Helmann J D, Márquez L M, Chamberlin M J
Department of Biochemistry, University of California, Berkeley 94720.
J Bacteriol. 1988 Apr;170(4):1568-74. doi: 10.1128/jb.170.4.1568-1574.1988.
Bacillus subtilis contains multiple forms of RNA polymerase holoenzyme, distinguished by the presence of different specificity determinants known as sigma factors. The sigma 28 factor was initially purified as a unique transcriptional activity in vegetatively growing B. subtilis cells. Purification of the sigma 28 protein has allowed tryptic peptides to be prepared and sequenced. The sequence of one tryptic peptide fragment was used to prepare an oligonucleotide probe specific for the sigma 28 structural gene, and the gene was isolated from a B. subtilis subgenomic library. The complete nucleotide sequence of the sigma 28 gene was determined, and the cloned sigma 28 gene was used to construct a mutant strain which does not express the sigma 28 protein. This strain also failed to synthesize flagellin protein and grew as long filaments. The predicted sigma 28 gene product is a 254-amino-acid polypeptide with a calculated molecular weight of 29,500. The sigma 28 protein sequence was similar to that of other sequenced sigma factors and to the flbB gene product of Escherichia coli. Since the flbB gene product is a positive regulator of flagellar synthesis in E. coli, it is likely that sigma 28 functions to regulate flagellar synthesis in B. subtilis.
枯草芽孢杆菌含有多种形式的RNA聚合酶全酶，它们通过存在不同的特异性决定因子（称为σ因子）来区分。σ28因子最初是作为在营养生长的枯草芽孢杆菌细胞中的一种独特转录活性而被纯化的。σ28蛋白的纯化使得能够制备并测序胰蛋白酶肽段。一个胰蛋白酶肽段的序列被用于制备针对σ28结构基因的寡核苷酸探针，并且该基因从枯草芽孢杆菌亚基因组文库中被分离出来。确定了σ28基因的完整核苷酸序列，并且使用克隆的σ28基因构建了一个不表达σ28蛋白的突变菌株。该菌株也无法合成鞭毛蛋白，并且长成了长丝。预测的σ28基因产物是一个254个氨基酸的多肽，计算分子量为29,500。σ28蛋白序列与其他已测序的σ因子以及大肠杆菌的flbB基因产物相似。由于flbB基因产物是大肠杆菌中鞭毛合成的正调控因子，因此σ28可能在枯草芽孢杆菌中发挥调节鞭毛合成的作用。
