Identification of sigma(s)-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri
Identification of sigma(s)-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri
复制标题
DOI：
10.1046/j.1365-2958.1996.00058.x
复制
发表时间：
1996-09-01
影响因子：
3.6
通讯作者：
Small, PLC
中科院分区：
文献类型：
--
作者：
Waterman, SR;Small, PLC
福氏志贺菌生长到固定相后，能在pH 2.5下存活数小时。这种耐酸性，可能与志贺氏菌病相关的低感染剂量有关，依赖于静止相特定的西格玛因子的表达(S)。采用随机TnPhoA和TnlacZ诱变方法，分离到5株对酸敏感的福氏志贺菌突变体，它们在体外pH 2.5条件下不能存活2 h。对每个带有福氏志贺氏菌DNA侧翼的转座子插入片段进行克隆和测序。数据库搜索表明，两个TnlacZ突变体在hdeA基因中有一个插入，这是hdeAB操纵子的第一个基因。通过携带完整hdeAB操纵子的质粒，在其中一个突变体中恢复了耐酸性。对其余TnlacZ和两个TnPhoA突变体的进一步序列分析表明，它们都在gadB基因下游的一个先前未鉴定的开放阅读框架(ORF)中插入了片段。这个假定的ORF编码一种与许多内膜氨基酸逆向转运蛋白具有同源性的蛋白质。克隆了一个含有该基因的1.8kb的聚合酶链式反应产物，该产物能够恢复每个突变体的耐酸性。这些融合是在进入指数后期时被诱导的，并受到rpos的正调控。我们证实，在酸化的微量培养基中，抗酸表型的表达依赖于谷氨酸的补充，并且这种谷氨酸依赖的系统是受rpos调控的。Southern杂交结果显示，沙门氏菌中没有GADC和hdeAB两个基因座。构建了福氏志贺氏菌rpos基因缺失突变体，以证实该基因在耐酸中的重要作用。这种rpos(-)衍生物对酸非常敏感。双向凝胶电泳法显示，该突变体在对数后期不再表达其等基因亲本中存在的27种蛋白质。这些数据表明，福氏志贺氏菌的耐酸性表达可能是多因素的，涉及位于不同亚细胞位置的蛋白质。
Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor sigma(s). Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS(-) derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.