The acquisition of genetic competence by Bacillus subtilis is repressed when the growth medium contains Casamino Acids. This repression was shown to be exerted at the level of expression from the promoters of the competence-regulatory genes srfA and comK and was relieved in strains carrying a null mutation in the codY gene. DNase I footprinting experiments showed that purified CodY binds directly to the srfA and comK promoter regions.When Bacillus subtilis cells reach stationary phase, they induce several adaptive responses. Among these is the acquisition of genetic competence, a physiological state that enables cells to take up exogenous DNA. The acquisition of competence requires the expression of at least 20 "late" com genes (7), whose transcription is regulated by a variety of nutritional, growth stage-, and cell type-specific mechanisms. These regulatory mechanisms involve the products of at least 15 additional genes (13). In the primary regulatory cascade, two extracellular signal peptides, competence-stimulating factor (39) and ComX (21), are detected by the products of the oligopeptide permease (opp) operon (29, 32) and comP, respectively, and as a result, the transcription factor ComA becomes phosphorylated. Phosphorylated ComA acts as a positive regulator for the srfA operon (14,26,27,45), one of whose products, ComS, is needed for expression of comK (5,6,18). ComK, in turn, activates transcription of the late com genes (e.g., comG [1,16,42,44]). The ComK activation pathway is counterbalanced by the MecA/MecB system, which inhibits ComK activity and can prevent the acquisition of competence under inappropriate environmental conditions (10,19,20,24).During exponential growth in minimal medium, competence is repressed by the addition of Casamino acids (9), an effect that appears to be exerted before srfA expression in the regulatory cascade (15). Such a repressive effect of amino acid mixtures has been described for other operons (2), including hut, the histidine utilization operon (3), dpp (37), which encodes a dipeptide transport system (23), and gsiA (25), which codes for a regulatory protein phosphatase (28). The product of the codY gene has been shown to be required for amino acid repression of the dpp operon (36, 38), and a codY mutation partially relieves amino acid control of hut (11, 38) and gsiA (35). Moreover, purified CodY binds to a part of the dpp promoter region within which mutations relieve amino acid repression (34). Since competence is also repressed by mixtures of amino acids, we tested whether the codY gene has any role in this repression. We show that CodY is required for the effect of Casamino Acids on competence and that CodY interacts with the srfA and comK promoter regions. MATERIALS AND METHODSBacterial strains. All B. subtilis strains used in this study (Table 1) were derived from FJS107 (36), a derivative of JH642 (30).Media. S7 minimal medium for competence assays and ␤-galactosidase assays contained S7 minimal salts (46) supplemented with 1% glucose, 0.1% glutamate, and ami...

A mutation (gltR24) that allows Bacillus subtilis glutamate synthase (gltAB) gene expression in the absence of its positive regulator, GltC, was identified. Cloning and sequencing of the gltR gene revealed that the putative gltR product belongs to the LysR family of transcriptional regulators and is thus related to GltC. A null mutation in gltR had no effect on gltAB expression under any environmental condition tested, suggesting that gltR24 is a gain-of-function mutation. GltR24-dependent transcription of gltAB, initiated at the same base pair as GltC-dependent transcription, was responsive to the nitrogen source in the medium and required the integrity of sequences upstream of the gltAB promoter that are also necessary for GltC-dependent expression. Expression of the gltC gene, transcribed divergently from gltA from an overlapping promoter, was not affected by GltR. Both wild-type GltR and GltR24 negatively regulated their own expression. The gltR gene was mapped to 233؇ on the B. subtilis chromosome, very close to the azlB locus.Glutamate synthase, encoded by the gltAB genes, synthesizes glutamate from 2-ketoglutarate and glutamine and is an essential component of the major pathway for ammonia assimilation in Bacillus subtilis (39). Glutamate synthase gene expression is positively regulated in a nitrogen source-dependent manner by the product of the gltC gene. In the absence of GltC protein, gltAB transcription is drastically reduced under normally activating growth conditions, e.g., when ammonium ions are available as nitrogen source (7,8). GltC may not be the only factor controlling gltAB expression, however, since the low, residual expression of gltA in a gltC mutant decreases further under nonactivating growth conditions, e.g., when glutamate or proline serves as sole nitrogen source (5). We sought to identify a second factor involved in gltAB regulation by looking for mutants with elevated expression of gltA despite the absence of GltC.GltC belongs to the LysR family of bacterial transcriptional regulators (20, 38), a family which now comprises about 100 members. The members of this family share some common properties (38): (i) they have near their N termini a conserved helix-turn-helix region that is thought to be responsible for DNA binding; (ii) their target genes are frequently linked to and transcribed divergently from the genes for the LysR-type regulators; (iii) binding sites for many LysR-type proteins have a consensus sequence T-N 11 -A, with elements of dyad symmetry around the two conserved nucleotides, often separated by an (AϩT)-rich core; centers of these sequences are located near position Ϫ65 with respect to the transcription start sites of the target genes; (iv) for some LysR proteins the binding site extends beyond the consensus site or they have an additional binding site between the consensus sequence and the transcription start site of the target gene; and (v) the genes encoding LysR-type proteins are frequently negatively autoregulated. All of these properties apply to GltC (7,8).Th...

