Rezuchova Bronislava, Skovierova Henrieta, Homerova Dagmar, Roberts Mark, Kormanec Jan
Institute of Molecular Biology, Slovak Academy of Science, Bratislava, Slovak Republik.
Mol Genet Genomics. 2009 Aug;282(2):119-29. doi: 10.1007/s00438-009-0450-x. Epub 2009 May 5.
The alternative sigma factor sigma(E) is critical for envelope stress response and plays a role in pathogenicity of a variety of different bacteria. We previously identified several critical nucleotides in the Salmonella enterica serovar Typhimurium (S. Typhimurium) sigma(E)-dependent rpoEp3 promoter that corresponded to the most conserved nucleotides in the sigma(E) consensus sequence of the -10 and -35 promoter elements. In the present study, we exploited a previously established Escherichia coli (E. coli) two-plasmid system with an error-prone PCR mutagenesis to identify mutants in the rpoE gene that suppress the mutation of the most conserved residue A-30G of the rpoEp3 promoter. This analysis identified amino-acid changes in the conserved arginine residue (R171G, R171C) located in the conserved region 4.2 of sigma(E) that enabled efficient recognition of the mutated rpoEp3 promoter. However, the change of this conserved arginine to alanine (R171A) resulted in an almost complete loss of sigma(E) activity. The activity of the mutant sigma(E) factors in directing transcription of the wild-type (WT) and the A-30G mutated rpoEp3 promoters was investigated by S1-nuclease mapping using RNA isolated from the E. coli two-plasmid system. In addition to suppression of the A-30G mutated rpoEp3 promoter, both mutant sigma factors (R171G, R171C) also efficiently directed transcription from the WT rpoEp3 promoter and from the rpoEp3 promoter with other mutations in the -35 element, indicating relaxed recognition of the sigma(E)-dependent promoters by both mutants. The activity of both mutant sigma(E) factors was confirmed in vivo in S. Typhimurium. In conclusion, replacement of the conserved R171 residue in sigma(E) by different amino-acid residues exhibited intriguingly different phenotypes; R171A almost completely abolished sigma factor activity, whereas R171G and R171C impart a relaxed recognition phenotype to sigma(E).
替代σ因子σ(E)对包膜应激反应至关重要，并在多种不同细菌的致病性中发挥作用。我们之前在鼠伤寒沙门氏菌（S. Typhimurium）的σ(E)依赖性rpoEp3启动子中鉴定了几个关键核苷酸，这些核苷酸对应于σ(E)共有序列中-10和-35启动子元件中最保守的核苷酸。在本研究中，我们利用先前建立的带有易错PCR诱变的大肠杆菌（E. coli）双质粒系统，来鉴定rpoE基因中的突变体，这些突变体可抑制rpoEp3启动子中最保守残基A-30G的突变。该分析确定了位于σ(E)保守区域4.2中的保守精氨酸残基（R171G、R171C）的氨基酸变化，这些变化能够有效识别突变的rpoEp3启动子。然而，将这个保守精氨酸替换为丙氨酸（R171A）导致σ(E)活性几乎完全丧失。通过使用从大肠杆菌双质粒系统中分离的RNA进行S1核酸酶图谱分析，研究了突变的σ(E)因子指导野生型（WT）和A-30G突变的rpoEp3启动子转录的活性。除了抑制A-30G突变的rpoEp3启动子外，两个突变的σ因子（R171G、R171C）还能有效地指导WT rpoEp增3启动子以及-35元件中带有其他突变的rpoEp3启动子的转录，这表明这两个突变体对σ(E)依赖性启动子的识别较为宽松。两种突变的σ(E)因子的活性在鼠伤寒沙门氏菌体内得到了证实。总之，用不同氨基酸残基替换σ(E)中保守的R171残基表现出了有趣的不同表型；R171A几乎完全消除了σ因子活性，而R171G和R171C赋予了σ(E)一种宽松识别表型。
