Negative regulation of L-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene.

The Bacillus subtilis araC locus, mapped at about 294 degrees on the genetic map, was defined by mutations conferring an Ara- phenotype to strains bearing the metabolic araA, araB, and araD wild-type alleles (located at about 256 degrees on the genetic map) and by mutants showing constitutive expression of the three genes. In previous work, it has been postulated that the gene in which these mutations lie exerts its effect on the ara metabolic operon in trans, and this locus was named araC by analogy to the Escherichia coli regulatory gene. Here, we report the cloning and sequencing of the araC locus. This region comprises two open reading frames with divergently arranged promoters, the regulatory gene, araC, encoding a 41-kDa polypeptide, and a partially cloned gene, termed araE, which most probably codes for a permease involved in the transport of L-arabinose. The DNA sequence of araC revealed that its putative product is very similar to a number of bacterial negative regulators (the GalR-LacI family). However, a helix-turn-helix motif was identified in the N-terminal region by its identity to the consensus signature sequence of another group of repressors, the GntR family. The lack of similarity between the predicted primary structure of the product encoded by the B. subtilis regulatory gene and the AraC regulator from E. coli and the apparently different modes of action of these two proteins lead us to propose a new name, araR, for this gene. The araR gene is monocistronic, and the promoter region contains -10 and -35 regions (as determined by primer extension analysis) similar to those recognized by RNA polymerase containing the major vegetative cell sigma factor sigmaA. An insertion-deletion mutation in the araR gene leads to constitutive expression of the L-arabinose metabolic operon. We demonstrate that the araR gene codes for a negative regulator of the ara operon and that the expression of araR is repressed by its own product.     

Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser-46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low-molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.     

A novel diffusible substance can overcome the apparent AbrB repression of the Bacillus subtilis fatR promoter

In this work we present evidence for a novel diffusible extracellular factor that modulates gene expression in Bacillus subtilis. The factor was found when studying the regulation of the fatR-cyp102A3 operon. In a Spo0A(-) mutant expression of the fatR-cyp102A3 operon was almost abolished. The fatR-cyp102A3 expression defect of a Spo0A(-) mutant could be overcome either by a mutation in the abrB gene or by a diffusible substance excreted by wild-type, abrB mutant and abrB-spo0A double mutant strains.     

Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene.

The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes. In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined. The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described lon gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli lon gene, the B. subtilis lon gene is induced by heat shock. Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by sigma 32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B. subtilis. A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.     

Sodium effect of growth on aspartate and genetic analysis of a Bacillus subtilis mutant with high aspartase activity.

Most strains of Bacillus subtilis, dervied from the 168 (Marburg) strain, grow slowly on aspartate as sole carbon source. We isolated a mutant (aspH) that grows rapidly on aspartate because it produces aspartase constitutively. Thus, aspartase is needed for rapid growth on aspartate, whereas aspartate-alpha-ketoglutarate aminotransferase is not needed, as was demonstrated by a mutant lacking that enzyme activity. By two--and three-factor crosses using PBSl transduction, the aspH mutation was located between the aroD and the lys markers of the genetic map. Although sodium ions do not affect growth on glucose or L-malate, they specifically stimulate growth on aspartate in both the parent and the aspH mutant strains. Enzyme activities of crude aspartase and fumarase and of purified aspartase do not increase in the presence of sodium. These results show that stimulation by sodium involves some reaction other than the enzymes catabolizing aspartate. The ease of purification from the aspH strain and the stability of aspartase suggest that the B. subtilis enzyme is particularly useful for aspartate determinations.     

Repression of sigK intervening (skin) element gene expression by the CI-like protein SknR and effect of SknR depletion on growth of Bacillus subtilis cells

The Bacillus subtilis phage DNA-like sigK intervening (skin) element (48 kb) is excised from the chromosome by DNA rearrangement, and a composite gene, sigK (spoIIIC and spoIVCB), is created on the chromosome during sporulation. In this study, we first focused on the role of sknR (skin repressor), which has homology with the gene encoding the Xre repressor of defective phage PBSX. The depletion of SknR caused overexpression of the region between yqaF and yqaN (the yqaF-yqaN operon) and a growth defect in B. subtilis. Point mutation analysis and an electrophoretic mobility shift assay (EMSA) suggested that SknR functions as a negative regulator of gene expression in the yqaF-yqaN operon of the skin element through direct interaction with operators of 2-fold symmetry located in the intergenic region between sknR and yqaF. Deletion analysis revealed that the lethal effect of depletion of SknR was related to overexpression of yqaH and yqaM, whose products were previously reported to associate with DnaA and DnaC, respectively. Furthermore, overexpression of either yqaH or yqaM caused cell filamentation and abnormal chromosome segregation, which suggested that overproduction of these proteins inhibits DNA replication. Moreover, overexpression of yqaM inhibited the initiation of replication. Taken together, these data demonstrate that the B. subtilis skin element carries lethal genes, which are induced by the depletion of sknR.