Transcriptional regulation and structure of the Bacillus subtilis sporulation locus spoIIIC.

The spoIIIC locus of Bacillus subtilis has been cloned from the lambda library of Ferrari et al. (E. Ferrari, D. J. HEnner, and J. A. Hoch, J. Bacteriol. 146:430-432, 1981) by using as an assay transformation of the mutant allele spoIIIC94 to the wild type. Regulation of the spoIIIC locus was studied by hybridization of cloned spoIIIC DNA to RNA pulse-labeled at various times during growth and sporulation. The relative rate of transcription of the spoIIIC locus was highest 3 h after the end of growth. The DNA sequence of the spoIIIC transcription unit indicated the coding capacity for a small protein (138 amino acids) having significant similarity with one domain of RNA polymerase sigma factors. Interruption of this coding sequence by an insertion mutation caused cells to become Spo-.

Relationship between aconitase gene expression and sporulation in Bacillus subtilis.

The citB of Bacillus subtilis codes for aconitase (D. W. Dingman and A. L. Sonenshein, J. Bacteriol. 169:3060-3065). By direct measurements of citB mRNA levels and by measurements of beta-galactosidase activity in a strain carrying a citB-lacZ fusion, we have examined the expression of citB during growth and sporulation. When cells were grown in nutrient broth sporulation medium, citB mRNA appeared in mid- to late-exponential phase and disappeared by the second hour of sporulation. This timing corresponded closely to the kinetics of appearance of aconitase enzyme activity. Decoyinine, a compound that induces sporulation in a defined medium, caused a rapid simultaneous increase in aconitase activity and citB transcription. After decoyinine addition, the rate of increase in aconitase activity in a 2-ketoglutarate dehydrogenase (citK) mutant and in a citrate synthase (citA) mutant was significantly less than in an isogenic wild-type strain. This is apparently due to a failure to deplete 2-ketoglutarate and accumulate citrate. These metabolites might act as negative and positive effectors of citB expression, respectively. Mutations known to block sporulation at an early stage (spo0H and spo0B) had no appreciable effect on citB expression or aconitase activity. These results suggest that appearance of aconitase is stimulated by conditions that induce sporulation but is independent of certain gene products thought to act at an early stage of sporulation.

Saccharomyces cerevisiae contains two functional citrate synthase genes.

The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed.

Transcriptional control of the Bacillus subtilis spoIID gene.

We cloned the wild-type allele of the spoIID locus of Bacillus subtilis. This DNA region was shown to be transcribed beginning within an hour after the onset of sporulation. The amount of spoIID mRNA present in cells at 1 h after the end of growth was more than 50-fold greater than it was growing cells; the pool of this mRNA decreased steadily after 1.5 h after the end of growth. spoIID mRNA was present in stationary-phase cells of sporulation mutants with lesions in the spo0J and spoIIB genes but was absent in cells carrying spo0B, spo0H, spoIIA, spoIIE, spoIIG, or spoIIIA mutations. In vitro runoff transcription with the E sigma 55, E sigma 37, E sigma 32, and E sigma 29 forms of RNA polymerase indicated that only the E sigma 29 form was able to transcribe the spoIID gene. This result is consistent with results of studies with the Spo- mutants, because only mutants that produced E sigma 29 were able to produce spoIID mRNA in vivo. In the course of this work, two additional transcription units were discovered in the DNA region neighboring the spoIID gene. One of these was expressed during vegetative growth; the other was expressed early during sporulation and corresponded to an in vitro transcript produced by the E sigma 29 forms of RNA polymerase.

Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine.

The activity of aconitase in Bacillus subtilis is greatly reduced in cells cultured in media containing rapidly metabolized carbon sources (e.g., glucose). Thus, expression of this enzyme appears to be subject to a form of catabolite repression. Since the product of the citB gene of B. subtilis is required for aconitase activity, we cloned the wild-type allele of this gene and used this DNA as a probe for transcription of citB in cells grown in various media. The steady-state level of RNA that hybridized to this probe was about 10-fold higher in B. subtilis cells grown in citrate-glutamine medium than in cells grown in glucose-glutamine medium. This result correlates well with the steady-state levels of aconitase activity. Two transcripts were shown to initiate within the cloned DNA; the steady-state level of one of these transcripts varied in the same way as did aconitase activity when cells were grown in media containing different carbon sources. This is the first demonstration of regulation by the carbon source of the level of a vegatative-cell transcript in B. subtilis.

Regulation of Bacillus subtilis glutamate synthase genes by the nitrogen source.

The wild-type alleles of the gltA292 and gltB1 mutations of Bacillus subtilis have been identified in banks of B. subtilis DNA cloned in phage lambda. These mutations are thought to define the genes for the two subunits of glutamate synthase. Sequences having transforming activity for each allele were subcloned in plasmids and used as hybridization probes for measurements of the rates of synthesis and steady-state levels of glt mRNAs under different growth conditions. For both gltA and gltB, the level of mRNA varied according to the nitrogen source in the growth medium, to an extent sufficient to explain the variation in glutamate synthase activity under the same conditions. Two start points for mRNA synthesis were detected within the cloned DNA, one of which corresponded to the gltA locus. The other start point appears to define a transcription unit, separate from gltA and gltB, within which mutations cause loss of glutamate synthase activity.

Glutamine synthetase gene of Bacillus subtilis.

The glutamine synthetase gene (glnA) of Bacillus subtilis was purified from a library of B. subtilis DNA cloned in phage lambda. By mapping the locations of previously identified mutations in the glnA locus it was possible to correlate the genetic and physical maps. Mutations known to affect expression of the glnA gene and other genes were mapped within the coding region for glutamine synthetase, as determined by measuring the sizes of truncated, immunologically cross-reacting polypeptides coded for by various sub-cloned regions of the glnA gene. When the entire B. subtilis glnA gene was present on a plasmid it was capable of directing synthesis in Escherichia coli of B. subtilis glutamine synthetase as judged by enzymatic activity, antigenicity, and ability to allow growth of a glutamine auxotroph. By use of the cloned B. subtilis glnA gene as a hybridization probe, it was shown that the known variability of glutamine synthetase specific activity during growth in various nitrogen sources is fully accounted for by changes in glnA mRNA levels.