Effect of l-methionine and vitamin B12 on methionine biosynthesis in Escherichia coli

Milner, Larry; Whitfield, Carolyn D.; Weissbach, Herbert

doi:10.1016/0003-9861(69)90470-6

Cited by 52 publications

(25 citation statements)

References 18 publications

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“…Interestingly, the release of the vitamin B12-dependent repression in lysogens 244XElac and 244XFlac was paralleled by lower expression of the metH gene in 244XHlac under similar growth conditions. Since the metH gene product has been shown to be involved in the vitamin B12-dependent repression of the metE and metF genes (6,11,22), it is plausible that the decreased production of metH gene product in GS244 is responsible for the disruption of the vitamin B12-dependent repression mechanism. In contrast, it is clear that the addition of L-methionine to the growth medium results in significant repression of all the met-lacZ fusions tested, including the GS244 derivatives.…”

Section: Methodsmentioning

confidence: 99%

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

Urbanowski¹,

Stauffer²,

Plamann³

et al. 1987

J Bacteriol

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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...

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Section: Methodsmentioning

confidence: 99%

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

Urbanowski¹,

Stauffer²,

Plamann³

et al. 1987

J Bacteriol

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“…This ultimately leads to a phenomenon known as folate trapping (Scott and Weir, 1994). In E. coli, which has both METE and METH, Met has a negative regulatory effect on METE expression (Milner et al, 1969;Weissbach and Brot, 1991), whereas homocysteine has a positive regulatory role in E. coli, S. typhimurium, and Ralstonia solanacearum Stauffer, 1989a, 1989b;Plener et al, 2012). Croft et al (2005) showed that, in the absence of B 12 , Met and folate can support the growth of the B 12 -dependent green alga Lobomonas rostrata when supplemented at concentrations of 10 mM and 1 mM, respectively.…”

Section: Exploring the Roles Of Other Factors Involved In Mete Regulamentioning

confidence: 99%

Unraveling Vitamin B₁₂-Responsive Gene Regulation in Algae

Helliwell

Scaife²,

Sasso³

et al. 2014

Plant Physiol.

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Photosynthetic microalgae play a vital role in primary productivity and biogeochemical cycling in both marine and freshwater systems across the globe. However, the growth of these cosmopolitan organisms depends on the bioavailability of nutrients such as vitamins. Approximately one-half of all microalgal species requires vitamin B 12 as a growth supplement. The major determinant of algal B 12 requirements is defined by the isoform of methionine synthase possessed by an alga, such that the presence of the B 12 -independent methionine synthase (METE) enables growth without this vitamin. Moreover, the widespread but phylogenetically unrelated distribution of B 12 auxotrophy across the algal lineages suggests that the METE gene has been lost multiple times in evolution. Given that METE expression is repressed by the presence of B 12 , prolonged repression by a reliable source of the vitamin could lead to the accumulation of mutations and eventually gene loss. Here, we probe METE gene regulation by B 12 and methionine/folate cycle metabolites in both marine and freshwater microalgal species. In addition, we identify a B 12 -responsive element of Chlamydomonas reinhardtii METE using a reporter gene approach. We show that complete repression of the reporter occurs via a region spanning 2574 to 290 bp upstream of the METE start codon. A proteomics study reveals that two other genes (S-Adenosylhomocysteine hydrolase and Serine hydroxymethyltransferase2) involved in the methionine-folate cycle are also repressed by B 12 in C. reinhardtii. The strong repressible nature and high sensitivity of the B 12 -responsive element has promising biotechnological applications as a cost-effective regulatory gene expression tool.

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“…This B12-mediated repression requires the metJ gene product and the metF gene product (the metF gene product provides the methyl donor for homocysteine methylation) (3,12), as well as the MetH-B12 holoenzyme. Although the mechanism of repression by the MetH-B12 holoenzyme requires the MetJ protein, this repression system is distinct from the repression system mediated by the MetJ protein and SAM (11).…”

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confidence: 96%

Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene

Urbanowski

Stauffer

1992

J Bacteriol

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The vitamin B12 (B.2)-mediated repression of the metE gene in Escherichia coli and Salmonella typhimurium requires the B12-dependent transmethylase, the metH gene product. It has been proposed that the MetH-B12 holoenzyme complex is involved directly in the repression mechanism. Using Escherichia coli strains lysogenized with a A phage carrying a metE-lacZ gene fusion, we examined B12-mediated repression of the metE-lacZ gene fusion. Although B12 supplementation results in a 10-fold repression of metE-lacZ expression, homocysteine addition to the growth medium overrides the B12-mediated repression. In addition, B12-mediated repression of the metE-lacZ fusion is dependent on a functional MetR protein. When a metB mutant was transformed with a high-copy-number plasmid carrying the metE gene, which would be expected to reduce intracellular levels of homocysteine, metE-lacZ expression was reduced and B12 supplementation had no further effect. In a metJ mutant, B12 represses metE-lacZ expression less than twofold. When the metJ mutant was transformed with a high-copy-number plasmid carrying the metH gene, which would be expected to reduce intracellular levels of homocysteine, B12 repression of the metE-lacZ fusion was partially restored. The results indicate that B12-mediated repression of the metE gene is primarily a loss of MetR-mediated activation due to depletion of the coactivator homocysteine, rather than a direct repression by the MetH-Bl2 holoenzyme.

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Effect of l-methionine and vitamin B12 on methionine biosynthesis in Escherichia coli

Cited by 52 publications

References 18 publications

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

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

Unraveling Vitamin B₁₂-Responsive Gene Regulation in Algae

Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene

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Effect of l-methionine and vitamin B12 on methionine biosynthesis in Escherichia coli

Cited by 52 publications

References 18 publications

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

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

Unraveling Vitamin B12-Responsive Gene Regulation in Algae

Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene

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Unraveling Vitamin B₁₂-Responsive Gene Regulation in Algae