Isolation and characterization of hemolysin mutants of Vibrio vulnificus

Abstract The importance of the cytolysin/hemolysin in the virulence of Vibrio vulnificus was investigated using both the naturally occuring virulent and avirulent colony variants and ethylmethane-sulfonate generated mutants. Both virulent and avirulent isogenic morphotypes produced similar amounts of hemolysin. Two mutants deficient in the production of hemolysin and negative for CHO cell activity were characterized and their virulence for mice was examined. Non-hemolytic mutants were found to be as virulent as their parent strain. It is concluded that the hemolysin produced by V. vulnificus is not required for the full virulence of this pathogen.     

Isolation and characterization of chitinase from a marine bacterium Vibrio sp.

A chitinase was separated from the culture broth of Vibrio sp. 11211 isolated from sediment from the South China Sea. The chitinase was purified 18.3-fold with 33% recovery by ammonium sulphate precipitation and chromatography. The subunit molecular weight of the enzyme was estimated by SDS-PAGE to be about 30kDa. The enzyme showed optimum pH at 6.5 and optimum temperature at 50^°C, and was stable in the pH range of 4 to 9 and at the temperature below 40^°C.     

Purification and characterization of a secreted protease from the pathogenic marine bacterium Vibrio anguillarum

Vibrio anguillarum is a pathogenic marine bacterium which causes the disease vibriosis in salmonid fish, which is characterized by a fatal hemorrhagic septicemia accompanied by massive tissue destruction. In this paper, the purification of the major caseinolytic extracellular protease from V. anguillarum is presented. The purification steps include ammonium sulfate precipitation, DEAE-Sepharose chromatography, Sephacryl S-200 chromatography, and DEAE high-pressure liquid chromatography. The purified protease migrates with Mr = 38,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A slightly larger protease of Mr 40,000 is also separated by this procedure, but accounts for only a minor fraction of the caseinolytic activity. The Mr 38,000 protease displays a broad pH activity profile in the neutral to basic range. It is not inhibited by serine, cysteine, or acid protease inhibitors, but is inhibited by EDTA and 1,10-phenanthroline, suggesting that it is a metalloprotease. The activity of the EDTA-inactivated protease could be partially restored by the addition of Ca2+ and Zn2+ together. The molecular weight and inhibition data show some similarities with proteases isolated from other Vibrio species such as Vibrio cholerae and Vibrio vulnificus.     

Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus

The viable but nonculturable (VBNC) suppression mutant formed platable cells at low temperature stress after inoculation in artificial seawater (ASW). Suppression subtractive hybridization was used to identify differentially expressed genes among cDNAs of the VBNC suppression mutant and the wild-type Vibrio vulnificus strain. Glutathione S-transferase was identified as a responsive gene of the VBNC suppression mutant in our assay, and was highly expressed from the VBNC suppression mutant at low temperature stress. Culturability tests revealed that the wild-type cells were sensitive to oxidative stress in the hydrogen peroxide (H(2)O(2)) and to 1-chloro-2,4-dinitrobenzene (CDNB) compared with the VBNC suppression mutant cells. Adding glutathione showed that many wild-type V. vulnificus cells maintained culturability in cold ASW. These results suggest that non-nutritional growth inhibitors, such as peroxide that accumulates at low temperatures, influence VBNC in V. vulnificus cells.     

Antacid increases survival of Vibrio vulnificus and Vibrio vulnificus phage in a gastrointestinal model.

Viable counts of three strains of Vibrio vulnificus and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37 degrees C. V. vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5). Viable V. vulnificus cells delivered from the gastric compartment during the first 30 min of exposure reached 10(6) to 10(8) CFU/ml in the intestinal compartment after 9 h (pH 7.0). Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5). Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0). When the gastric compartment contained antacid V. vulnificus counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 10(7) to 10(9) CFU/ml. Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h. Presence of antacid in the gastric compartment of the model greatly increased the ability of both V. vulnificus and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness.     

Isolation and preliminary characterization of bacteriocins produced by Vibrio vulnificus

AIMS: The aim of this work was to isolate bacteriocins from the environment that would be effective in neutralizing Vibrio vulnificus in seafood. METHODS AND RESULTS: Water samples from Wilmington (NC, USA) were plated to determine total viable counts and to isolate presumptive Vibrio spp. Isolates containing plasmids were checked for antimicrobial activity which was not due to lytic bacteriophage or small, non-specific molecules. Three bacteriocin producers were detected and their inhibitory spectra determined: IW1 inhibited few strains of V. vulnificus; BC1 inhibited several strains of V. vulnificus, V. cholerae and V. parahaemolyticus and BC2 inhibited all tested Vibrio spp., Plesiomonas shigelloides and Escherichia coli. Loss of inhibitory activity coincided with loss of the bacteriocinogenic plasmid. The bacteriocins were found to be between 1.3 and 9.0 kDa. IW1 was heat labile, while BC1 was moderately stable except at extreme temperatures. BC2 was very stable and maintained its activity when frozen, autoclaved or exposed to extreme pH values. CONCLUSIONS: Bacteriocins have been isolated from environmental isolates of V. vulnificus and V. cholerae. BC2, with its broad spectrum and stability, may be useful in neutralizing V. vulnificus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to reducing the occurrence of food poisoning caused by V. vulnificus.     

Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus.

In this study, we have reevaluated the taxonomic position of biotype 2 of Vibrio vulnificus. For this purpose, we have biochemically and serologically characterized 83 biotype 2 strains from diseased eels, comparing them with 17 biotype 1 strains from different sources. Selected strains were also molecularly analyzed and tested for eel and mouse pathogenicity. Results have shown that biotype 2 (i) is biochemically homogeneous, indole production being the main trait that distinguishes it from biotype 1, (ii) presents small variations in DNA restriction profiles and outer membrane protein patterns, some proteins being immunologically related to outer membrane proteins from biotype 1, (iii) expresses a common lipopolysaccharide (LPS) profile, which is immunologically identical among strains and distinct from that of LPS of tested biotype 1 strains, and (iv) contains at least two high-Mr plasmids. Regarding host range, we have confirmed that both biotypes are pathogenic for mice but only biotype 2 is pathogenic for eels. On the basis of these data, we propose that biotype 2 of V. vulnificus constitutes an LPS-based O serogroup which is phenotypically homogeneous and pathogenic for eels. In this article, the serogroup is designated serogroup E (for eels).     

Cloning and characterization of a blue fluorescent protein from Vibrio vulnificus

The blue fluorescent protein (BFPVV) gene bfpvv from Vibrio vulnificus CKM-1 was cloned and sequenced. The transformants exhibited blue fluorescence when irradiated by UV source. Nucleotide sequence analysis predicted an ORF of 717 bp encoding a 239-amino-acid polypeptide with a calculated molecular mass of 25.8 kDa. The nucleotide sequence of the bfpvv gene and its deduced amino acid sequence showed significant homology to those of the short-chain dehydrogenase/reductase (SDR) family proteins from various organisms. Some functionally important residues in SDR were strictly conserved in BFPVV, such as an active-site Tyr145, a catalytic site Lys149, and a common GlyXXXGlyXGly pattern in the N-terminal part of the molecule. By changing three amino acid residues, Tyr145, Lys149, and Gly9 to Phe, Ile, and Val, respectively, it was found that the G9V mutant did not generate blue fluorescence, while mutants Y145F and K149I have 126 and 68.5% fluorescence compared with the wild-type BFPVV.     

Isolation and Characterization of Vibrio vulnificus from Two Florida Estuaries

Vibrio vulnificus was enumerated in seawater and shellfish from two Florida estuaries at selected seasonal intervals. There were significant fluctuations in the presence and numbers of V. vulnificus. Relatively high seawater temperature and salinity favored the presence of V. vulnificus in both seawater and shellfish samples.     

Identification of the cadBA operon from Vibrio vulnificus and its influence on survival to acid stress

By a transposon-tagging method, cadBA genes encoding a lysine/cadaverin antiporter and a lysine decarboxylase were identified and cloned from Vibrio vulnificus. The deduced amino acid sequences of cadBA were 64-97% similar to those reported from other Enterobacteriaceae. Functions of cadBA genes on acid tolerance were assessed by comparing acid tolerances of V. vulnificus and its isogenic mutants, whose cadBA genes were separately inactivated by allelic exchanges. The results demonstrated that gene products of cadBA contribute to acid tolerance of V. vulnificus, and that their contribution is dependent on prior exposure of cells to moderately acidic pH.     

Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan

The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.     

一株锰氧化细菌的分离、鉴定及其锰氧化特性

【目的】获得锰氧化细菌,对锰矿周边土壤中生物所参与的锰氧化过程进行初探。【方法】依据细菌是否能氧化Mn(Ⅱ),形成棕褐色锰氧化物进行筛选。利用染料LBB对生成的锰氧化物进行检测。通过考察分离菌株的形态、生理特征和16S r RNA基因、gyr B基因、gyr A基因序列的同源性对分离菌株进行鉴定。分析筛选菌与所在属已知锰氧化菌的亲缘关系。利用LBB显色法检测氧化锰的动态生成,通过扫描电镜-能谱分析和X射线衍射技术分析生物氧化锰的表征。【结果】获得1株锰氧化细菌菌株,命名为CP133,综合形态、生理及分子分析结果,鉴定为蜡样芽孢杆菌(Bacillus cereus),分离菌株与多株分离自海洋及土壤的芽孢类锰氧化菌在进化上具有一定的差异。与其他菌株比较菌株CP133具有较强的锰氧化能力,进入稳定期后可生成紧密结合在菌体周围的无定形态生物氧化锰。【结论】从锰矿周边土壤分离出1株具有较强锰氧化功能的蜡样芽孢杆菌,丰富了土壤芽孢类锰氧化菌的资源,同时也为锰矿周围土壤与锰氧化菌间的生物地球化学循环提供了线索及材料。     

