Analysis of the gene of Vibrio hollisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus

The gene (designated as Vh-tdh) of Vibrio hollisae 9041 encoding a hemolysin similar to the thermostable direct hemolysin (TDH) of V. parahaemolyticus contained a 567-base-pair open reading frame (ORF), which was 93.3-93.5% homologous to those of the tdh genes of V. parahaemolyticus, V. cholerae non-01, and V. mimicus encoding TDH or similar hemolysins. Comparative analysis of the nucleotide sequence containing the Vh-tdh ORF with published nucleotide and amino acid sequences suggested that the Vh-tdh gene and other tdh genes diverged from a common ancestral gene, that the divergence was closely associated with the evolutionary divergence of V. hollisae from other species of genus Vibrio, and that strain-to-strain variation of the Vh-tdh gene exists in V. hollisae.     

Expression, characterization and immunogenicity of a major outer membrane protein from Vibrio alginolyticus

Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals.During infection and induction of the host immune response,outer membrane proteins of bacteria play animportant role.In this study,an outer membrane protein gene(ompW)was cloned from V.alginolyticus andexpressed in Escherichia coli.The 645 bp open reading frame(ORF)encodes a protein of 214 amino acidresidues with a predicted molecular weight of 23.3 kDa.The amino acid sequence showed a high identitywith that of Photobacterium damselae(96.2%)and Vibrio parahaemolyticus(94.4%).The alignment analy-sis indicated that OmpW was highly conserved.Sodium dodecyl sulfate-polyacrylamide gel electrophoresisshowed that the gene was over-expressed in E.coli BL21(DE3).Western blot analysis revealed that theexpressed protein had immunoreactivity.The recombinant protein was purified by affinity chromatographyon Ni-NTA Superflow resin.Large yellow croaker vaccinated with the purified OmpW showed significantlyincreased antibody to OmpW,which could resist the infection by V.alginolyticus.A specific antibody wasdetected by enzyme-linked immunosorbent assay.This study suggested that the conserved OmpW could bean effective vaccine candidate against infection by V.alginolyticus.     

溶藻弧菌HY9901 ahr基因克隆与序列分析

TouchdownPCR扩增溶藻弧菌依赖ATP的DNA解旋酶rep基因部分序列,得一1．2kb片段,再以反向PCR和巢式PCR联合扩增其侧翼序列,拼接得一由2016bp组成,共编码671aa的完整基因。该基因演绎的氨基酸序列与几种弧菌的同源性都比较高,与副溶血弧菌RIMD2210633、溶藻弧菌12G01、Vibriosp．Ex25、创伤弧菌YJ016、霍乱弧菌RC385、灿烂弧菌12801、费氏弧菌ES114及矿angustumS14分别为96％、95%、95%、94%、92％、91%、89％、86％。     

溶藻弧菌HY9901 AcrA基因克隆与序列分析

Touchdown PCR扩增溶藻弧菌HY9901 AcrA基因部分序列,得一460bp片段,再以反向PCR和巢式PCR联合扩增其侧翼序列,拼接得一由1101 nt组成,共编码366 aa的完整基因.该基因演绎的氨基酸序列与几种弧菌的同源性都比较高,与创伤弧菌YJ016、副溶血弧菌RIMD 2210633、灿烂弧菌12B01、霍乱弧菌O1 N16961同源性分别为76%、73%、71%和70%.     

Cloning and expression of two genes encoding highly homologous hemolysins from a Kanagawa phenomenon-positive Vibrio parahaemolyticus T4750 strain

We have cloned and sequenced the gene encoding thermostable direct hemolysin (TDH), a possible virulence factor in Vibrio parahaemolyticus gastroenteritis, from a Kanagawa-phenomenon-positive strain, T4750. This strain was found to contain two sequences (tdhA and tdhS) homologous to the tdh gene previously reported by Nishibuchi and Kaper [J. Bacteriol 162 (1985) 558-564] and Taniguchi et al. [Microb. Pathog. 1 (1986) 425-432]. Sequence homology of the coding regior between tdhA and tdhS was 97.2%. The deduced amino acid (aa) sequence of TdhA, excluding the putative signal peptide was identical to that of TDH protein purified from V. parahaemolyticus [Tsunasawa et al., J. Biochem. 101 (1987) 111-121] except for Glu118 instead of Gln118. Although the aa sequence deduced from the second gene, tdhS, differed in eight residues from the TDH protein, it agreed with the sequence of Tdh deduced from the previously cloned tdh gene. Both tdhA and tdhS expressed biologically active hemolysins in Escherichia coli. While the apparent molecular size of TDH purified from a culture supernatant of V. parahaemolyticus T4750 was identical to TdhA protein synthesized in E. coli, it was larger than TdhS. Only one band was detected in the culture supernatant of V. parahaemolyticus T4750 by Western blotting; its mobility was indistinguishable from that of purified TDH. These data suggest that tdhA is the structural gene for TDH found in the culture supernatant of V. parahaemolyticus T4750, and that there was only partial, if any, tdhS expression in the strain T4750 under the test conditions employed.     

