Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone.

An Escherichia coli mutant lacking HSP70 function, delta dnaK52, is unable to grow at both high and low temperatures and, at intermediate temperature (30 degrees C), displays defects in major cellular processes such as cell division, chromosome segregation and regulation of heat shock gene expression that lead to poor growth and genetic instability of the cells. In an effort to understand the roles of molecular chaperones such as DnaK in cellular metabolism, we analyzed secondary mutations (sid) that suppress the growth defects of delta dnaK52 mutants at 30 degrees C and also permit growth at low temperature. Of the five suppressors we analyzed, four were of the sidB class and mapped within rpoH, which encodes the heat shock specific sigma subunit (sigma 32) of RNA polymerase. The sidB mutations affected four different regions of the sigma 32 protein and, in one case, resulted in a several fold reduction in the cellular concentration of sigma 32. Presence of any of the sidB mutations in delta dnaK52 mutants as well as in dnaK+ cells caused down-regulation of heat shock gene expression at 30 degrees C and decreased induction of the heat shock response after shift to 43.5 degrees C. These findings suggest that the physiologically most significant function of DnaK in the metabolism of unstressed cells is its function in heat shock gene regulation.     

腾冲嗜热厌氧菌热休克蛋白60及其基因表达的热激动态变化分析

热休克蛋白60(HSP60)是细菌体内一种非常重要的分子伴侣,其可以协助蛋白质或肽链的正确折叠和构型,防止变性和降解。基于本实验室的早期观察,腾冲嗜热厌氧菌的HSP60是一个典型的温度相关蛋白质,在80℃的表达水平最高。为了进一步了解嗜热菌应急的分子机制,继续进行了在热激后HSP60基因表达的动态研究。将最适温度(75℃)下培养的腾冲嗜热厌氧菌迅速地转移至80℃继续培养,然后在不同的时间点上分别取样,并通过双向电泳、Western blot和Real_time PCR等方法,分析了HSP60在mRNA和蛋白质水平上的表达量的改变。试验结果表明,在80℃热处理4h内的短期应急过程中,HSP60蛋白水平一直呈上升趋势,而它的mRNA水平则表现为先升高后下降的一个非对称性的峰形变化。HSP60的mRNA和蛋白质的对温度的应答快慢程度是不同的。HSP60的mRNA水平的显著变化在1h内便可观察到,而蛋白质水平的显著改变要延迟3h左右。此外,HSP60的mRNA和蛋白质对温度的应答量变大小也是不同的。     

Role of RpoH, a heat shock regulator protein, in Escherichia coli carbon starvation protein synthesis and survival.

Escherichia coli starvation proteins include several heat shock proteins whose induction by heat is controlled by the minor sigma factor, sigma 32. The level of sigma 32 increased in wild-type E. coli upon starvation, and three sigma 32-controlled heat shock proteins (DnaK, GroEL, and HtpG) were not induced during starvation in an isogenic delta rpoH strain, which is unable to synthesize sigma 32. Thus, sigma 32 plays a role in the induction of these proteins during both heat shock and starvation. The delta rpoH strain was more sensitive to starvation but could develop starvation-mediated cross protection against heat and oxidation.