The major citrate synthase of Bacillus subtilis (CS-II) was purified to near homogeneity and shown to correspond to the product of the citZ gene. Accumulation of CS-II during exponential growth and stationary phases paralleled expression of the citZ gene. The physical and kinetic properties of CS-II were similar to those of citrate synthase enzymes from Bacillus megaterium and from eukaryotic cells but differed from those of citrate synthases from many gram-negative bacteria.Citrate synthase (CS), the first enzyme of the Krebs cycle, is required for synthesis of glutamate from glycolytic intermediates and is, therefore, a critical enzyme in cellular biosynthesis (23). Moreover, the role of the Krebs cycle in production of ATP and reducing power makes this enzyme a major contributor to cellular energetics. In Bacillus subtilis, CS is needed for both growth (in the absence of a source of glutamate) and spore formation (5-7).B. subtilis has two distinct, homologous CS genes, citA and citZ, expressed during growth and early stationary phase (7,8). A null mutation in citA has little effect on growth, CS enzyme activity, or sporulation, but an in-frame deletion mutation in citZ causes partial glutamate auxotrophy, a Ͼ90% loss of CS enzyme activity, and a significant defect in sporulation. A citA citZ double mutant is an absolute glutamate auxotroph, has undetectable CS activity, and sporulates more poorly than does either a citA or citZ single mutant. These results, along with comparative measurements of citA and citZ mRNA levels, have indicated that citZ encodes the major CS of B. subtilis (8).Here we report the biochemical and kinetic properties of purified CS-II, the citZ gene product. Previous studies of B. subtilis CS activity were based on crude extracts (5, 9), now known to contain a mixture of CS-I, the product of citA, and CS-II. Several CSs from gram-negative bacteria have been purified and their kinetic and biochemical properties have been studied in detail (14,16,17,21,22), but only one CS enzyme from a gram-positive bacterium (Bacillus megaterium) has been studied previously in its homogeneously purified form (18). Bacillus sp. strain C4 CS has been studied after partial purification (19).To purify B. subtilis CS-II without contamination by CS-I, we used a citA null mutant strain, SJB9, a derivative of JH642 (7). Purification followed the method of Robinson et al. (18) with minor modifications (see the legend to Fig. 1 and Table 1). B. subtilis CS activity in crude extracts and in pure preparations was found to be rapidly lost at 4ЊC unless protected by 20% glycerol. In the presence of 20% glycerol and 100 mM KCl, the purified B. subtilis enzyme retained its activity for more than 6 months at 4ЊC and for about 2 years at Ϫ20ЊC.The N-terminal amino acid sequence (MTATRGLEGV VATTSSVSSII) and the sequence of an internal tryptic peptide (MLTEIGEVEN) were identical to those predicted from the DNA sequence of the citZ gene (7). These results very strongly suggest that citZ encodes CS-II. Molecular mass and olig...