Transcriptional Regulation of the rsbV Promoter Controlling Stress Responses to Ethanol, Carbon Limitation, and Phosphorous Limitation in Bacillus subtilis

Abstract

The sigma(B)-dependent promoter in front of the rsbV gene of Bacillus subtilis is induced approximately 5-fold in response to (1) the addition of 4% ethanol, (2) carbon starvation, and (3) phosphorous starvation. Binding sites for the global carbon and nitrogen regulators, CcpA and TnrA, were mutated, and the consequences of their loss and that of CcpA or TnrA were studied using rsbV-lacZ fusions. These responses proved to be dependent on CcpA, TnrA, and their putative binding sites upstream of the promoter. Induction in response to glucose limitation was largely abolished by loss of CcpA or the upstream region, while induction in response to phosphorous limitation was largely abolished only by the upstream mutations. The results suggest that CcpA directly influences the carbon starvation response and that both proteins exert indirect effects on all three stress responses. The integrity of the DNA sequence is important for all three responses.

Figure 1

Schematic depiction of the rsb or sigB operon in B. subtilis (top) as well as the constructs made in order to determine the involvement of putative CcpA and TnrA binding sites in control of σ ^B promoter activity in response to various stresses. The octacistronic operon as well as the two promoters, one dependent on σ ^A-RNA polymerase in front of the rsbR gene and one dependent on σ ^B-RNA polymerase in front of the rsbV gene, are shown. The wild-type control region (nucleotides −330 to +282) including the putative TnrA binding site (open arrow), the putative CcpA binding site (closed arrow), and the σ ^B promoter was fused to the lacZ gene encoding β-galactosidase as the reporter gene. The wild-type construct is designated by rsbV1. In the second construct, rsbV1m1, the CcpA binding site (CRE) was mutated, changing the central conserved C to an A (see sequence on right-hand side of the construct). In the rsbV3 construct, the control region started at position −238, and the TnrA binding site (TRE) was thereby deleted. In construct rsbV5, the control region started at position −215, and consequently the CRE site as well as the TnrA binding site was thereby deleted. All constructs were inserted in the chromosome at the amyE locus.

Figure 2

Effects of various mutations and conditions on the ethanol stress response of the σ ^B-dependent rsbV promoter with cells growing exponentially in LB medium. (a) At time = 0, ethanol was added to a final volume of 4%, giving rise to a 4-5-fold enhancement of promoter activity in the wild-type genetic background (rsbV1) (■). The mutations (Figure 1) decreased the basal as well as the induced activities of the promoter. Wild type (■); ccpA mutant (); hprK mutant (×); the rsbV1m1 mutant (◆); rsbV3 (lacking the TnrA binding site) (▾) rsbV5 (lacking the TnrA and CcpA binding sites) (▴). (b) Effects of the loss of CcpA (), TnrA (▴), or both transcription factors (◆) on the ethanol stress response of the σ ^B-dependent promoter in the rsb operon. (c) Effect of glucose on the ethanol stress response of the rsbV promoter. The experiment was conducted in LB medium with (closed symbols) and without (open symbols) 1% glucose. The wild type (squares) and ccpA mutant (circles) were examined. (d) Response of the σ ^B-dependent ctc gene to ethanol stress. A lacZ fusion was made to the ctc gene, and the response to 4% ethanol was measured in the wild-type background (■), the ccpA mutant background (), the rsbV1m1 genetic background (◆), and the rsbV5 genetic background (▴). The experiment was conducted in LB medium under standard conditions. Ethanol (4%) was added at t = 0.

Figure 3

Effects of various mutations and conditions on the glucose starvation response of the rsbV promoter. (a) Effects of the rsbV1m1 (◆), rsbV3 (▴), and rsbV5 (×) mutations in the rsbU (upstream) region in front of the rsbV promoter on promoter activity in response to glucose limitation as a function of time. The wild-type response (rsbV1, ■) is shown at the top. (b) The effects of ccpA (), tnrA (◆), and ccpA tnrA (▴) double knockout mutants on the glucose starvation response as a function of time. The wild-type response is represented by square symbols (■). (c) Effect of glucose limitation on expression of the ctc gene which is under sigma B (σ ^B) control, in the wild-type (■) or ccpA () genetic backgrounds.

Figure 4

Effects of various mutations and conditions on the phosphorous starvation response. (a) Wild-type response (rsbV1, ■) to phosphate limitation, and the effects of the rsbV1m1 (◆), rsbV3 (▴), and rsbV5 (×) mutations on this response as a function of time. (b) Effects of the loss of CcpA (), TnrA (◆), and both transcription factors (▴) as compared with wild type (■) on the phosphate limitation response. (c) Lack of effect of the loss of CcpA () on the phosphate limitation response of the ctc gene which is under sigma B (σ ^B) control. The wild-type response is represented by ■.