During aerobic respiration, Bacillus subtilis utilizes three terminal oxidases, cytochromes aa 3 , caa 3 , and bd. Cytochrome bd is encoded by the cydABCD operon. We report here the first identification of a regulator for the cydABCD operon, YdiH. While working with ⌬resDE mutant strains, we identified colonies which contained suppressor mutations (cmp) which bypassed the requirement for ResD for all phenotypes not associated with cytochrome aa 3 or caa 3 . Mapping identified a class of Tn10 insertions which were close to the cmp locus (Tn10-2) and a second class (Tn10-1) which was inserted in cydD, a gene which appears to be essential to the cmp phenotype. Sequencing of the cmp loci from four independent ⌬resDE cmp isolates yielded four loss-offunction alleles of ydiH, a gene encoding a protein with homology to AT-rich DNA-binding proteins. Additionally, we determined that cytochrome bd was aberrantly expressed in the ⌬resDE cmp background. Together these data led to the hypothesis that YdiH serves as a negative regulator of cydABCD expression, a hypothesis supported by both gel-shift and DNase I footprinting analyses. YdiH protected the cydA promoter region at three 22-bp repeats located in the long 5 untranslated region (193 bp). Induction of the cydABCD operon in a ⌬resDE background showed that expression of the terminal oxidase bd was responsible for the bypass phenotype observed in a ⌬resDE cmp strain, indicating that cytochrome bd expression complemented the loss of cytochromes aa 3 and caa 3 in the ⌬resDE strain.Bacillus subtilis utilizes a branched electron transport chain under aerobic conditions. To date, three terminal oxidases have been identified in B. subtilis. Both cytochromes aa 3 (26) and caa 3 (5) have been identified as heme-copper oxidases. The third oxidase has been shown to be a member of the cytochrome bd family (35). The cydABCD operon of B. subtilis encodes cytochrome bd and a putative ABC transporter required for the production of functional cytochrome bd (35). This oxidase is produced under conditions of low oxygen tension and in cells grown in the presence of glucose (35). A single cydA transcriptional start site with a putative Ϫ10 and Ϫ35 consensus for a A promoter has been found in cells grown to stationary phase in nutrient sporulation medium with phosphate buffer and glucose (NSMPG) (35). A perfect 16-bp palindromic sequence, upstream of the translation start site for cydA, was proposed as a potential operator binding site for a regulatory protein (35). To date, no regulators have been reported for the cydABCD operon. It was originally reported that the quinol oxidases (either cytochrome aa 3 or bd), are required for aerobic growth in B. subtilis (34). However, further evidence has shown that a strain deficient in the production of both cytochrome aa 3 and cytochrome bd, a derivative of the B. subtilis 168 strain (24), can be constructed and grown aerobically (37). A putative fourth terminal oxidase, YthAB, has been found in B. subtilis and is a member of the cytochrome bd famil...
The Bacillus subtilis Pho signal transduction network, which regulates the cellular response to phosphate starvation, integrates the activity of three signal transduction systems to regulate the level of the Pho response. This signal transduction network includes a positive feedback loop between the PhoP/PhoR and ResD/ResE two-component systems. Within this network, ResD is responsible for 80% of the Pho response. To date, the role of ResD in the generation of the Pho response has not been understood. Expression of two terminal oxidases requires ResD function, and expression of at least one terminal oxidase is needed for the wild-type Pho response. Previously, our investigators have shown that strains bearing mutations in resD are impaired for growth and acquire secondary mutations which compensate for the loss of the a-type terminal oxidases by allowing production of cytochrome bd. We report here that the expression of cytochrome bd in a ⌬resDE background is sufficient to compensate for the loss of ResD for full Pho induction. A ctaA mutant strain, deficient in the production of heme A, has the same Pho induction phenotype as a ⌬resDE strain. This demonstrates that the production of a-type terminal oxidases is the basis for the role of ResD in Pho induction. Terminal oxidases affect the redox state of the quinone pool. Reduced quinones inhibit PhoR autophosphorylation in vitro, consistent with a requirement for terminal oxidases for full Pho induction in vivo.The Bacillus subtilis phosphate starvation response (Pho response) is under the control of a complex regulatory network that allows the cell to respond to the level of inorganic phosphate (P i ) in the environment. This system is critical to survival because phosphate is the limiting nutrient in soil (33), the natural environment for B. subtilis.Central to the B. subtilis Pho response is the PhoP/PhoR two-component signal transduction system. The phoPR operon (23, 43) is subject to activation by PhoP under phosphate starvation conditions (34). PhoP/PhoR directly regulates the expression of genes involved in the cellular response to phosphate starvation. The histidine kinase, PhoR, is autophosphorylated in response to an environmental signal and then phosphorylates its cognate response regulator, PhoP. PhoPϳP activates the transcription of the alkaline phosphatases (19), phoA (formerly phoAIV) (20), and phoB (formerly phoAIII) (7); phosphodiesterases, phoD (11), and glpQ (1); a high-affinity phosphate transport system, pstS (37); teichuronic acid synthetic genes (teichuronic acid is a cell wall polymer lacking phosphate), tuaABCDEFGH (27, 46); and a gene encoding a 60-residue peptide of unknown function, ykoL (38). PhoPϳP has been shown to repress the expression of the tagAB and tagDEF genes responsible for the production of teichoic acid (a cell wall polymer containing phosphate) (26). The collective action of these products allows the cell to scavenge extracellular phosphate and to release additional P i from the cell wall.The regulation of the Pho respon...
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...
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