The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic‐binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA–phoA and dppA–lacZ gene fusions. Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

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When Escherichia coli was grown in medium containing both inosine and glycine, the PurR repressor protein was shown to be responsible for a twofold reduction from the fudly induced glycine cleavage enzyme levels. This twofold repression was also seen by measuring PI-galactosidase levels in cells carrying a AgcvT-lacZ gene fusion. In this fusion, the synthesis of 13-galactosidase is under the control of the gcv regulatory region. A DNA friaent carrying thegcv control region was shown by gel mobility shift assay and DNase I footprinting to bind purified PurR protein, suggesting a direct involvement of the repressor in gcv regulation. A separate mechanism of purine-mediated regulation of gcv was shown to be independent of the purR gene product and resulted in an approximately 10-fold reduction of 13-galactosidase levels when cells were grown in medium containing inosine but lacking the inducer glycine. This additional repression was dependent upon a functional gcvA gene, a positive activator for the glycine cleavage enzyme system. A dual role for the GcvA protein as both an activator in the presence of glycine and a repressor in the presence of inosine is suggested.

We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF gene product. Transduction analysis indicates that (i) the mutant carries normal metE, metH, and metF genes; (ii) the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and (iii) the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmidborne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitamin B12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitamin B12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whose product acts in trans to activate the metE and metH genes.The methylation of homocysteine to form methionine can be carried out by either of two transmethylases in Salmonella typhimurium and Escherichia coli (for a review, see reference 15). The first is a vitamin B12-independent enzyme, the product of the metE gene; the second is a vitamin B12-dependent enzyme, the product of the metH gene. The methyl donor for both enzymes is 5-methyltetrahydrofolate, produced by the metF gene product at a point of convergence of two major pathways, the methionine biosynthetic pathway and the C1 pathway (Fig. 1). The cell regulates the flow of C1 units through this convergence point on several levels to balance the requirements for protein synthesis, methylation reactions, and nucleic acid synthesis.The genes in the nonfolate branch of the methionine pathway (metA, metB, metC, and metK) and those in the folate branch of the pathway (metF, metE, and, to a small extent, metH) are all negatively controlled by the metJ repressor system. In addition, the metH gene product is involved in repression of the metE and metF genes when the cells are grown in medium containing vitamin B12. We report here the finding of a third regulatory mechanism at the methionine-C, convergence point, namely, the positive activation of the metE and metH genes. MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophages. All bacterial strains used are derivatives of E. coli K-12 and are described in Table 1. Plasmids pGS47 and pGS69, and their metE::Tn5 derivatives have been described previously (16). Plasmid pMC1403 (4) was from M. Casadaban. Bacteriophage Xgt2 (13) was from R. Davis. Plasmid pBR322 has been described previously (9). Plasmid pGS191, the lacZ * Corresponding author. fusion plasmids, and la...