainly by UV-B and slightly by UV-A. UV-B radiation induced filaments containing multiple genome copies with low cyclobutane pyrimidine dimers. These cells did not show modifications in cellular fatty acid composition in comparison with dark control cultures and decreased in size by division after subsequent incubation in the dark. A large portion of the bacterial population grown under visible light showed an alteration in cellular DNA fluorescence as measured by flow cytometry after SYBR-Green I staining. This alteration was not aggravated by UV-A and was certainly due to a change in DNA topology rather than DNA deterioration because all the cells remained viable and their growth was not impaired. Ecological consequences of these observations are discussed.

RpoS induces expression of the Vibrio anguillarum quorum-sensing regulator VanT.

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.

Effects of Ultraviolet Radiation on the Gram-positive marine bacterium Microbacterium maritypicum.

Although extensive information is available on the effect ultraviolet (UV) radiation has on Gram-negative marine bacteria, there is a scarcity of data concerning UV radiation and Gram-positive marine bacteria. The focus of this paper is on Microbacterium maritypicum, with the Gram-negative Vibrio natriegens being used as a standard of comparison. M. maritypicum exhibited growth over a NaCl range of 0-1000 mM: , with optimum growth occurring between 0 and 400 mM: NaCl. In contrast, V. natriegens grew over a NaCl span of 250-1000 mM: , with best growth being observed between 250 and 600 mM: NaCl. UV radiation experiments were done using the medium with 250 mM: NaCl. For solar (UV-A and B) radiation and log-phase cells, M. maritypicum was determined to be three times more resistant than V. natriegens. For germicidal (UV-C) radiation, the pattern of resistance of the log-phase cells to the lethal effects of the radiation was even more pronounced, with the Gram-positive bacterium being more than 12 to 13 times more resistant. Similar data to the solar and germicidal log-phase UV kill curves were obtained for stationary-phase cells of both organisms. Photoreactivation was observed for both types of cells exposed to UV-C but none for cells treated with UV-A and B. When log phase cells of M.maritypicum were grown at 0.0 and 0.6 M: NaCl and exposed to UV-C radiation, no difference in survivorship patterns was noted from that of 0.25 M: NaCl grown cells. Although this study has only focused on two marine bacteria, our results indicate that the Gram-positive M. maritypicum could have a built-in advantage for survival in some marine ecosystems.

A furanosyl-carbonate autoinducer in cell-to-cell communication of V. harveyi.

An autoinducer arising from reaction of cyclized S-DPD and carbonate is shown to induce light in V. harveyi and thus may play a previously unknown role in quorum sensing.

Irradiation in the production, processing, and handling of food. Final rule.

The Food and Drug Administration (FDA) is amending the food additive regulations to provide for the safe use of ionizing radiation for control of Vibrio species and other foodborne pathogens in fresh or frozen molluscan shellfish (e.g., oysters, mussels, clams, etc.). This action is in response to a petition filed by the National Fisheries Institute and the Louisiana Department of Agriculture and Forestry.

Involvement of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi.

CgtA is a member of the Obg/Gtp1 subfamily of small GTP-binding proteins. CgtA homologues have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Nevertheless, despite the fact that cgtA is an essential gene in most bacterial species, its function in the regulation of cellular processes is largely unknown. Here it has been demonstrated that in two bacterial species, Escherichia coli and Vibrio harveyi, the cgtA gene product enhances survival of cells after UV irradiation. Expression of the cgtA gene was found to be enhanced after UV irradiation of both E. coli and V. harveyi. Moderate overexpression of cgtA resulted in higher UV resistance of E. coli wild-type and dnaQ strains, but not in uvrA, uvrB, umuC and recA mutant hosts. Overexpression of the E. coli recA gene in the V. harveyi cgtA mutant, which is very sensitive to UV light, restored the level of survival of UV-irradiated cells to the levels observed for wild-type bacteria. Moreover, the basal level of the RecA protein was lower in a temperature-sensitive cgtA mutant of E. coli than in the cgtA(+) strain, and contrary to wild-type bacteria, no significant increase in recA gene expression was observed after UV irradiation of this cgtA mutant. Finally, stimulation of uvrB gene transcription under these conditions was impaired in the V. harveyi cgtA mutant. All these results strongly suggest that the cgtA gene product is involved in DNA repair processes, most probably by stimulation of recA gene expression and resultant activation of RecA-dependent DNA repair pathways.

