Isolation and characterization of mutants of a marine Vibrio strain that are defective in production of extracellular proteins

A marine Vibrio strain, Vibrio sp. strain 60, produces several extracellular proteins, including protease, amylase, DNase, and hemagglutinin. Mutants of Vibrio sp. strain 60 (epr mutants) pleiotropically defective in production of these extracellular proteins were isolated. They fell into two classes, A and B. In class A, no protease activity was detected in the cells either, whereas in class B, considerable protease activity was detected in the cells. Gel electrophoretic analysis revealed that the protease detected in class B mutant cells was similar to the protease excreted by the parent strain. In addition, the protease in class B mutant cells was found to be localized in the periplasmic space. These results suggest that the passage of the protease through the outer membrane is blocked in class B mutants. Comparison of membrane protein profiles by polyacrylamide gel electrophoresis revealed that all the epr mutants contained an increased amount of a 94,000-Mr protein that may be an outer membrane protein. Four epr mutations were mapped in two different regions of the Vibrio chromosome by transduction; two class A mutations and one class B mutation were located close to each other, whereas another class B mutation was located in a different region of the chromosome.     

Type II secretion by Aeromonas salmonicida: evidence for two periplasmic pools of proaerolysin

Aeromonas salmonicida containing the cloned gene for proaerolysin secretes the protein via the type II secretory pathway. Here we show that altering a region near the beginning of aerA led to a dramatic increase in the amount of proaerolysin that was produced and that a large amount of the protein was cell associated. All of the cell-associated protein had crossed the cytoplasmic membrane, because the signal sequence had been removed, and all of it was accessible to processing by trypsin during osmotic shock. Enlargement of the periplasm was observed by electron microscopy in overproducing cells, likely caused by the osmotic effect of the very large concentrations of accumulated proaerolysin. Immunogold electron microscopy localized nearly all of the proaerolysin in the enlarged periplasm; however, only half of the protoxin was released from the cells by osmotic shocking. Cross-linking studies showed that this fraction contained normal dimeric proaerolysin but that proaerolysin in the fraction that was not shockable had not dimerized, although it appeared to be correctly folded. Both periplasmic fractions were secreted by the cells; however, the nonshockable fraction was secreted much more slowly than the shockable fraction. We estimated a rate for maximal secretion of proaerolysin from the bacteria that was much lower than the rates that have been estimated for inner membrane transit, which suggests that transit across the outer membrane is rate limiting and may account for the periplasmic accumulation of the protein. Finally, we show that overproduction of proaerolysin inhibited the release of the protease that is secreted by A. salmonicida.     

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板, 采用PCR技术, 扩增气溶素基因(aerA)的DNA片段, 将其克隆到pMD18-T载体上。通过序列测定, 分析结果表明：所克隆的1393 bp片段为aerA部分序列, 编码产生464个氨基酸。BZ与NK之间aerA核苷酸同源性为97.6%, 氨基酸同源性为98.3%, 与其它分离物核苷酸同源性为71.6%~97.5%, 氨基酸同源性为68.0%~98.9%。利用邻接法构建了aerA分子树状图, 树状图分析表明：气单胞菌属各分离物聚为三支, 其中嗜水气单胞菌各菌株之间关系密切, 被聚类为同一支。     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板,采用PCR技术,扩增气溶素基因(aerA)的DNA片段,将其克隆到pMDl8-T载体上.通过序列测定,分析结果表明:所克隆的1393 bp片段为aerA部分序列,编码产生464个氨基酸.BZ与NK之间aerA核苷酸同源性为97.6%.氨基酸同源性为98.3%,与其它分离物核苷酸同源性为71.6%～97.5%,氨基酸同源性为68.0%～98.9%.利用邻接法构建了aerA分子树状图,树状图分析表明:气单胞菌属各分离物聚为三支,其中嗜水气单胞菌各菌株之间关系密切,被聚类为同一支.     

