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.

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.

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.

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.

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.

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.

Studies on the mechanism of bacteria photosensitization by meso-substituted cationic porphyrins.

Cationic porphyrins have been shown to photoinduce the direct inactivation of Gram-positive (G+) and Gram-negative (G-) bacteria, thereby differing from anionic or neutral porphyrins which can photosensitize the G- bacteria only after permeabilization of their outer membrane. The present data show that the differences between these positively and negatively charged porphyrins are not related by a difference in the intrinsic photosensitizing efficiency, as determined by the photo-oxidation of model substrates or the yield of 1O2 generation; moreover, there are only minor differences in the quantum yield of porphyrin photobleaching. Rather, it appears that the positive charge promotes an electrostatic binding of the porphyrin to the outer cell surface inducing an initial limited damage which favours the penetration of the photosensitizer. Actually, the overall photoprocess is inhibited by the preincorporation of the porphyrin into liposomes, while it is enhanced by using amphiphilic dicationic porphyrins which bind to endocellular sites in larger amounts and in a more stable form.

Meso-substituted cationic porphyrins as efficient photosensitizers of gram-positive and gram-negative bacteria.

Previous studies on the photosensitization of bacterial cells with different neutral or negatively charged porphyrins and phthalocyanines have demonstrated that, although Gram-positive bacteria are efficiently photoinactivated, Gram-negative bacteria become photosensitive only after modification of the permeability of their outer membrane. The results described in this paper show that two meso-substituted cationic porphyrins, namely tetra(4N-methyl-pyridyl)porphine tetraiodide and tetra(4N,N,N-trimethyl-anilinium)porphine, efficiently photosensitize the inactivation of Gram-negative bacteria, such as Vibrio anguillarum and Escherichia coli. A negatively charged meso-substituted porphyrin, tetra(4-sulphonatophenyl)porphine, has no appreciable photosensitizing activity towards Gram-negative bacteria, although all three porphyrins exhibit a similar subcellular distribution pattern, being mainly localized in the protoplasts or spheroplasts. Moreover, the three porphyrins show similar efficiency in the photoinactivation of the Gram-positive bacterium Entorecoccus seriolicida.

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.

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.

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.