Virulence factors of fish pathogenic bacteria

1.底物SDS-PAGE分析表明，114和152 kDa的蛋白酶对酪蛋白和明胶都有活性，32 ~ 86 kDa的蛋白酶对明胶有活性，但对酪蛋白没有活性。研究的29个分离物根据蛋白酶形成四个组。在幼鲑的感染性实验中，蛋白酶组与毒力之间存在一定的联系。6株嗜冷梭菌产蛋白酶的最适温度为13.3（SY+-[）1.5 ℃，生长最适温度为19.6（SY+-[）0.5 ℃.从鳗弧菌（Vibrioanguillarum）中克隆了一个5 kb的溶血素基因片段。溶血素基因（VAH 1）的开放阅读框为2，253 bp。用VAH 1在高严谨条件下进行DNA杂交分析，结果表明VAH 1与28株鳗弧菌中的25株有杂交，而与其他弧菌没有杂交.采用PCR-随机诱变技术，从嗜水气单胞菌AHH 3中分离到3株溶血活性阴性的突变株（AHH 3-m1-3）。当将AHH 3的氨基酸序列与AHH 3-m3的氨基酸序列进行比较时，9个氨基酸残基被替换。His 355-Arg 355的置换突变体丧失了溶血活性，His 291-Gln 291的突变体降低了溶血活性.对EDDHA的MIC可以区分迟缓爱德华氏菌的强毒株和无毒毒株。在含有EDDHA的HI肉汤中，强毒株产生铁载体样物质，而无毒毒株则不产生。利用转座子Tn 5获得铁吸收能力降低的突变株。突变株的毒力低于原始菌株.克隆了迟缓艾美耳球虫溶血素基因（ETH）的一个位点并进行了序列测定。该区域编码两个开放阅读框，分别命名为ethA和ethB，ethA为溶血素基因，长度为4782 bp，ethB为激活/分泌蛋白基因，长度为1677 bp。EthB蛋白是激活EthA蛋白（溶血素）所必需的。ethB基因的转录受铁的调控。

1. The ECPs of Cytophaga phychrophila isolates were analyzed by substrate SDS-PAGE.Proteases of 114 and 152 kDa had activity against both casein and gelatin, and proteases from 32 to 86 kDa were active against gelatin but not casein. The 29 isolates studied formed four groups based on the proteases. In infectivity experiments with juvenile salmonids indicated some association between protease group and virulence.2. The optimum temperatures for protease production of six C.psychrophila isolates was 13.3(]SY+-[)1.ﾟC,while 19.6(]SY+-[)0.5ﾟC for growth.3. A 5 kb DNA fragment encoding a hemolysin was cloned from Vibrio anguillarum. An open reading frame of the hemolysin gene (VAH1) was 2,253 bp. DNA hybridization analysis under high-stringent conditions using VAH1, demonstrated that VAH1 hybridized with 25 out of 28 strains of V.anguillarum, but did not with other species of Vibrio.4. Three hemolytic activity negative mutants of Aeromonas hydrophila AHH3 were isolated by the PCR-random mutagenesis (AHH3-m1-3). When compared the amino acid sequences of AHH3 with those of AHH3-m3, nine amino acid residues replaced. The replacement mutant of His355-Arg355 lost the hemolytic activity, and mutant of His291-Gln291 decreased the hemolytic activity.5. MICs against EDDHA may distinguish virulent strains from avirulent ones of Edwardsiella tarda. In HI broth containing EDDHA,virulent strains produced siderophore-like substance (s), while avirulent ones did not. Mutant strains with lower iron acquisition ability were produced by using transposon (Tn5). This mutants had less virulence than the original strain.6. A hemolysin gene locus from E.tarda (ETH) was cloned and sequenced. This region coded two open reading frames, designated ethA and ethB.The ethA is a hemolysin gene consisting of 4782bp and ethB is an activation/secretion protein gene of 1677 bp. The EthB protein was necessary for activation of EthA protein (hemolysin). Transcription of the ethB gene was regulated by iron.