Soft-agar-coated filter method for early detection of viable and thermostable direct hemolysin (TDH)- or TDH-related hemolysin-producing Vibrio parahaemolyticus in seafood

A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in seafood was developed. The method involved (i) enrichment culture, selective for viable, motile cells penetrating a soft-agar-coated filter paper, and (ii) a multiplex PCR assay targeting both the TDH gene (tdh) and TRH gene (trh) following DNase pretreatment on the test culture to eradicate any incidental DNAs that might have been released from dead cells of tdh- or trh-positive (tdh+ trh+) strains and penetrated the agar-coated filter. A set of preliminary laboratory tests performed on 190 ml of enrichment culture that had been inoculated simultaneously with ca. 100 viable cells of a strain of tdh+ trh+ V. parahaemolyticus and dense populations of a viable strain of tdh- and trh-negative V. parahaemolyticus or Vibrio alginolyticus indicated that the method detected the presence of viable tdh+ trh+ strains. Another set of preliminary tests on 190 ml of enrichment culture that had been initially inoculated with a large number of dead cells of the tdh+ trh+ strain together with dense populations of the tdh- and trh-negative strains confirmed that the method did not yield any false-positive results. Subsequent quasi-field tests using various seafood samples (ca. 20 g), each of which was experimentally contaminated with either or both hemolysin-producing strains at an initial density of ca. 5 to 10 viable cells per gram, demonstrated that contamination could be detected within 2 working days.     

Analysis of the tdh gene cloned from a tdh gene- and trh gene-positive strain of Vibrio parahaemolyticus

A variant of the gene (tdh) encoding thermostable direct hemolysin (TDH) was cloned from the chromosome of Vibrio parahaemolyticus AQ3860, which gave positive results in the hybridization tests with the tdh gene probe and the trh (tdh-related hemolysin) gene probe and showed a low level of reaction in an enzyme-linked immunosorbent assay for TDH. Nucleotide sequence analysis of the cloned gene (tdh5) provided no evidence that tdh5 is evolutionally closer to the trh gene than the other tdh genes. The tdh5 gene was flanked by 40 base-pair sequences constituting perfect inverted repeats, which may suggest association of the tdh5 gene with insertion sequence-like structure. These results suggest that the tdh5 gene and the trh gene were not originally produced by gene duplication in AQ3860 but rather that one of the two genes moved into AQ3860 from an external source.     

Vibrio parahaemolyticus thermostable direct hemolysin can induce an apoptotic cell death in Rat-1 cells from inside and outside of the cells

Rat-1 cells exposed to Vibrio parahaemolyticus thermostable direct hemolysin (TDH) developed morphological changes including shrinkage of the cells and reduction in the size of nuclei. Cells either microinjected with TDH or transfected with the tdh gene also showed morphological changes similar to those induced by externally added toxin. Furthermore, TDH-exposed or tdh-transfected cells both showed chromatin condensation and DNA fragmentation which suggest cells undergoing apoptosis. In contrast, expression of a TDH mutant (R7) did not reveal any cytotoxic effects. We demonstrate that expressed TDH was distributed in the cytoplasm. The interleukin-1beta-converting enzyme-related protease inhibitor ZVAD-FMK did not inhibit TDH cytotoxicity. Our results suggest that TDH can induce its cytotoxicity both from outside and from inside the cells and killed the cells through apoptosis.     

Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China

AIMS: To investigate the distribution of the virulence of two Vibrio species among different strains obtained from the mariculture systems on the coast of Guangdong in China and the correlation between the virulence strains and the virulence genes among Vibrio alginolyticus. METHODS: Besides three strains, 72 V. alginolyticus strains and seven Vibrio parahaemolyticus strains were examined by PCR or semi-nested PCR for the virulence genes (tlh, trh, tdh, toxR, toxRS, ctxA, VPI). Additionally, the virulence of 18 V. alginolyticus strains was tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulence genes homologous to those in the V. parahaemolyticus and Vibrio cholerae are widely distributed among V. alginolyticus and V. parahaemolyticus in the coastal mariculture systems in Guangdong, China. Some of the V. alginolyticus strains are pathogenic to aquatic animals, and might have derived their virulence genes from V. parahaemolyticus or V. cholerae, representing a possible reservoir of these genes. However, there is no correlation between presence and absence of the virulence genes used to investigate V. alginolyticus and its virulent strains. In this report, we also show that tlh is distributed among V. alginolyticus.     

Characterization of a new beta-lactamase gene from isolates of Vibrio spp. in Korea

PCR was performed to analyze the beta-lactamase genes carried by ampicillin-resistant Vibrio spp. strains isolated from marine environments in Korea between 2006 and 2009. All 36 strains tested showed negative results in PCR with the primers designed from the nucleotide sequences of various known beta-lactamase genes. This prompted us to screen new beta-lactamase genes. A novel beta-lactamase gene was cloned from Vibrio alginolyticus KV3 isolated from the aquaculture water of Geoje Island of Korea. The determined nucleotide sequence (VAK-3 beta-lactamase) revealed an open reading frame (ORF) of 852 bp, encoding a protein of 283 amino acids (aa), which displayed low homology to any other beta-lactamase genes reported in public databases. The deduced 283 aa sequence of VAK-3, consisting of a 19 aa signal peptide and a 264 aa mature protein, contained highly conserved peptide segments specific to class A beta-lactamases including the specific amino acid residues STFK (62-65), SDN (122-124), E (158), and RTG (226-228). Results from PCR performed with primers specific to the VAK-3 beta-lactamase gene identified 3 of the 36 isolated strains as V. alginolyticus, Vibrio cholerae, and Photobacterium damselae subsp. damselae, indicating the utilization of various beta-lactamase genes including unidentified ones in ampicillin-resistant Vibrio spp. strains from the marine environment. In a mating experiment, none of the isolates transfered the VAK-3 beta-lactamase gene to the Escherichia coli recipient. This lack of mobility, and the presence of a chromosomal acyl-CoA flanking sequence upstream of the VAK-3 beta- lactamase gene, led to the assumption that the location of this new beta-lactamase gene was in the chromosome, rather than the mobile plasmid. Antibiotic susceptibility of VAK-3 beta-lactamase was indicated by elevated levels of resistance to penicillins, but not to cephalosporins in the wild type and E. coli harboring recombinant plasmid pKV-3, compared with those of the host strain alone. Phylogenetic analysis showed that VAK-3 beta-lactamase is a new and separate member of class A beta-lactamases.     

基于原核表达的副溶血性弧菌类毒素疫苗对海水鱼类免疫保护作用

目的:实现副溶血性弧菌溶血毒素基因tdh克隆与表达,并以表达产物TDH作免疫原,研究其对海水鱼免疫活性的影响.方法:PCR扩增tdh;构建重组质粒(pET-28-TDH);IPTG诱导表达;SDS-PAGE检测:以纯化的TDH融合蛋白脱毒为类毒素免疫原免疫健康真鲷,测定其血清中过氧化氢酶(CAT)、酸性磷酸酶(ACP)、超氧化物歧化酶(SOD)和碱性磷酸酶(AKP)活性变化;用副溶血性弧菌病原攻毒,检测免疫保护作用.结果:tdh基因成功克隆并表达;表达蛋白相对分子量为21 kDa,证实为TDH蛋白;类毒素免疫真鲷后,其血清中总超氧化物歧化酶(T- SOD)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)和过氧化氢酶(CAT)活力在注射后48h、24h、24h、2Ah达到最高,最高点分别高于对照组77％、328％、75％、381％;72 h后均恢复至对照组水平.结论:类毒素对真鲷免疫系统具有明显的刺激作用;类毒素免疫后对攻毒的真鲷保护率达50％.     

