Regulation of Methionine Biosynthesis in Escherichia coli: Mapping of the metJ Locus and Properties of a metJ ⁺ / metJ ^- Diploid

Abstract

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)

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

“…the relative sensitivity of met gene promoters to induction under SAM-limiting conditions (Table 4; Greene et al, 1973;Holloway et al, 1970); and in our metJ : : Tn5 (no aporepressor) and metK : : Tn5 (limited SAM synthesis) cells (Table 5; Greene et al, 1973;Su & Greene, 1971). …”

“…Mutants of the more common type have decreased in vivo pools of SAM as a result of impaired SAM synthetase activity, and the defects map to metK (Greene et al, 1973;Hafner et al, 1977). Mutations of the second class map to metJ, and genetic and biochemical evidence identified the corresponding small protein as a DNAbinding aporepressor of met gene transcription (Su & Greene, 1971). These results suggested that a complex of MetJ and SAM binds a common nucleotide sequence in the promoters of target genes to prevent their transcription (Greene et al, , 1973Hobson & Smith, 1973).…”

Methionine Regulatory Defects in Salmonella typhimurium Arising from Amber-suppressible Mutations

“…Assuming that S-adenosylmethionine (SAM) is the co-repressor, Chater felt that the available evidence was in favour of metK rather than metJ coding for SAM synthetase, and recently Hobson (personal communication) has shown that SAM synthetase levels in some metK mutants vary from o to 25 % of those in wild-type or metJ mutants. Certainly the derepressed state of methionine enzymes in mutants of Escherichia coli K 1 2 with low levels of SAM synthetase and in others constitutive for the same enzyme (and apparently analogous to the Salmonella typhimurium metJ mutants -Holloway, Greene & Su, 1970; Su & Greene, 1971), also clearly implicate SAM in repression control of methionine synthesis in E. coli. However, the existence of SAM synthetase-deficient mutants implies that this enzyme may be dispensable which seems unlikely because of the direct involvement of SAM in critical intracellular methylation processes.…”

Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene

“…This protein interacts with S-adenosylmethionine to form an active complex that presumably functions as a classical repressor (13,17). The metJ gene of S. typhimurium has been cloned and its nucleotide sequence has been determined (18,19).…”

“…Promoter Pjl 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 pJl.In both Salmonella typhimurium and Escherichia coli the metJ gene codes for a protein that is involved in the regulation of the methionine pathway (5,13,17). This protein interacts with S-adenosylmethionine to form an active complex that presumably functions as a classical repressor (13,17).…”