Deora R, Tseng T, Misra T K
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612, USA.
J Bacteriol. 1997 Oct;179(20):6355-9. doi: 10.1128/jb.179.20.6355-6359.1997.
A homolog of the multiple-stress-responsive transcription factor sigmaB of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter. A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated sigmaSB, indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis sigmaB-dependent PB promoter of the sigB operon was stimulated by sigmaSB in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted sigmaB-dependent PB promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins. Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary sigma factor sigmaSA, while that of the P3 promoter is dependent on sigmaSB. As determined by primer extension studies, the 5' end of the sigmaSB-initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5' end of the cellular RNA.
通过对金黄色葡萄球菌染色体区域的DNA序列分析，预测出了枯草芽孢杆菌多重应激反应转录因子σB的一个同源物。构建了噬菌体T7基因10前导肽前11个氨基酸的编码序列（T7.Tag）与金黄色葡萄球菌假定的sigB基因之间的杂种，并将其克隆到大肠杆菌BL21(DE3)pLysS中，以便从T7启动子进行过量表达。通过用与琼脂糖偶联的T7.Tag单克隆抗体进行亲和层析，获得了过量产生蛋白的纯品。纯化蛋白前22个残基的氨基末端氨基酸序列与从核苷酸序列推导的序列相符。纯化后的蛋白命名为σSB，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明它以约39 kDa的多肽形式迁移。当与金黄色葡萄球菌核心RNA聚合酶（RNAP）重组时，σSB以浓度依赖的方式刺激枯草芽孢杆菌sigB操纵子中依赖于σB的PB启动子的启动子特异性转录。在径流转录反应中，重组的RNAP从金黄色葡萄球菌sigB操纵子预测的依赖于σB的PB启动子获得了特异性转录本。金黄色葡萄球菌的sar操纵子包含三个启动子元件（P1、P2和P3），已知它部分控制多种细胞外毒素和几种细胞壁蛋白的合成。我们的体外研究表明，P1启动子的转录依赖于主要的σ因子σSA，而P3启动子的转录则依赖于σSB。通过引物延伸研究确定，体外从sar P3启动子合成的由σSB起始的mRNA的5'端与细胞RNA的5'端一致。