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin,hemolysin,and extracellular serine protease (i.e.,aerA,hlyA,and ahpA,respectively).These genes were investigated using polymerase chain reaction (PCR)with specific primers for each gene.And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed.PCR results demonstrated that the distribution patterns of aerA,hlyA,and ahpA were different in these strains.6/9 of A.hydrophila strains were aer A positive,8/9 of strains hly A positive,7/9 of strains ahp A positive,respectively.However,the assay for pathogenesis showed that two strains (A.hydrophila XS91-4-1 and C2)were strong virulent,two strains (A.hydrophila ST78-3-3 and 58-20-9)avirulent and the rest middle virulent was to the fish.In conclusion,there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio.All strong virulent A.hydrophila strains were aerA+hlyA+ahpA+genotype,and all aerA+hlyA+ahpA+strains were virulent.Strains with the genotype of aerA-hlyA-ahpA+have middle pathogenicity.In the present study,we found for the first time that all A.hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio.Additionally,there was a positive correlation between the virulence of A.hydrophila and the presence of aerA and ahpA.     

Mutagenesis by insertion of drug resistance transposon Tn7 into a vibrio species

A halotolerant, collagenolytic strain of Vibrio sp. was conjugated with an Escherichia coli strain carrying plasmid RP4. The plasmid was transferred to and maintained in the Vibrio and could be subsequently transferred in matings to suitably marked stains of the same species. After conjugation with an E. coli carrying the cointegrate plasmid RP4::Mu cts61::Tn7, Vibrio transconjugants were selected that carried Tn7 inserted into the bacterial chromosome. A large proportion of these transconjugants were auxotrophic, showing that plasmid suicide by Mu can be used to isolate Tn7-derived mutants in Vibrio. Approximately half of the auxotrophs isolate Tn7-derived mutants in Vibrio. Approximately half of the auxotrophs isolated were ilv mutants, all of which exhibited the same phenotype. Thus, although Tn7 insertion can induce auxotrophy, including trp, thy, his and ura, in Vibrio, there does appear to be a hot spot for integration in the ilv operon.     

Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation

Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp. in separate steps. To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida , as well as Vibrio cholerae . Replacing Met-41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge. A double mutant was also made, in which Gly-202 and Ile-445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded. The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants. Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted. We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur. The proton ionophore CCCP reduced release of the folded proteins. Unoxidized protein was secreted by cells grown in β-mercaptoethanol and by a dsbA mutant of V. cholerae , indicating that disulphide bond formation may not be essential for release.     

High temperature-induced changes in exopolysaccharides, lipopolysaccharides and protein profile of heat-resistant mutants of Rhizobium sp. (Cajanus)

A thermosensitive wild-type strain (PP201) of Rhizobium sp. (Cajanus) and its 14 heat-resistant mutants were characterized biochemically with regard to their cell surface (exopolysaccharides (EPSs) and lipopolysaccharides (LPSs)) properties and protein profile. Differences were observed between the parent strain and the mutants in all these parameters under high temperature conditions. At normal temperature (30 °C), only half of the mutant strains produced higher amounts of EPSs than the parent strain, but at 43 °C, all the mutants produced higher quantities of EPS. The LPS electrophoretic pattern of the parent strain PP201 and the heat-resistant mutants was almost identical at 30 °C. At 43 °C, the parent strain did not produce LPS but the mutants produced both kinds of LPSs. The protein electrophoretic pattern showed that the parent strain PP201 formed very few proteins at high temperature, whereas the mutants formed additional new proteins. A heat shock protein (Hsp) of 63–74 kDa was overproduced in all mutant strains.     

The induction of stress proteins in three marine Vibrio during carbon starvation

Abstract The induction of DnaK and GroEL homologous proteins by heat-shock and long-term carbon starvation was studied in Vibrio vulnificus, Vibrio sp. strain S14, and Vibrio sp. strain DW1. In each Vibrio strain one protein (60 kDa) reacted with antibodies against Escherichia coli -GroEL and two proteins, DnaK (69 kDa) and Sis1 (62-60 kDa), reacted with antibodies against E. coli -Dnak. The carbon starvation elicited induction of the stress proteins was strain-specific, suggesting that the induction of stress proteins like DnaK and GroEL in marine Vibrios might not be a uniform starvation response. It appears as of these proteins, only DnaK in Vibrio sp. strain S14 remains induced after long-term carbon starvation in the three marine bacterial strains that were tested.     

