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.     

Expression of the hut operons of Salmonella typhimurium in Klebsiella aerogenes and in Escherichia coli.

The normal hut (histidine utilization) operons, as well as those with mutations affecting the regulation of their expression, of Salmonella typhimurium were introduced on an F' episome into cells of S. typhimurium and Klebsiella aerogenes whose chromosomal hut genes had been deleted and into cells of Escherichia coli, whose chromosome does not carry hut genes. The episomal hut operons respond in a manner very similar to induction and catabolite repression in all three organisms. The small differences found reflect both different abilities to take up inducers from the medium and different degrees of catabolite repression exerted by glucose.     

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.     

Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the alpha subunit in transcription activation of the Escherichia coli rhaBAD operon

The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar L-rhamnose. Full rhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the -35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at -92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on rhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions. We also expressed a derivative of the alpha subunit of RNA polymerase deleted for the entire C-terminal domain (alpha-Delta235) and assayed expression from rhaBAD promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the alpha subunit (alpha-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on alpha-CTD. Overall, our results suggest that CRP interaction with alpha-CTD may not be required for rhaBAD activation; however, alpha-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.     

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.     

Regulation of the hut operons of Salmonella typhimurium and Klebsiella aerogenes by the heterologous hut repressors.

In merodiploid strains of Klebsiella aerogenes with chromosomal hut genes of K. aerogenes and episomal hut genes of Salmonella typhimurium, the repressor of either species can regulate the hut operons of the other species. The repression exerted by the homologous repressor on the left-hand hut operon is, in both organisms, stronger than that exerted by the heterologous repressor.     

Regulation of the galactose-inducible lac operon and the histidine utilization operons in pts mutants of Klebsiella aerogenes.

Galactose appears to be the physiological inducer of the chromosomal lac operon in Klebsiella aerogenes. Both lactose and galactose are poor inducers in strains having a functional galactose catabolism (gal) operon, but both are excellent inducers in gal mutants. Thus the slow growth of K. aerogenes on lactose reflects the rapid degradation of the inducer. Several pts mutations were characterized and shown to affect both inducer exclusion and permanent catabolite repression. The beta-galactosidase of pts mutants cannot be induced at all by lactose, and pts mutants appear to have a permanent and constitutive inducer exclusion phenotype. In addition, pts mutants show a reduced rate of glucose metabolism, leading to slower growth on glucose and a reduced degree of glucose-mediated permanent catabolite repression. The crr-type pseudorevertants of pts mutations relieve the constitutive inducer exclusion for lac but do not restore the full level of glucose-mediated permanent catabolite repression and only slightly weaken the glucose-mediated inducer exclusion. Except for weakening the glucose-mediated permanent catabolite repression, pts and crr mutations have no effect on expression of the histidine utilization (hut) operons.     

Activation of the Bacillus subtilis hut operon at the onset of stationary growth phase in nutrient sporulation medium results primarily from the relief of amino acid repression of histidine transport.

During growth of Bacillus subtilis in nutrient sporulation medium containing histidine (DSM-His medium), the expression of histidase, the first enzyme in the histidine-degradative pathway (hut), is derepressed 40- to 200-fold at the onset of stationary phase. To identify the gene products responsible for this regulation, histidase expression was examined in various hut regulatory mutants as well as in mutants defective in stationary-phase gene regulation. Histidase expression during growth in DSM-His medium was significantly altered only in a strain containing the hutC1 mutation. The hutC1 mutation allows the hut operon to be expressed in the absence of its inducer, histidine. During logarithmic growth in DSM-His medium, histidase levels were 25-fold higher in the HutC mutant than in wild-type cells. Moreover, histidase expression in the HutC mutant increased only four- to eightfold after the end of exponential growth in DSM-His medium. This suggests that histidine transport is reduced in wild-type cells during exponential growth in DSM-His medium and that this reduction is largely responsible for the repression of hut expression in cells growing logarithmically in this medium. Indeed, the rate of histidine uptake in DSM-His medium was fourfold lower in exponentially growing cells than in stationary-phase cells. The observation that the degradation of histidine is inhibited when B. subtilis is growing rapidly in medium containing a mixture of amino acids suggests that a hierarchy of amino acid utilization may be present in this bacterium.     

Cloning, expression, and carbon catabolite repression of the bamM gene encoding beta-amylase of Bacillus megaterium DSM319

The bamM gene from Bacillus megaterium DSM319 encoding an extracellular beta-amylase was isolated and completely sequenced. Chromosomal inactivation by deletion mutagenesis resulted in total loss of amylolytic activity, indicative of a single starch-degrading enzyme. Functional characterization of the expressed protein revealed a maltogenic enzyme exhibiting optimal activities at pH 7.5 and 50 degrees C. Amylase expression is subject to catabolite repression by glucose. A putative cis-acting catabolite-responsive element (CRE) was identified; it is located within the bamM coding region, matching the position of the predicted signal peptide processing site. Base substitutions introduced by site-directed mutagenesis within the bamM-CRE--retaining unchanged the amino acid sequence--provoked a remarkable relief from carbon catabolite repression (CCR), thereby proving functionality of the CRE for CCR.