Antelmann H, Bernhardt J, Schmid R, Hecker M
Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universität, Greifswald, Germany.
J Bacteriol. 1995 Jun;177(12):3540-5. doi: 10.1128/jb.177.12.3540-3545.1995.
In Bacillus subtilis, general stress proteins (Gsps) are induced in response to different stresses (heat, salt, or ethanol) or after nutrient starvation. The majority of the genes for the Gsps are organized in a very large stationary-phase or stress regulon which is controlled by alternative sigma factor sigma B. The most striking spots on Coomassie-stained two-dimensional gels belong to GsiB and GspA, which are synthesized at extremely high levels in response to different stresses. Therefore, we determined the N-terminal protein sequence of GspA, which exhibited total identity to a hypothetical 33.5-kDa protein of B. subtilis encoded by open reading frame 2 (ipa-12d) in the sacY-tyrS1 intergenic region. The GspA-encoding gene gspA and the upstream and downstream regions were cloned with the aid of the PCR technique. By primer extension experiments, one sigma B-dependent promoter immediately upstream of the coding region was identified. A putative factor-independent terminator closely followed the coding region. By Northern (RNA) blot analysis, a 0.95-kb transcript was detected which indicates a monocistronic transcriptional unit. The gspA mRNA was strongly induced by different stimuli like heat or salt stress and starvation for glucose. Analysis of RNA isolated from a sigma B deletion mutant revealed that the transcription of gspA is sigma B dependent. Insertional inactivation of the B. subtilis chromosomal gspA gene confirmed that the gspA gene is not essential for either vegetative growth or growth under the influence of different stresses. In gspA mutant cells, the level of flagellin was increased severalfold over that in wild-type cells.
在枯草芽孢杆菌中，一般应激蛋白（Gsps）会在响应不同应激（热、盐或乙醇）或营养饥饿后被诱导产生。大多数Gsps基因被组织在一个非常大的稳定期或应激调节子中，该调节子由替代sigma因子sigma B控制。考马斯亮蓝染色的二维凝胶上最明显的斑点属于GsiB和GspA，它们在响应不同应激时以极高水平合成。因此，我们确定了GspA的N端蛋白序列，该序列与枯草芽孢杆菌在sacY-tyrS1基因间区域由开放阅读框2（ipa-12d）编码的一个假定的33.5 kDa蛋白完全相同。借助PCR技术克隆了编码GspA的基因gspA及其上下游区域。通过引物延伸实验，在编码区上游立即鉴定出一个依赖sigma B的启动子。一个假定的不依赖因子的终止子紧跟在编码区之后。通过Northern（RNA）印迹分析，检测到一个0.95 kb的转录本，这表明是一个单顺反子转录单元。gspA mRNA在热或盐应激以及葡萄糖饥饿等不同刺激下被强烈诱导。对从sigma B缺失突变体中分离的RNA的分析表明，gspA的转录依赖于sigma B。枯草芽孢杆菌染色体gspA基因的插入失活证实，gspA基因对于营养生长或在不同应激影响下的生长都不是必需的。在gspA突变体细胞中，鞭毛蛋白的水平比野生型细胞中增加了几倍。