Stimulation of DNA repair as an evolutionary drive for bacterial luminescence.

It was demonstrated recently that luminescence of a free-living marine bacterium, Vibrio harveyi, stimulates DNA repair, most probably by activation of the photoreactivation process. Here, we ask whether the stimulation of DNA repair could be an evolutionary drive that ensured maintenance and development of early bacterial luminescent systems. To test this hypothesis, we cultivated V. harveyi lux(+) bacteria and luxA mutants in mixed cultures. Initial cultures were mixed to obtain a culture consisting of roughly 50% lux(+) cells and 50% luxA mutants. Then bacteria were cultivated for several days and ratio of luminescent to dark bacteria was measured. Under these conditions, luxA mutants became highly predominant within a few days of cultivation. This indicates that, without a selective pressure, the luminescence is a disadvantage for bacteria, perhaps due to consumption of significant portion of cell energy. However, when the same experiments were repeated but cultures were irradiated with low UV doses, luminescent bacteria started to predominate shortly after the irradiation. Therefore, we conclude that stimulation of photoreactivation may be an evolutionary drive for bacterial bioluminescence.

Induction of light emission by luminescent bacteria treated with UV light and chemical mutagens.

Intensity of light emission by luminescent bacteria in response to UV irradiation and chemical mutagens was tested. We demonstrated that luminescence of six strains of marine bacteria (belonging to four species: Photobacterium leiognathi, P. phosphoreum, Vibrio fischeri and V. harveyi) is significantly increased by UV irradiation relatively shortly after dilution of cultures. Such a stimulation of luminescence was abolished in cells treated with chloramphenicol 15 min before UV irradiation, indicating that effective gene expression is necessary for UV-mediated induction of light emission. These results suggest that stimulation of luminescence in UV-irradiated bacterial cells may operate independently of the quorum sensing regulation. A significant induction of luminescence was also observed upon treatment of diluted cultures of all investigated strains with chemical mutagens: sodium azide (SA), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine x 2HCl (ICR-191), 4-nitro-o-phenylenediamine (NPD), 4-nitroquinolone-N-oxide (NQNO), 2-aminofluorene (2-AF), and benzo[alpha]pyrene. These results support the proposal that genes involved in bioluminescence belong to the SOS regulon. The use of bacterial luminescence systems in assays for detection of mutagenic compounds is discussed in the light of this proposal.

Impact of irradiation and polycyclic aromatic hydrocarbon spiking on microbial populations in marine sediment for future aging and biodegradability studies.

Experiments were carried out to develop methods to generate well-characterized, polycyclic aromatic hydrocarbon (PAH)-spiked, aged but minimally altered sediments for fate, biodegradation, and bioavailability experiments. Changes in indigenous bacterial populations were monitored in mesocosms constructed of relatively clean San Diego Bay sediments, with and without exposure to gamma radiation, and then spiked with five different PAHs and hexadecane. While phenanthrene and chrysene degraders were present in the unspiked sediments and increased during handling, PAH spiking of nonirradiated sediments led to dramatic increases in their numbers. Phenotypic characterization of isolates able to grow on phenanthrene or chrysene placed them in several genera of marine bacteria: Vibrio, Marinobacter or Cycloclasticus, Pseudoalteromonas, Marinomonas, and HALOMONAS: This is the first time that marine PAH degraders have been identified as the latter two genera, expanding the diversity of marine bacteria with this ability. Even at the highest irradiation dose (10 megarads), heterotrophs and endospore formers reappeared within weeks. However, while bacteria from the unirradiated sediments had the capacity to both grow on and mineralize 14C-labeled phenanthrene and chrysene, irradiation prevented the reappearance of PAH degraders for up to 4 months, allowing spikes to age onto the sediments, which can be used to model biodegradation in marine sediments.

