Physical mapping of the scattered methionine genes on the Escherichia coli chromosome.

Methionine is an important amino acid which acts not only as a substrate for protein elongation but also as the initiator of protein synthesis. The genes of the met regulon, which consists of 10 biosynthetic genes (metA, metB, metC, metE, metF, metH, metK, metL, metQ, and metX), two regulatory genes (metJ and metR), and the methionyl tRNA synthetase gene (metG), are scattered throughout the chromosome. The only linked genes are metK and metX at 63.6 min, metE and metR at 86.3 min, and the metJBLF gene cluster at 89 min. metBL form the only met operon.     

Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions.

Fusions of the lac genes to the promoters of four structural genes in the methionine biosynthetic pathway, metA, metB, metE, and metF, were obtained by the use of the Mu d(Ap lac) bacteriophage. The levels of beta-galactosidase in these strains could be derepressed by growth under methionine-limiting conditions. Furthermore, growth in the presence of vitamin B12 repressed the synthesis of beta-galactosidase in strains containing a fusion of lacZ to the metE promoter, phi(metE'-lacZ+). Mutations affecting the regulation of met-lac fusions were generated by the insertion of Tn5. Tn5 insertions were obtained at the known regulatory loci metJ and metK. Interestingly, a significant amount of methionine adenosyltransferase activity remained in the metK mutant despite the fact that the mutation was generated by an insertion. Several Tn5-induced regulatory mutations were isolated by screening for high-level beta-galactosidase expression in a phi(metE'-lacZ+) strain in the presence of vitamin B12. Tn5 insertions mapping at the btuB (B12 uptake), metH (B12 dependent tetrahydropteroylglutamate methyltransferase), and metF (5,10-methylenetetrahydrofolate reductase) loci were obtained. The isolation of the metH mutant was consistent with previous suggestions that the metH gene product is required for the repression of metE by vitamin B12. The metF::Tn5 insertion was of particular interest since it suggested that a functional metf gene product was also needed for repression of metE by vitamin B12.     

Characterization of two mutant metJ proteins with reduced, temperature-dependent capacity to regulate Escherichia coli K-12 met regulon elements.

At 28 degrees C, but not at 34 or 42 degrees C, strains with the metJ193 allele repressed chromosomal met genes but not a plasmid-borne met promoter. Increasing the metJ193 gene dosage to two copies resulted in overrepression of chromosomal and plasmid-borne met promoters at 28 degrees C. Suppressing the metJ185 amber mutation with supF (tRNATyr) produced the MetJ185F protein. Strains producing MetJ185F repressed chromosomal met promoters but not a plasmid-borne met promoter at 42 degrees C. These are the first known defective MetJ proteins with documented temperature-dependent function.     

Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine

The metJ gene encoding the methionine aporepressor was placed under the control of a strong and inducible promoter, ptac. Bacterial strains carrying the recombinant plasmid pIP35 overproduced the regulatory protein by a factor of 200 over the wild type strain as determined by the immunoblot technique. The purified metJ gene product negatively controls the expression of the metF gene, in a cell-free system as shown by repression of beta-galactosidase synthesis under the control of the metF promoter. The metJ protein binds to a DNA fragment containing the potential operator of the metF gene with an affinity which is 10 times greater in the presence of S-adenosylmethionine than in its absence. Equilibrium dialysis experiments showed that the met aporepressor binds 2 mol of S-adenosylmethionine per mol of dimer with a dissociation constant of 200 microM.     

Insertion mutagenesis of the metJBLF gene cluster of Escherichia coli K-12: evidence for an metBL operon.

The effects of Mu or transposon 5 insertions on the expression of genes of the metJBLF cluster show that metB and metL form an operon, transcribed from metB to metL, and that metF and metJ are independently transcribed.     

Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.     

Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster.

Secondary attachment site lysogens of Deltaatt(lambda)Deltappc-argECBH strains of Escherichia coli with lambdacI857 integrated into the bfe gene (88 min) were isolated. Of 20 such lysogens examined, 2 produce lysates with transducing phage containing the metBJF gene cluster (87 min). Reintroduction of the ppc-argECBH chromosome segment (which lies between the bfe and met genes) into these strains virtually abolishes the production of met transducing phage. All of the phage examined have lost essential genes from the left arm of the lambda chromosome. Approximately 85% of the phage appear to have the same genetic composition, containing the metBJF gene cluster, but not the closely linked gene cytR, and having lost phage genes G and J. Analytical CsCl density gradient centrifugation of five representatives of this major class of phage shows four of them to have identical densities (lighter than lambda), while the fifth cannot be resolved from lambda. The four apparently identical phage were isolated from three separate lysates, which suggests the existence of preferred sites for illegitimate recombination on the bacterial and phage chromosomes. Three specialized transducing phage that carry cytR in addition to metB, metJ, and metF have also been studied. Each of these viruses has a different amount of phage deoxyribonucleic acid. Two of them have less deoxyribonucleic acid than lambda, whereas the third has about the same amount. The metB, metF, and cytR genes of the transducing phage have been shown to function in vivo. The phage-borne metB and metF genes are subject to metJ-mediated repression.     

Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene.

Regulation of the Salmonella typhimurium metJ gene was examined by measuring beta-galactosidase activity in Escherichia coli strains lysogenic for a phage carrying a metJ-lacZ gene fusion. The results indicated that the metJ gene is regulated by its own gene product and by methionine supplementation to the growth medium. This autoregulatory mechanism involved two tandem promoters, pJ1 and pJ2, separated by approximately 65 base pairs. Deletion analysis permitted the assessment of the activity of promoters pJ1 and pJ2 individually. Promoter Pj1 was negatively regulated by the metJ gene product and by methionine. Although Pj2 regulation remained unclear, evidence is presented which suggests that it is not negatively regulated like pJ1.     

Mapping of the structural genes of the three aspartokinases and of the two homoserine dehydrogenases of Escherichia coli K-12

Mutants requiring threonine plus methionine (or homoserine), or threonine plus methionine plus diaminopimelate (or homoserine plus diaminopimelate) have been isolated from strains possessing only one of the three isofunctional aspartokinases. They have been classified in several groups according to their enzymatic defects. Their mapping is described. Several regions of the chromosome are concerned: thrA (aspartokinase I-homoserine dehydrogenase I) is mapped in the same region as thrB and thrC (0 min). lysC (aspartokinase III) is mapped at 80 min, far from the other genes coding for diaminopimelate synthesis. metLM (aspartokinase II-homoserine dehydrogenase II) lies at 78 min closely linked to metB, metJ, and metF.     

Mutations affecting the regulation of the metB gene of Salmonella typhimurium LT2.

We isolated and characterized cis-acting mutations that affect the regulation of the metB gene of Salmonella typhimurium LT2. The mutations were isolated in an Escherichia coli lac deletion strain lysogenized with lambda bacteriophage carrying a metB-lacZ gene fusion (lambda JBlac) in which beta-galactosidase production is dependent upon metB gene expression. The mutant lysogens show elevated, poorly regulated beta-galactosidase production. The altered regulation is a result of disruption of the methionine control system mediated by the metJ repressor. The mutations are located in a region of dyad symmetry centered near the -35 sequence of the metB promoter. We propose that these mutations alter the repressor binding site and define the metB operator sequence. In addition, we discuss a highly conserved, nonsymmetric DNA sequence of unknown function which occurs in the control regions of the metA, metC, metE, metF, metG, and metJB genes of both S. typhimurium and E. coli.     

Cloning of the methionine regulatory gene, metJ, of Escherichia coli K12 and identification of its product

Both wild-type and mutant forms of the methionine regulatory gene, metJ, of Escherichia coli K12 have been cloned in derivatives of pBR322. In cells carrying plasmids with a functional copy of metJ, the methionine regulon appears to be repressed even under conditions of methionine limitation. Maxicell labeling experiments show that the plasmids code for a small peptide (12 kilodaltons) only when they carry a functional copy of metJ. The lesions in five independently isolated metJ mutants are located in, or slightly upstream from, a coding sequence proposed to be metJ by Saint-Girons, I., Duchange, N., Cohen, G. N., and Zakin, M. M. [1984) J. Biol. Chem. 259, 14282-14285).     

Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid.

The repression of MetE synthesis in Escherichia coli by vitamin B12 is known to require the MetH holoenzyme (B12-dependent methyltransferase) and the metF gene product. Experiments using trimethoprim, an inhibitor of dihydrofolate reductase, show that the MetF protein is not directly involved in the repression, but that N5-methyltetrahydrofolic acid (N5-methyl-H4-folate), the product of the MetF enzymatic reaction is required. Since the methyl group from N5-methyl-H4-folate is normally transferred to the MetH holoenzyme to form a methyl-B12 enzyme, the present results suggest that a methyl-B12 enzyme is involved in the vitamin B12 repression of metE expression. Other results argue against the possibility that a methyl-B12 enzyme functions in this repression solely by decreasing the cellular level of homocysteine, which is required for MetR activation of metE expression. Experiments with metJ mutants show that the MetJ protein mediates about 50% of the repression of metE expression by B12 but is totally responsible for the regulation of metF expression by vitamin B12.     

Structure and autoregulation of the metJ regulatory gene in Escherichia coli

The nucleotide sequence of the Escherichia coli metJ regulatory gene (312 nucleotides) has been determined as well as that of two mutations located within the gene. Analysis of the sequence downstream from the metJ gene has revealed inverted repeats homologous to several intercistronic regions, also reported to occur between operons. A hybrid protein that contains the 55 first amino acid residues of the metJ protein substituting for the 8 amino acid residues at the NH2 terminus of beta-galactosidase was produced by gene fusion. The hybrid protein retaining beta-galactosidase activity was purified. Its amino-terminal sequence was determined and this allowed us to locate the translational start codon of the metJ gene. Evidence was provided for autoregulation by repression of the metJ gene. By sequencing upstream from metJ, the region situated between the metJ and metB genes was found to contain putative operator structures that we propose to call "Met boxes."