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

The -dependent promoter in front of the rsbV gene of Bacillus subtilis is induced 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. 1. Introduction In Gram-positive bacteria such as species of Bacillus, the complex rsb operon encodes the stress sigma factor, σB, and many of its regulatory proteins [1, 2]. This operon includes two promoters, a σA-dependent promoter in front of the rsbR gene at the beginning of the operon, and a σB-dependent promoter in front of the rsbV gene, the fifth gene in this 8-cistron operon [3]. Much is known about the biochemical mechanism of sigma B control which involves an anti-σB, an anti-anti-σB, and protein phosphorylation [4–6]. Additionally, induction of the σBpromoter in part mediates adaptation to heat shock, (40–54 ), cold shock (4–12 ), NaCl (10%) shock, ethanol (4–10%) shock, and various starvation (carbon, phosphorous, and nitrogen) stresses [5, 7]. It has been argued that a drop in ATP levels may trigger σB activation [8]. Elements of this regulatory system are found in a wide range of bacteria which in addition to low and high G？+？C, Gram-positive bacteria include bacteroidetes, cyanobacteria, proteobacteria, and deinococci [2]. We have investigated the dependencies of various stress responses (ethanol stress and limitation for carbon or phosphorous), showing that these responses are dependent on the principal carbon-limitation transcription factor in B. subtilis, CcpA [9–11], as well as the principal nitrogen regulator TnrA [12]. However, we show that point mutations and deletions in the region upstream of the rsbV promoter, within the rsbU gene, largely abolish all of these stress responses. Because these studies were conducted with an rsbV-lacZ fusion reporter system integrated at the amyE locus, we can conclude that RsbU