翘嘴鳜病原嗜水气单胞菌分子特征及LAMP检测方法的建立

嗜水气单胞菌(Aeromonas hydrophila)是一种危害鳜鱼养殖生产的重要病原细菌, 为进一步明确该病原菌的分子特征及建立快速检测技术, 实验对引起翘嘴鳜(Siniperca chuatsi)暴发性死亡的病原嗜水气单胞菌进行了致病性、菌株毒力特征研究, 同时以嗜水气单胞菌气溶素基因aerA为分子靶标设计引物, 利用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立了病原嗜水气单胞菌的快速检测方法。结果表明, 本次引起翘嘴鳜暴发性死亡的病原嗜水气单胞菌半致死浓度为1.6×106 CFU/mL, 携带aerA等14种毒力基因, 此14种毒力基因可用于其致病性分析及分子检测。以气溶素基因aerA设计引物进行的环介导恒温扩增, 结果显示可扩增出阶梯状条带, 加入SYBR Green I染色后呈现绿色的阳性反应, 而对照组均未出现任何扩增条带且反应体系呈现橙色, 表明LAMP检测方法对于嗜水气单胞菌检测具有很好的特异性; 灵敏度检测的最低检测限为4.6×101 CFU/mL; 10种经人工感染的淡水养殖鱼虾组织匀浆增菌液, 提取DNA后进行LAMP方法检测, 结果均可获得阳性扩增结果, 而对照未染菌组呈阴性, 表明该方法具有较好的应用性, 可应用于嗜水气单胞菌引起的水生动物疾病的检测。     

Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model

Previous studies have shown that two hemolytic toxins, HlyA and AerA, contribute to the virulence of Aeromonas hydrophila. A survey was performed to gauge the distribution of hlyA and aerA genes in clinical and environmental Aeromonas isolates. For A. hydrophila, A. veronii biotype sobria and A caviae, 96%, 12% and 35% of strains, respectively, were hlyA positive, whereas, 78%, 97%, 41%, respectively, were aerA positive. All virulent A. hydrophila isolates were hlyA+ aerA+. This genotype was most common in A. hydrophila (75.4%) followed by A. caviae (29.4%) and A. veronii biotype sobria (9.6%). For A. hydrophila, a two-hemolytic toxin model of virulence provides the best prediction of virulence in an animal model.     

Vibrio cholerae persistence in aquatic environments and colonization of intestinal cells: involvement of a common adhesion mechanism

Forty-one Tnpho A mutants of Vibrio cholerae O1 classical strain CD81 were analyzed for their ability to interact with chitin particles, Tigriopus fulvus copepods and the Intestine 407 cell line compared to the parent strain. Thirteen mutants were less adhesive than CD81; in particular, T21, T33 and T87 were less adhesive towards all substrates and insensitive to inhibition by N-acetyl glucosamine (GlcNAc). By SDS-PAGE analysis of sarkosyl-insoluble membrane proteins (siMPs) isolated from mutants and parent, it was found that a 53 kDa siMP is missing in T21, T33 and T87 mutants. It is hypothesized that this protein might have the function to mediate adherence to GlcNAc-containing substrates both in the aquatic environment and in human intestine.     

Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway

Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

和厚朴酚对嗜水气单胞菌气溶素表达的抑制作用

为了研究不同浓度和厚朴酚对嗜水气单胞菌(Aeromonas hydrophila)致病力的影响, 筛选抗嗜水气单胞菌感染的天然化合物, 通过溶血试验、免疫印记试验、荧光定量PCR试验和动物试验进行了研究。结果发现, 和厚朴酚能在亚抑菌浓度下降低嗜水气单胞菌培养物上清中的溶血活性; 蛋白免疫印迹试验发现和厚朴酚能降低嗜水气单胞菌气溶素的分泌; 荧光定量PCR试验进一步表明和厚朴酚与嗜水气单胞菌共培养后降低了气溶素编码基因aerA的转录而降低气溶素的分泌。此外, 通过动物试验发现和厚朴酚治疗能显著提高斑点叉尾鮰( Ictalurus punctatus )嗜水气单胞菌感染模型的存活率。以上研究表明, 和厚朴酚能通过降低气溶素编码基因aerA的转录而降低嗜水气单胞菌的致病力, 和厚朴酚是一种潜在的新型抗嗜水气单胞菌感染的先导化合物。     

湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究

为了调查引起鱼类运动型气单胞菌败血症(俗称暴发病)的嗜水气单胞菌的遗传多样性和毒力特征, 阐明其流行规律, 研究于2006-2009年度从湖北省内3个不同地区的6个发病鱼塘中分离了30株嗜水气单胞菌, 其中20株为临床株(分离自血液、肝脏、肾脏或腹水), 6株为肠道株, 4株为池水株。基于所有菌株gyrB基因序列, 构建了系统发育树; 通过ERIC (Enterobacterial repetitive intergenic consensus, 肠道细菌基因间重复序列)指纹图谱进行菌株的遗传分型; 用PCR方法检测了7个毒力基因在菌株中的分布模式。这7个基因包括气溶素(aerA)、溶血素(hlyA)、热不稳定性细胞兴奋性肠毒素(alt)、热稳定性细胞兴奋性肠毒素(ast)、弹性蛋白酶(ahpB)、脂酶(lip)和鞭毛基因(fla)。此外, 以斑马鱼为感染对象, 通过腹腔注射测定了15株代表菌株的毒力。结果表明: 不同来源的20株临床株、1株肠道株和3株池水株具有相同的遗传特性, 体现为在系统树上聚为一枝, 序列相似性为100%, 具有相同的ERIC指纹图谱, 毒力基因分布模式为: aerA+hlyA+alt+ast+ahpB+lip+fla+, 且均为强毒株(LD50 9.74104cfu/尾)。与临床株相比, 其余5株肠道株和1株池水株或具有不同的ERIC指纹图谱或具有不同的毒力基因分布模式, 显示出了遗传多样性, 且毒力均弱于临床株(LD501.01106cfu/尾)。这说明在一定时间、一定区域内, 作为暴发病病原的嗜水气单胞菌为同一克隆系在流行, 不存在明显的变异或遗传多样性。此结果有助于阐明嗜水气单胞菌引起的暴发病的流行规律, 制定相应的防御措施。多种毒力基因在致病性菌株中的联合流行为发病机理的解析奠定了基础。此外, 鉴于毒力基因谱与致病性之间的相关性, 表明毒力基因可作为标记基因, 用于致病性菌株的检测。       

罗尼氏弧菌Vibrio shilonii BY新琼胶酶基因的克隆、序列分析及表达

【目的】从海洋来源的罗尼氏弧菌菌株BY中克隆得到一个具有琼胶酶活性的新基因,并对其进行重组表达。【方法】对实验室保藏的产琼胶酶菌株BY进行16S rRNA基因序列分析,并构建系统发育树。根据已报道的琼胶酶基因序列的同源性,设计简并引物,利用降落PCR (Touch-down PCR)及染色体步移技术扩增琼胶酶基因序列全长,对基因序列进行生物信息学分析。将目的基因插入pET22a(+)载体,转化大肠杆菌BL21(DE3),对重组酶进行表达,利用DNS法测定了重组酶的酶活,对该重组琼胶酶酶学性质进行研究。【结果】克隆得到一条新的琼胶酶基因,命名为Vibrio sp. BY (GenBank登录号：AIW39921.1),Vibrio sp. BY基因序列全长2 232 bp,编码744个氨基酸,理论分子量为85 kD,Vibrio sp. BY的氨基酸序列基因库中与已知的琼胶酶氨基酸序列Vibrio sp. EJY3的相似度为86%。发酵液琼胶酶酶活力为71.73 U/mL,证明表达的蛋白为琼胶酶。酶学性质研究表明重组琼胶酶的最适温度及pH分别为50 °C和7.0,并且具有较好的稳定性。【结论】利用染色体步移技术克隆得到一条新的琼胶酶基因,并在大肠杆菌BL21(DE3)中实现了重组表达,为琼胶酶的应用奠定了基础。