Effects of potassium halides on bacterial bioluminescence.

The effects of potassium halides KCl, KBr and KI on NADH:FMN-oxidoreductase-luciferase bioluminescent coupled enzyme system were studied. The influence of salt additions on bioluminescence intensity and bioluminescence light yield was investigated. The inhibition and activation parameters of the salts were calculated using their dependencies on concentration of the salts. The correlation between the inhibition of bioluminescence intensity and the halide mass was demonstrated: the inhibiting ability of the salts increases with the increase of atomic weight of the anions. The inhibition parameters increase and the activation parameters decrease, accordingly.

Role of spoT-dependent ppGpp accumulation in the survival of light-exposed starved bacteria.

In bacteria, cytoplasmic levels of the effector nucleotide ppGpp are regulated in response to changes in growth conditions. This study describes the involvement of SpoT-mediated ppGpp accumulation in the survival of light-exposed bacteria during fatty acid starvation. In contrast to isogenic wild-type strains and relA mutants, the 'Vibrio angustum' S14 spoT and Escherichia coli relA spoT mutants displayed significant losses in viability in response to cerulenin-induced fatty acid starvation under cool-white fluorescent light. However, when starvation experiments were performed in complete darkness, or under light filtered through a UV-resistant perspex sheet, only a minor decline in viability was observed for the wild-type and mutant strains. This finding indicated that the lethal effect was mediated by weak UV emission. In contrast to the E. coli relA spoT mutant, which lacks ppGpp, the 'V. angustum' S14 spoT mutant exhibited higher ppGpp levels and lower RNA synthesis rates during fatty acid starvation, features that might be correlated with its lethality. In agreement with this finding, fatty acid starvation lethality also occurred upon induction of ppGpp overaccumulation in E. coli. These data suggest that the precise regulation of ppGpp levels in the stressed cell is crucial, and that both the absence and the overaccumulation of ppGpp impair fatty acid starvation survival of light-exposed cells. Moreover, the UV-induced lethal effect during fatty acid starvation was also observed for E. coli strains mutated in rpoS and dps, which, in the wild-type, are regulated directly or indirectly by ppGpp, respectively. The restoration of viability of fatty-acid-starved spoT mutant cells through the addition of exogenous catalase suggested that the observed light-dependent lethal effect was, at least in part, caused by UV-imposed oxidative stress. Based on these results, it is proposed that fatty acid starvation adaptation of light-exposed bacterial cells depends on the development of resistance to UV-induced oxidative stress. This stress resistance was found to require appropriate ppGpp levels, ppGpp-induced RpoS expression and, hence, upregulation of RpoS-regulated stress-defending genes, such as dps.

Vibrio harveyi bioluminescence plays a role in stimulation of DNA repair.

Although the genetics and biochemistry of bacterial luminescence have been investigated extensively, the biological role of this phenomenon remains unclear. Here it is shown that luxA, luxB and luxD mutants (unable to emit light) of the marine bacterium Vibrio harveyi are significantly more sensitive to UV irradiation when cultivated in the dark after irradiation than when cultivated under a white fluorescent lamp. This difference was much less pronounced in the wild-type (luminescent) V. harveyi strain. Survival of UV-irradiated Escherichia coli wild-type cells depended on subsequent cultivation conditions (in the dark or in the presence of external light). However, after UV irradiation, the percentage of surviving E. coli cells that bear V. harveyi genes responsible for luminescence was significantly higher than that of non-luminescent E. coli, irrespective of the subsequent cultivation conditions. Moreover, it is demonstrated that luminescence of V. harveyi can be stimulated by UV irradiation even in diluted cultures, under conditions when light emission by these bacteria is normally impaired due to quorum sensing regulation. It is proposed that luminescent bacteria have an internal source of light which could be used in DNA repair by a photoreactivation process. Therefore, production of internal light ensuring effective DNA repair seems to be at least one of the biological functions of bacterial luminescence.