Addition of methionine to the growth medium of Escherichia coli K-12 leads to a reduction in the specific activity of S-adenosylmethionine (SAM) synthetase. Thus the enzyme appears to be repressible rather than inducible. Mutant strains (probably metJ-) are constitutive for SAM synthetase as well as for the methionine biosynthetic enzymes, suggesting that the regulatory systems for these enzymes have at least some elements in common. Cells grown to stationary phase in complete medium, which have low specific activities of the enzymes, were routinely used for derepression experiments. The lag in growth and derepression when these cells are incubated in minimal medium is shortened by threonine. Ethionine, norleucine, and a-methylmethionine are poor substrates or nonsubstrates for SAM synthetase and are ineffective repressors. Selenomethionine, a better substrate for SAM synthetase than methionine, is also slightly more effective at repression than methionine. Although SAM is considered to be a likely candidate for the corepressor in the control of the methionine biosynthetic enzymes, addition of SAM to the growth medium does not cause repression. Measurement of SAM uptake shows that too little is taken into the cells to have a significant effect, even if it were active in the control system.
MetJ-mutants of Escherichia coli have elevated nonrepressible levels of the enzymes of methionine biosynthesis and S-adenosylmethionine synthetase (ATP :L-methionine S-adenosyltransferase, EC 2.5.1.6). In E. coli, as in Salmonella typhimurium, the metJ locus is close to metB (95% cotransduction of metB and metJ markers), but in E. coli the order is reversed, with metJ mapping clockwise to metB. A stable merodiploid, heterozygous for metJ, is subject to repression by methionine. Thus, metJ functions via a diffusible product. MetJ could either be a regulatory locus or could code for an enzyme required for the synthesis of a methionine metabolite that functions in the control system.One of the first amino acid regulatory mutants described in Escherichia coli was constitutive for the biosynthesis of methionine (1). This and similar mutants have been used to demonstrate that the several enzymes of the methionine synthetic pathway are regulated by the same system (1-3), but the mechanism of regulation has not been extensively investigated. Lawrence, Smith, and Rowbury (4) have described three classes of mutants in Salmonella typhimurium resistant to various analogs of methionine. Mutants at the metl locus produce homoserine transsuccinylase which is not subject to feedback inhibition (5), but have normal levels of the biosynthetic enzymes. On the other hand, mutants at the metJ and metK loci produce elevated levels of these enzymes (4). We have isolated two classes of ethionine-resistant mutants of E. coli, both of which overproduce methionine biosynthetic enzymes. One class (which we believe to be analogous to the metK mutants of S. typhimurium) has low levels of S-adenosylmethionine (SA.M) synthetase (6). The derepressed condition of these strains may be indicative of the participation of SAM or one of its metabolites in control of the enzymes of methionine biosynthesis. In the other class of derepressed strains, which appear to be metJ mutants, the level of SAM synthetase, as well as that of the methionine biosynthetic enzymes, is elevated and nonrepressible (7). Although the evidence is not definitive, metJ may be a regulatory locus.In this communication we report mapping of the met. locus in E. coli and the construction and regulatory properties of a stable merodiploid heterozygous for met.J.
MATERIALS AND METHODS
MethodsGrowth of cells and assay of their cystathioniney-synthetase, cystathionase, and SAM synthetase (EC 2.5.1.6) activities have been described (7). Defined growth media are based on a modified Davis and Mingioli minimal medium (8)
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