Isolation and characterization of actinomycetes antagonistic to pathogenic Vibrio spp. from nearshore marine sediments

Summary A total of 94 actinomycete strains were isolated from the marine sediments of a shrimp farm, 87.2% belonged to the genus Streptomyces, others were Micromonospora spp. Fifty-one percent of the actinomycete strains showed activity against the pathogenic Vibrio spp. strains. Thirty-eight percent of marine Streptomyces strains produced siderophores on chrome azurol S (CAS) agar plates. Seven strains of Streptomyces were found to produce siderophores and to inhibit the growth of Vibrio spp. in vitro. Two of them belonged to the Cinerogriseus group, the most frequently isolated group of Streptomyces. The results showed that streptomycetes could be a promising source for biocontrol agents in aquaculture.     

Improved Isolation of Vibrio vulnificus from Seawater and Sediment with Cellobiose-Colistin Agar

An improved selective medium, cellobiose-colistin (CC) agar, gave a significantly higher (P < 0.05) isolation rate of Vibrio vulnificus from water and sediment samples than did modified cellobiose-polymyxin B-colistin (mCPC) agar. In a total of 446 alkaline peptone water preenrichments amended with polymyxin B, V. vulnificus was isolated from 154 preenrichments (35%) with mCPC agar and from 179 preenrichments (40%) with CC agar. CC agar gave a higher plating efficiency of V. vulnificus cells than did cellobiose-polymyxin B-colistin (CPC) agar, mCPC agar, or thiosulfate-citrate-bile salts-sucrose (TCBS) agar; the only significant difference was observed with TCBS agar, which gave much lower plating efficiencies than the other selective media. Determination of MICs demonstrated that the concentrations of colistin and polymyxin B in CPC agar inhibit growth of a proportion of V. vulnificus strains.     

Vibrio vulnificus RTX toxin plays an important role in the apoptotic death of human intestinal epithelial cells exposed to Vibrio vulnificus

During Vibrio vulnificus infection, V. vulnificus reaches the intestine and then invades the bloodstream by crossing the intestinal mucosal barrier of the host, which results in systemic septicemia. Previously, we reported that the RtxA toxin secreted through the RtxE transporter contributes to the cytotoxicity of V. vulnificus against intestinal epithelial cells. Here, we used gene mutants of rtxE and rtxA to determine the role that V. vulnificus RtxA toxin plays in the apoptotic death of human intestinal epithelial cells. The levels of DNA fragmentation were lower in human epithelial cells infected with an rtxE mutant of V. vulnificus than in those that were infected with the wild type. In addition, the rtxE mutant was found to induce lower levels of TUNEL positive cells and cell cycle arrest at the subG(1) than the wild type V. vulnificus. Furthermore, the decreased levels of DNA fragmentation, TUNEL positive cells and subG(1) arrest by the rtxE gene mutation were restored by the complementation of an rtxE gene into the rtxE mutant V. vulnificus. Finally, the rtxA mutant induced significantly lower levels of apoptotic cell death than the wild type. The levels of the PARP, cytochrome c, caspase-3, and mitochondrial membrane depolarization were lower in human epithelial cells infected with the rtxE and rtxA mutants, compared with the wild type and rtxE gene-complemented strains of V. vulnificus. Taken together, these results indicate that V. vulnificus RtxA toxin induces the apoptotic death through a mitochondria-dependent pathway in human intestinal epithelial cells exposed to V. vulnificus.     

Purification and characterization of a catalase from the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 and its role in the oxidative stress response

When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth. Upon preincubation with 100 μM H₂O₂, catalase activity increased fivefold. Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol treatment, and stationary-phase conditions. Only one band of catalase activity was detected after native and denaturing PAGE. The enzyme was purified 304-fold with a yield of 7%. The purified enzyme displayed a heterodimeric structure with subunits of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme. The subunits had almost identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases. The enzyme exhibited an apparent K _m of 40 mM and a V _max of 285,000 U (mg protein)^–1. Spectroscopic analysis indicated the presence of protoheme IX. The heme content calculated from pyridine hemochrome spectra was 0.43 mol per mol of enzyme. The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol, and sodium dithionite. These data indicate that this catalase belongs to the class of monofunctional catalases. Received: 15 October 1997 / Accepted: 2 February 1998