Role of bkdR, a transcriptional activator of the sigL-dependent isoleucine and valine degradation pathway in Bacillus subtilis

Abstract

A new gene, bkdR (formerly called yqiR), encoding a regulator with a central (catalytic) domain was found in Bacillus subtilis. This gene controls the utilization of isoleucine and valine as sole nitrogen sources. Seven genes, previously called yqiS, yqiT, yqiU, yqiV, bfmBAA, bfmBAB, and bfmBB and now referred to as ptb, bcd, buk, lpd, bkdA1, bkdA2, and bkdB, are located downstream from the bkdR gene in B. subtilis. The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1alpha (dehydrogenase), E1beta (decarboxylase), and E2 (dihydrolipoamide acyltransferase). Isoleucine and valine utilization was abolished in bcd and bkdR null mutants of B. subtilis. The seven genes appear to be organized as an operon, bkd, transcribed from a -12, -24 promoter. The expression of the bkd operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family. Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene bkdR. Deletion analysis showed that upstream activating sequences are involved in the expression of the bkd operon and are probably the target of bkdR. Transcription of the bkd operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions.

FIG. 1

Proposed pathway for the degradation of branched-chain amino acids in B. subtilis.

FIG. 2

Organization of the structural genes of the bkd operon of B. subtilis. The proposed functions of the gene products are based on similarities to the corresponding genes from Enterococcus faecalis (36). CoA, coenzyme A.

FIG. 3

Reverse transcriptase mapping of the transcriptional start site of the ptb gene in B. subtilis 168 grown in the presence (lane A) or absence (lane B) of 20 mM isoleucine. The positions of the cDNA-extended fragments identified with oligonucleotide O₁ were compared with those obtained by sequencing of an M13 recombinant phage containing the promoter region with the same oligonucleotide used as a primer (lanes at left, TCGA, respectively, from left to right). Transcriptional start sites are indicated by the asterisks.

FIG. 4

Nucleotide sequences of promoter regions of the levanase operon (plev), the rocABC operon (procABC), the rocDEF operon (procDEF), and the bkd operon (pbkd). The transcriptional start sites are indicated by arrows. Boxes indicate strictly conserved DNA sequences around positions −12 and −24 with respect to the transcriptional start sites.

FIG. 5

Nucleotide sequence of the ptb upstream region. Deletion end points are indicated by arrows and numbered with respect to the transcriptional start site (indicated by asterisks). The sequences at positions −12 and −24 are indicated. Boxed regions indicate putative UAS. The effects of upstream deletions on the expression of the ptb′-′lacZ translational fusions are expressed as β-galactosidase specific activities, which were determined with extracts prepared from cells growing exponentially in MM minimal medium containing glucose and 20 mM isoleucine as the inducer. In the absence of isoleucine, the basal level was about 5 U/mg of protein for each strain. For deletion end points ΔA (−232), ΔB (−122), ΔC (−107), ΔD (−92), and ΔE (−77), β-galactosidase activities were 2,350, 2,135, 205, 5, and 10 U/mg of protein, respectively.

FIG. 6

Alignment of PAS-like domains of RocR and BkdR from B. subtilis. Conserved amino acid residues are boxed. Asterisks indicated the locations of constitutive missense mutations in RocR.