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.     

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.     

A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium.

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 the mutant carries normal metE, metH, and metF genes; the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and 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 plasmid-borne 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.     

Activity changes in lac repressor with cysteine oxidation.

The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein.     

Localization of the Leptospira interrogans metF gene on the CII secondary chromosome

An open reading frame of 885 nucleotides was identified as the Leptospira interrogans metF gene. The deduced amino acid sequence (294 amino acids) showed similarities with Escherichia coli methylene tetrahydrofolate reductase (MetF or MTHFR) (33% identity) and with the N-terminal part of human MTHFR (33% identity). The L. interrogans metF gene complements an E. coli metF mutant to prototrophy, suggesting the functionality of the folate branch converging to form methionine. In addition, the L. interrogans MetF was found to be thermolabile. The metF gene belonged to the CII secondary chromosome, in contrast to the previously isolated metY and metX genes, which have been localized to the CI chromosome of Leptospira sp.     

Two Aspergillus nidulans genes encoding methylenetetrahydrofolate reductases are up-regulated by homocysteine

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate in the synthesis of methionine from homocysteine. We have cloned and characterized two Aspergillus nidulans genes encoding MTHFRs: metA and metF. Mutations in either gene result in methionine requirement; the metA-encoded enzyme is responsible for only 10-15% of total MTHFR activity. These two enzymes belong to different classes of MTHFRs. Mutations in metA but not in the metF gene are suppressed by mutations resulting in enhancement of homocysteine synthesis. The expression of both genes is up-regulated by homocysteine.     

Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain.

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.     

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.     

Sequence analysis of operator mutants of the phase-1 flagellin-encoding gene, fliC, in Salmonella typhimurium

In phase-2 cells of diphasic Salmonella strains, expression of the phase-1 flagellin-encoding gene, fliC, is repressed by the repressor encoded by the fljA gene. Nine operator-constitutive (Oc) mutants of fliC were isolated from S. typhimurium by selecting those which could express fliC in the presence of the repressor. Among them, eight mutants could express fliC both in the presence and the absence of the repressor, whereas the ninth one could express only in the presence of the repressor. Nucleotide sequence analysis revealed that the Oc mutations of the former type were all located between bp 7 and 20 upstream from the coding region of fliC, which suggests that this region may correspond to the operator for fliC. The latter mutant was found to have a tandem duplication of 28 bp which contains a part of the operator sequence, and seems to require the repressor to activate fliC expression.     

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.