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 exerted by two catabolite-responsive elements

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-LacI family of bacterial regulatory proteins, and the seryl-phos-phorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (cre_down), this operon contains another ere located in the promoter region (cre_up). A translational gntR-lacZ fusion expressed under the control of various combinations of wild-type and mutant cre_down and cre_up was integrated into the chromosomal amyE locus, and then catabolite repression of p-galac-tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by cre_up was probably independent of catabolite repression exerted by cre_down; both creup and cre_down catabolite repression involved CcpA. Catabolite repression exerted by cre_up was independent of P-ser-HPr, and catabolite repression exerted by cre_down was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize cre_up, in contrast to its specific recognition of cre_down. However, CcpA complexed with glucose-6-phosphate specifically recognized cre_up as well as cre_down, but the physiological significance of this complexing is unknown.     

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.     

Insight into the stability of the hydrophobic binding proteins of Escherichia coli: assessing the proteins for use as biosensors

Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins. Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model. Ligand binding causes significant stabilization of both proteins. There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein. A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors.     

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.     

Evaluation of in situ valine production by Bacillus subtilis in young pigs

Mutants of Bacillus subtilis can be developed to overproduce Val in vitro. It was hypothesized that addition of Bacillus subtilis mutants to pig diets can be a strategy to supply the animal with Val. The objective was to investigate the effect of Bacillus subtilis mutants on growth performance and blood amino acid (AA) concentrations when fed to piglets. Experiment 1 included 18 pigs (15.0±1.1 kg) fed one of three diets containing either 0.63 or 0.69 standardized ileal digestible (SID) Val: Lys, or 0.63 SID Val: Lys supplemented with a Bacillus subtilis mutant (mutant 1). Blood samples were obtained 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding and analyzed for AAs. In Experiment 2, 80 piglets (9.1±1.1 kg) were fed one of four diets containing 0.63 or 0.67 SID Val: Lys, or 0.63 SID Val: Lys supplemented with another Bacillus subtilis mutant (mutant 2) or its parent wild type. Average daily feed intake, daily weight gain and feed conversion ratio were measured on days 7, 14 and 21. On day 17, blood samples were taken and analyzed for AAs. On days 24 to 26, six pigs from each dietary treatment were fitted with a permanent jugular vein catheter, and blood samples were taken for AA analysis 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding. In experiment 1, Bacillus subtilis mutant 1 tended (P<0.10) to increase the plasma levels of Val at 2 and 3 h post-feeding, but this was not confirmed in Experiment 2. In Experiment 2, Bacillus subtilis mutant 2 and the wild type did not result in a growth performance different from the negative and positive controls. In conclusion, results obtained with the mutant strains of Bacillus subtilis were not better than results obtained with the wild-type strain, and for both strains, the results were not different than the negative control.     

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.     

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.