荧光假单胞菌M18的rpoS基因克隆及其功能分析  

Cloning, Sequencing and Functional Studies of rpoS Gene from Pseudomonas Fluorescent sp. M18

作　　者：徐汪节[1] 朱栋华[1] 张雪洪[1] 许煜泉[1] 

机构地区：[1]上海交通大学生命科学技术学院,上海200240

出　　处：《微生物学报》2004年第3期309-314,共6页Acta Microbiologica Sinica

基　　金：国家"十五"科技攻关项目 ( 2 0 0 1BA3 0 8A0 2 14 ) ;国家自然科学基金项目 ( 3 0 3 70 0 41)~~

摘　　要：从荧光假单胞菌 (Pseudomonasfluorescentsp .)M1 8基因组中克隆了RNA聚合酶的稳定期σs 因子编码基因rpoS ,推测其氨基酸序列与铜绿假单胞菌、荧光假单胞菌和恶臭假单胞菌的同源性分别为 99 1 %、87 35 %和87 8%。利用体外定点插入突变和同源重组技术 ,构建了M1 8的rpoS突变株M1 8R- 。对突变株M1 8R- 合成抗生素吩嗪 1 羧酸 (PCA)和藤黄绿菌素 (Plt)的动力学分析结果表明 ,在KB或PPM培养基中 ,突变株合成PCA的能力比野生型分别提高了 2 5或 5 78倍 ,但Plt的积累量不受影响。与野生型相比 ,突变株对碳源饥饿的耐性下降。同时 ,在碳源饥饿条件下对过氧化氢、乙醇和和氯化钠等环境胁迫的交叉保护性减小 。The rpoS gene encoding the stationary phase sigma factor σ s from fluorescent Pseudomonas sp. M18 was cloned and sequenced. The deduced RpoS protein of M18 strain showed 99 1%、87 35% and 87 7% identity with that of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas putida respectively but only 63 6% identity with that of E.coli. The rpoS gene was inactivated by inserting a Km cassette at the site of blunted BamHⅠ in vitro and then the resulting reconstruct was introduced into the M18 genome using homologous recombination technique to obtain the null mutant M18R -. Several pleiotropic effects of rpoS - mutation in M18R - were observed. The M18R - could overproduce phenazine-1-carboxylic acid 25 and 5 fold more in KB and PPM medium respectively but produce the same amount of pyoluteorin in KB medium in comparison with the wild type strain M18 The M18R - also showed reduced survival of carbon starvation and cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol and hydrogen peroxide.

关 键 词：荧光假单胞菌M18 rpoS突变株 吩嗪-1-羧酸 藤黄绿菌素 环境胁迫 

分 类 号：Q785[生物学—分子生物学]