Bacillus subtilis YdiH Is a Direct Negative Regulator of the cydABCD Operon

Abstract

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...

Direct Regulation ofBacillus subtilis phoPRTranscription by Transition State Regulator ScoC

“…Reduced quinones were shown to inhibit autophosphorylation of the PhoR in vitro, suggesting that it was the ResD role in terminal oxidase production that positively modulates the PhoR signal (35) upstream of PhoPR. Consistent with this idea, resD mutants containing a spontaneous mutation in rex (formerly ydiH), a repressor of cydABCD encoding bd oxidase (34), allowed expression of cydABCD during Pho induction, which bypassed the requirement for ResD for full Pho induction (35). Together, these data indicate that the terminal oxidase bd, encoded by cydABCD, was sufficient to replace the loss of caa 3 and aa 3 in the resD mutant strain by restoring the terminal oxidase function of oxidation of reduced quinones that inhibit PhoR autophosphorylation.…”

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“…Rex homologues exist in most gram-positive bacteria. In B. subtilis, a Rex homolog, YdiH, encoded by the ydiH gene acts as a repressor for cydABCD transcription under aerobic growth conditions (27 of B. subtilis acts as a negative regulator of cydABCD, ldh-lctP, and ywcJ and coordinates the expression of these genes during the transition from aerobic to anaerobic growth (15). DNase I footprinting analysis revealed three binding sites of YdiH in the cydA promoter region, and a consensus sequence was proposed.…”

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