Role of CodY in regulation of the Bacillus subtilis hut operon.

Bacillus subtilis mutants deficient in amino acid repression of the histidine utilization (hut) operon were isolated by transposon mutagenesis. Genetic characterization of these mutants indicated that they most likely contained transposon insertions within the codVWXY operon. The codY gene is required for nutritional regulation of the dipeptide permease (dpp) operon. An examination of hut expression in a delta codY mutant demonstrated that amino acid repression exerted at the hutOA operator, which lies immediately downstream of the hut promoter, was defective in a delta codY mutant. The codY gene product was not required for amino acid regulation of either hut induction or the expression of proline oxidase, the first enzyme in proline degradation. This indicates that more than one mechanism of amino acid repression is present in B. subtilis. An examination of dpp and hut expression in cells during exponential growth in various media revealed that the level of CodY-dependent regulation appeared to be related to the growth rate of the culture.     

Catabolite repression of the Bacillus subtilis gnt operon mediated by the CcpA protein.

Inducer exclusion was not important in catabolite repression of the Bacillus subtilis gnt operon. The CcpA protein (also known as AlsA) was found to be necessary for catabolite repression of the gnt operon, and a mutation (crsA47, which is an allele of the sigA gene) partially affected this catabolite repression.     

Mechanism of CcpA-mediated glucose repression of the resABCDE operon of Bacillus subtilis

The resABCDE operon of Bacillus subtilis encodes a three-protein complex involved in cytochrome c biogenesis as well as the ResE sensor kinase and the ResD response regulator that control electron transfer and other functions in response to oxygen availability. We have investigated the mechanism of CcpA-mediated control of res operon expression which occurs maximally in the stationary phase of growth. Two CcpA-binding (CRE) sites were found in the res operon, one (CRE1) in the control region in front of the resA promoter, the other (CRE2) in the resB structural gene. Both CRE sites proved to be essential for full CcpA-mediated glucose repression of res operon expression. We propose that both looping and road block mechanisms are involved in res operon control by CcpA.     

trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis

In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.     

A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon

An insertion mutation was isolated that resulted in derepressed expression of the Bacillus subtilis dipeptide transport operon (dpp) during the exponential growth phase in rich medium. DNA flanking the site of insertion was found to encode an operon (codVWXY) of four potential open reading frames (ORFs). The deduced product of the codV ORF is similar to members of the λ Int family; CodW and CodX are homologous to HsIV and HsIU, two putative heat-shock proteins from Escherichia coli, and to LapC and LapA, two gene products of unknown function from Pasteurella haemolytica. CodX also shares homology with a family of ATPases, including CIpX, a regulatory subunit of the E. coli ClpP protease. CodY does not have any homologues in the databases. The insertion mutation and all previously isolated spontaneous cod mutations were found to map In codY. In-frame deletion mutations in each of the other cod genes revealed that only codY is required for repression of dpp in nutrient-rich medium. The cody mutations partially relieved amino acid repression of the histidine utilization (hut) operon but had no effect on regulation of certain other early stationary phase-induced genes, such as spoVG and gsiA.     

The inhibition of translation in Bacillus subtilis via the repression by the secreted enzyme of its synthesis]

It has been shown that inhibition of Bacillus subtilis alpha-amylase formation by the level of the active enzyme in the cultural medium leads to the decrease of translation accompanied by reduction of level of [14C] valin transport into the cells.     

Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase

In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.     

Characterization of the proteins encoded by the Bacillus subtilis yoxA-dacC operon

In Bacillus subtilis , the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An aldose-1-epimerase or mutarotase activity was measured with the YoxA protein that we propose to rename as GalM by analogy with its counterpart in E. coli . The peptide d -Glu-δ- m -A₂pm- d -Ala- m -A₂pm- d -Ala mimicking the B. subtilis and E. coli interpeptide bridge was synthesized and incubated with the purified dacC product, the PBP4a. A clear dd -endopeptidase activity was obtained with this penicillin-binding protein, or PBP. The possible role of this class of PBP, present in almost all bacteria, is discussed.     

Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.     

Phosphorylation of HPr and Crh by HprK, early steps in the catabolite repression signalling pathway for the Bacillus subtilis levanase operon

Carbon catabolite repression (CCR) of Bacillus subtilis catabolic genes is mediated by CcpA and in part by P-Ser-HPr. For certain operons, Crh, an HPr-like protein, is also implicated in CCR. In this study we demonstrated that in ptsH1 crh1 and hprK mutants, expression of the lev operon was completely relieved from CCR and that both P-Ser-HPr and P-Ser-Crh stimulated the binding of CcpA to the cre sequence of the lev operon.     

Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immunoprecipitation and genome-wide transcript analysis

Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.