When confronted with a stress factor, bacteria react with a specific stress response, a genetically encoded programme resulting in the transiently enhanced expression of a subset of genes. One of these stress factors is a sudden increase in the external pH. As a first step to understand the response of Bacillus subtilis cells towards an alkali shock at the transcriptional level, we attempted to identify alkali‐inducible genes using the DNA macroarray technique. To define the appropriate challenging conditions, we used the ydjF gene, the orthologue of the Escherichia coli pspA, as a model gene for an alkali‐inducible gene. Hybridization of 33P‐labelled cDNA to a DNA macroarray revealed induction of more than 80 genes by a sudden increase in the external pH value from 6.3 to 8.9. It was discovered that a large subset of these genes belong to the recently described σW regulon, which was confirmed by the analysis of a sigW knockout. A comparison of B. subtilis wild type with the congenic sigW knockout also led to the discovery of new members of the σW regulon. In addition, we found several genes clearly not belonging to that regulon. This analysis represents the first report of an extracellular stimulus inducing the σW regulon.

To study the influence of the htpG gene on thermal stress management in Bacillus subtilis, two different kinds of htpG mutation were constructed. In one case, the gene was inactivated by insertion of a cat cassette in to the coding region; htpG was thus found to be non-essential. In the second case, the htpG gene was fused to a xylose-dependent promoter, allowing expression of the gene to be controlled. In the absence of HtpG protein, recovery of cells from a heat shock at 53 degrees C was retarded, and this delay could be eliminated by overproduction of HtpG. While htpG is not involved in the development of induced thermotolerance, DnaK and GroE proteins are absolutely required. Overproduction of class I heat-shock proteins prior to shifting cells to a lethal temperature is important but not sufficient for the development of intrinsic thermotolerance. It could be shown that the HtpG protein does not act as a cellular thermometer in B. subtilis.

We show that the htpG gene of Bacillus subtilis is induced by heat, as has been reported for the Escherichia coli homolog. Analysis of different mutants revealed that the htpG gene belongs to class III heat shock genes in B. subtilis. An about 10-fold induction after thermal upshock was found at the levels of both transcription and translation, and this induction resulted from enhanced synthesis of mRNA. By primer extension, we identified one potential transcription start site immediately downstream of a putative A -dependent promoter which became activated after thermal upshift. Northern blot analysis revealed that htpG is part of a monocistronic transcriptional unit. An operon fusion where the complete region between htpG and its upstream gene was fused to the bgaB reporter gene accurately reflected htpG expression. Analysis of this fusion revealed that, in contrast to other class III heat shock genes, htpG was not induced by osmotic upshock, by ethanol, or by oxygen limitation, suggesting that it belongs to a subgroup within class III. Deletion of the region upstream of the putative promoter resulted in an enhanced basal level of htpG expression, but the 10-fold induction was retained, suggesting that the upstream sequences are involved in the regulation of expression in the absence of heat shock.Organisms as diverse as bacteria, animals, and plants respond to elevated temperatures and to a variety of chemical and physiological stresses by a rapid and transient increase in the synthesis of a set of conserved polypeptides collectively referred to as heat shock proteins (Hsps). The conservation of Hsps between bacteria and eukaryotic organisms suggests that they had an ancient function that was essential for survival throughout evolution. Indeed, most members of the Hsp family are synthesized and accumulate as abundant proteins in the cell even under normal conditions of growth and have essential functions as molecular chaperones involved in protein folding, translocation, higher-order assembly, and protein degradation (4,8,9,12,25).Besides elucidating the action of Hsps, another effort focuses on the regulation of the heat shock genes which are tightly controlled at the level of transcription. In Escherichia coli, most Hsp genes are under the control of a specific transcription factor, 32 , which directs the bacterial core RNA polymerase to heat shock promoters, and these genes constitute the sigma-32 regulon (for recent reviews, see references 3 and 45).32 is an unstable protein under normal conditions, and its concentration is transiently increased by changes in translational efficiency and protein stability during heat shock.In Bacillus subtilis, three classes of heat shock genes have been identified and found to be regulated by different mechanisms (for recent reviews, see references 11, 32, and 33). Class I heat shock genes are negatively regulated at the level of transcription, and their regulation involves the HrcA protein (30) interacting with an inverted repeat DNA sequence (6, 44) that we have design...

The heat shock genes of Bacillus subtilis are assigned to four classes on the basis of their regulation mechanisms. While classes I and III are negatively controlled by two different transcriptional repressors, class II is regulated by the alternative sigma factor B . All heat shock genes with unidentified regulatory mechanisms, among them htpG, constitute class IV. Here, we show that expression of htpG is under positive control. We identified a DNA sequence (GAAAAGG) located downstream of the A -dependent promoter of htpG. The heat inducibility of the promoter could be destroyed by inversion, nucleotide replacements, or removal of this DNA sequence. Fusion of this sequence to the vegetative lepA promoter conferred heat inducibility. Furthermore, we were able to show that the heat induction factor is dependent on the absolute temperature rather than the temperature increment and that nonnative proteins within the cytoplasm fail to induce htpG.