霍乱毒素B亚单位基因（CtxB）的克隆及其表达

从霍乱弧菌中抽提基因组ＤＮＡ,用ＰＣＥＲ方法获取霍乱毒素Ｂ亚单位基因（ＣｔｘＢ）。序列分析结果表明,ＣｔｘＢ基因编码１２４个氨基酸,其中编码６２位Ｔｈｒ的密码子与文献报道有差异。将ＣｔｘＢ基因插入质粒ｐＧＥＸ－４Ｔ－２,构建ｐＧＥＸ－ＣＴＸＢ表达质粒,转化大肠相菌ＢＬ２１（ＤＥ３０,筛选表达菌株ＣＴＸＢ／ＢＬ２１。工程株经ＩＰＴＧ诱导表达,可产生大量的表达蛋白,经ＳＤＳ－ＰＡＧＥ分析,融合蛋白分子     

弧菌三联融合毒素基因表达及ELISA检测方法建立

采用柔性Linker(GGGGS)和重叠延伸PCR方法将三个毒素基因, 即副溶血性弧菌的耐热型直接溶血素基因tdh、创伤弧菌的溶细胞毒素基因vvhA和拟态弧菌的热不稳定型溶血素基因vmhA的成熟蛋白基因片段进行串联, 得到三联融合毒素基因tdh-vvhA-vmhA(命名为FT)。一致性比对分析与预计融合的基因序列一致性为99.6%。FT的开放阅读框架全长3225 bp, 编码1074个氨基酸残基, 预测分子量为120.4 kDa。将FT亚克隆至表达载体pET-22b(+)中, 再转化至E. coli BL21 (DE3)中进行表达, 经SDS-PAEG分析, 融合蛋白分子量与预测的相符。终浓度为1mmol/L的IPTG, 37 ℃诱导4 h, 融合毒素蛋白表达量最高, 经薄层扫描分析, 此时融合毒素蛋白占全菌体蛋白的11.49%。融合毒素蛋白包涵体经纯化, 免疫豚鼠制备血清抗体, 确定间接ELISA的最佳工作条件, 并进行敏感性、特异性、重复性及模拟样品检测试验, 建立了食物中毒性弧菌广谱、快速、特异的同步免疫学检测方法。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾(Litopenaeus vannamei)幼体中分离到两株病原菌zouA和zouB, 常规形态和生理生化试验表明均为弧菌属菌种, 弧菌编码鉴定系统分别鉴定为溶藻弧菌(Vibrio alginolyticus)和副溶血弧菌(V. parahaemolyticus)。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98%, 相互间不能区分; HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98%以上, 而与所有其它弧菌的相似性不到92%。结合表型和分子特征的鉴定结果, 菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾（Litopenaeus vannamei）幼体中分离到两株病原菌zouA和zouB,常规形态和生理生化试验表明均为弧菌属菌种,弧菌编码鉴定系统分别鉴定为溶藻弧菌（Vibrioatginolyticus）和副溶血弧菌（V.parahaemolyticus）。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98％,相互间不能区分;HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98％以上,而与所有其它弧菌的相似性不到92％。结合表型和分子特征的鉴定结果,菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus.

The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.     

Characterization of the H(+)-pumping F1F0 ATPase of Vibrio alginolyticus.

The F1F0 ATPase of Vibrio alginolyticus was cloned from a chromosomal lambda library. The unc operon, which contains the structural genes for the ATPase, was sequenced and shown to have a gene organization of uncIBEFHAGDC. The sequence of each subunit was compared with those of other eubacterial ATPases. The V. alginolyticus unc genes exhibited greater similarity to the Escherichia coli unc genes than to any of the other bacterial unc genes for which the sequence is available. The ATPase was expressed in an E. coli unc deletion strain, and the ATP hydrolytic activity was characterized. It has a pH optimum of 7.6 and is stimulated by the addition of Triton X-100 or any of a variety of salts. The recombinant F1F0 was purified 30.4-fold and reconstituted into proteoliposomes. This enzyme catalyzed the pumping of protons coupled to ATP hydrolysis as measured in fluorescence quenching experiments but would not pump Na+ ions under similar conditions.     

副溶血弧菌海产品分离株tdh基因及其临近区域结构分析

摘要：【目的】初步探索副溶血弧菌海产品分离株tdh基因区域的结构特征。【方法】采用长距离PCR和基因步移技术进行tdh基因侧翼序列扩增,测序验证后拼接成疑似毒力基因片段,将所获序列与NCBI数据库进行比较,初步明确tdh基因侧翼序列的结构与功能。【结果】海产品分离株ZS34与参考菌株 RIMD2210633的tdh基因区域（VPA1310-VPA1327）结构基本一致,核苷酸同源性达98.3%;而FJ14与WZ64株基因组中的tdh基因均与tdh3的同源性最高,在基因组中的位置也不同于ZS34株和参考菌株     

Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis.

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.