No abstract

SummaryThe molecular mechanisms for regulation of the genes involved in the biosynthesis of methionine and cysteine are poorly characterized in Bacillus subtilis. Analyses of the recently completed B. subtilis genome revealed 11 copies of a highly conserved motif. In all cases, this motif was located in the leader region of putative transcriptional units, upstream of coding sequences that included genes involved in methionine or cysteine biosynthesis. Additional copies were identified in Clostridium acetobutylicum and Staphylococcus aureus, indicating conservation in other Gram-positive genera. The motif includes an element resembling an intrinsic transcriptional terminator, suggesting that regulation might be controlled at the level of premature termination of transcription. The 5Ј portion of all of the leaders could fold into a conserved complex structure. Analysis of the yitJ gene, which is homologous to Escherichia coli metH and metF, revealed that expression was induced by starvation for methionine and that induction was independent of the promoter and dependent on the leader region terminator. Mutation of conserved primary sequence and structural elements supported a model in which the 5Ј portion of the leader forms an anti-antiterminator structure, which sequesters sequences required for the formation of an antiterminator, which, in turn, sequesters sequences required for the formation of the terminator; the anti-antiterminator is postulated to be stabilized by the binding of some unknown factor when methionine is available. This set of genes is proposed to form a new regulon controlled by a global termination control system, which we designate the S box system, as most of the genes are involved in sulphur metabolism and biosynthesis of methionine and cysteine.

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNA Gln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNA Gln through the transamidation of misacylated Glu-tRNA Gln , functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNA Gln in B. subtilis and that glutamyl-tRNA Gln amidotransferase is a novel and essential component of the translational apparatus.

Expression of the alpha-amylase gene of Bacillus subtilis is controlled at the transcriptional level, and responds to the growth state of the cell as well as the availability of rapidly metabolizable carbon sources. Glucose-mediated repression has previously been shown to involve a site near the transcriptional start-point of the amyE gene. In this study, a transposon insertion mutation was characterized which resulted in loss of glucose repression of amyE gene expression. The gene affected by this mutation, which was localized near 263 degrees on the B. subtilis chromosomal map, was isolated and its DNA sequence was determined. This gene, designated ccpA, exhibited striking homology to repressor genes of the lac and gal repressor family. The ccpA gene was found to be allelic to alsA, previously identified as a regulator of acetoin biosynthesis, and may be involved in catabolite regulation of other systems as well.