Varón D, Boylan S A, Okamoto K, Price C W
Department of Food Science and Technology, University of California, Davis 95616.
J Bacteriol. 1993 Jul;175(13):3964-71. doi: 10.1128/jb.175.13.3964-3971.1993.
Transcription factor sigma B of Bacillus subtilis controls a large stationary-phase regulon, but in no case has the physiological function of any gene in this regulon been identified. Here we show that transcription of gtaB is partly dependent on sigma B in vivo and that gtaB encodes UDP-glucose pyrophosphorylase. The gtaB reading frame was initially identified by a sigma B-dependent Tn917lacZ fusion, csb42. We cloned the region surrounding the csb42 insertion, identified the reading frame containing the transposon, and found that this frame encoded a predicted 292-residue product that shared 45% identical residues with the UDP-glucose pyrophosphorylase of Acetobacter xylinum. The identified reading frame appeared to lie in a monocistronic transcriptional unit. Primer extension and promoter activity experiments identified tandem promoters, one sigma B dependent and the other sigma B independent, immediately upstream from the proposed coding region. A sequence resembling a factor-independent terminator closely followed the coding region. By polymerase chain reaction amplification of a B. subtilis genomic library carried in yeast artificial chromosomes, we located the UDP-glucose pyrophosphorylase coding region near gtaB, mutations in which confer phage resistance due to decreased glycosylation of cell wall teichoic acids. Restriction mapping showed that the coding region overlapped the known location of gtaB. Sequence analysis of a strain carrying the gtaB290 allele found an alteration that would change the proposed initiation codon from AUG to AUA, and an insertion-deletion mutation in this frame conferred phage resistance indistinguishable from that elicited by the gtaB290 mutation. We conclude that gtaB encodes UDP-glucose pyrophosphorylase and is partly controlled by sigma B. Because this enzyme is important for thermotolerance and osmotolerance in stationary-phase Escherichia coli cells, our results suggest that some genes controlled by sigma B may play a role in stationary-phase survival of B. subtilis.
枯草芽孢杆菌的转录因子σB控制着一个大型的稳定期调节子，但该调节子中任何基因的生理功能都尚未被确定。在此我们表明，gtaB的转录在体内部分依赖于σB，并且gtaB编码UDP-葡萄糖焦磷酸化酶。gtaB的阅读框最初是通过一个依赖于σB的Tn917lacZ融合体csb42鉴定出来的。我们克隆了csb42插入位点周围的区域，确定了包含转座子的阅读框，并发现该阅读框编码一个预测的292个氨基酸的产物，它与木醋杆菌的UDP-葡萄糖焦磷酸化酶有45%的相同氨基酸残基。所鉴定的阅读框似乎位于一个单顺反子转录单元中。引物延伸和启动子活性实验确定了串联启动子，一个依赖于σB，另一个不依赖于σB，就在拟编码区的上游。一个类似于不依赖因子的终止子的序列紧跟在编码区之后。通过对酵母人工染色体中携带的枯草芽孢杆菌基因组文库进行聚合酶链反应扩增，我们将UDP-葡萄糖焦磷酸化酶编码区定位在gtaB附近，该区域的突变由于细胞壁磷壁酸糖基化减少而赋予噬菌体抗性。限制性图谱分析表明，编码区与gtaB的已知位置重叠。对携带gtaB290等位基因的菌株进行序列分析发现了一个改变，该改变会将拟起始密码子从AUG变为AUA，并且该阅读框中的一个插入缺失突变赋予的噬菌体抗性与gtaB290突变所引发的抗性无法区分。我们得出结论，gtaB编码UDP-葡萄糖焦磷酸化酶并且部分受σB控制。因为这种酶对稳定期大肠杆菌细胞的耐热性和耐渗透压性很重要，我们的结果表明，一些受σB控制的基因可能在枯草芽孢杆菌的稳定期存活中发挥作用。
