van Schaik Willem, Tempelaars Marcel H, Wouters Jeroen A, de Vos Willem M, Abee Tjakko
Wageningen Centre for Food Sciences. Laboratory of Food Microbiology, Wageningen University, Wageningen, The Netherlands.
J Bacteriol. 2004 Jan;186(2):316-25. doi: 10.1128/JB.186.2.316-325.2004.
A gene cluster encoding the alternative sigma factor sigma(B), three predicted regulators of sigma(B) (RsbV, RsbW, and RsbY), and one protein whose function is not known (Orf4) was identified in the genome sequence of the food pathogen Bacillus cereus ATCC 14579. Western blotting with polyclonal antibodies raised against sigma(B) revealed that there was 20.1-fold activation of sigma(B) after a heat shock from 30 to 42 degrees C. Osmotic upshock and ethanol exposure also upregulated sigma(B), albeit less than a heat shock. When the intracellular ATP concentration was decreased by exposure to carbonyl cyanide m-chlorophenylhydrazone (CCCP), only limited increases in sigma(B) levels were observed, revealing that stress due to ATP depletion is not an important factor in sigma(B) activation in B. cereus. Analysis of transcription of the sigB operon by Northern blotting and primer extension revealed the presence of a sigma(B)-dependent promoter upstream of the first open reading frame (rsbV) of the sigB operon, indicating that transcription of sigB is autoregulated. A second sigma(B)-dependent promoter was identified upstream of the last open reading frame (orf4) of the sigB operon. Production of virulence factors and the nonhemolytic enterotoxin Nhe in a sigB null mutant was the same as in the parent strain. However, sigma(B) was found to play a role in the protective heat shock response of B. cereus. The sigB null mutant was less protected against the lethal temperature of 50 degrees C by a preadaptation to 42 degrees C than the parent strain was, resulting in a more-than-100-fold-reduced survival of the mutant after 40 min at 50 degrees C.
在食源性病原体蜡样芽孢杆菌ATCC 14579的基因组序列中，鉴定出一个基因簇，该基因簇编码替代σ因子σ(B)、三个预测的σ(B)调节因子（RsbV、RsbW和RsbY）以及一个功能未知的蛋白质（Orf4）。用针对σ(B)产生的多克隆抗体进行蛋白质印迹分析表明，在从30℃热激至42℃后，σ(B)有20.1倍的激活。渗透上调和乙醇暴露也会上调σ(B)，尽管上调程度低于热激。当通过暴露于羰基氰化物间氯苯腙（CCCP）降低细胞内ATP浓度时，仅观察到σ(B)水平有限增加，这表明ATP耗竭引起的应激不是蜡样芽孢杆菌中σ(B)激活的重要因素。通过Northern印迹和引物延伸分析sigB操纵子的转录，发现在sigB操纵子第一个开放阅读框（rsbV）上游存在一个依赖于σ(B)的启动子，这表明sigB的转录是自我调节的。在sigB操纵子最后一个开放阅读框（orf4）上游鉴定出第二个依赖于σ(B)的启动子。sigB基因缺失突变体中致病因子和非溶血肠毒素Nhe的产生与亲本菌株相同。然而，发现σ(B)在蜡样芽孢杆菌的保护性热激反应中起作用。与亲本菌株相比，sigB基因缺失突变体通过预先适应42℃对50℃致死温度的保护作用较小，导致该突变体在50℃下40分钟后的存活率降低了100倍以上。
