Regulators of the Bacillus subtilis cydABCD Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD

Abstract

The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions ؊58 and ؊107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions ؊4 to ؊33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.The cydABCD operon of Bacillus subtilis is responsible for the production of intact cytochrome bd oxidase (36). cydAB encodes the structural proteins of the bd oxidase, while cydCD is proposed to encode an ABC transporter which is required for the assembly of cytochrome bd. A single cydA transcriptional start site was identified for this operon from cells grown to stationary phase in nutrient sporulation medium with phosphate buffer (pH 7.0) and glucose (NSMPG) (36). Candidate sequences for the E A Ϫ10 and Ϫ35 elements were identified. Expression of cytochrome bd was first observed under conditions of low oxygen availability and in cells grown in the presence of glucose (36). The role of YdiH (Rex) in the regulation of the cydABCD operon was determined through analysis of a suppressor of poor growth of a ⌬resDE mutant. We showed that YdiH (Rex) binds downstream of the transcriptional start site in a long untranslated sequence and appears to negatively regulate the operon (28). It was recently proposed that YdiH (Rex) is a redox sensor whose activity is regulated by the levels of NAD ϩ and NADH in the cell (11,17). YdiH has...

The Bacillus subtilis Response Regulator Gene degU Is Positively Regulated by CcpA and by Catabolite-Repressed Synthesis of ClpC

“…Both types of CcpA protein at similar concentrations were also reported to achieve protection from DNase I digestion in the ilvB promoter (52,53). In addition, there are several reports of specific DNA binding of His-CcpA to target sequences (36,(48)(49)(50), indicating the full functionality of in vitro DNA binding of His-CcpA. In fact, the His tag in His-CcpA increases DNA binding about 6-fold over that of CcpA in the absence of P-Ser-Hpr, while there is no stimulatory effect of P-SerHpr addition (57).…”

“…This result was consistent with the results of EMSA using the H4 probe. In previous binding studies ( [48][49][50][51], CcpA protected the relatively narrow regions containing the cre sequence, except for the cre sequence in the lev gene, for which a long footprint pattern was observed (about 65 bases). Thus, our results showed some differences from those of previous studies.…”

“…In most cases, CcpA does not bind specifically to its target if it is not associated with P-Ser-Hpr (2, 47). In several experiments, however, His-tagged CcpA bound specifically to its target without P-Ser-Hpr, probably because of the positive charge of histidines and/or structural changes in the protein (36,(48)(49)(50). Thus, we used His-CcpA for the analysis.…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…28) Moreover, the cydABCD operon, encoding cytochrome bd oxidase, was found to be directly repressed by CcpA. 57) Transcription of the resABCDE operon requires the PhoP/PhoR two-component system. 116) The PhoP-P response regulator directly binds to the cis-element of the res promoter and is essential for transcriptional activation of the resABCDE operon, as well as being involved in repression of the internal resDE promoter during phosphate-limited growth.…”

Genome-Wide Analysis of ResD, NsrR, and Fur Binding in Bacillus subtilis during Anaerobic Fermentative Growth by In Vivo Footprinting

“…As shown in Table 1, most of genes in group A, to which ResD binds both the promoter and coding regions, function in aerobic or anaerobic respiration and are controlled positively by ResD. These genes include hmp (8), nasD (19), narG (4), ctaA (heme A synthase gene) (20), ctaB (major heme O synthase gene) (21), qox (cytochrome aa 3 quinol oxidase gene) (22), and cydA (cytochrome bd ubiquinol oxidase gene) (23,24). Therefore, we speculated that ResD activates the transcription of all genes in this class.…”