Journal of General Microbiology (1991), 137, 685-691. Printed in Great Britain 685 Control of methionine biosynthesis in Escherichia coli K12: a closer study with analogue= resistant mutants M. K. CHATTOPADHYAY, A. K. GHOSH and S. SENGUPTA" Department of Applied Biochemistry, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India (Received 16 February 1990 ; revised 28 September I990 ; accepted 3 December 1990) Control of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue- resistant mutants. Norleucine (NL) and a-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization. Adenosylation of Et to generate S- adenosylethionine (AdoEt) by cell-free enzyme from E. coli K12 was demonstrated. Tolerance of increasing concentrationsof NL by E. coli K12 mutants is expressed serially as phenotypes NLR,NLR EtR,NLR MMRand finally NLR EtR MMR.All spontaneous NLRmutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metX and met.! genes. The kinetics of methionine adenosylation by the E. coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture. NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet. Introduction In Escherichia coli, the genes of the methionine regulon are distributed throughout the chromosome (Bachmann, 1983). The regulatory functions of S-adenosylmethionine (AdoMet) as co-repressor and of the rnetJgene product as aporepressor have been confirmed by studying in vitro expression of some of the E. coli methionine biosynthesis genes (Shoeman et a!., 1985a, 6). However, none of the metJ and metK (see Fig. 1) regulatory mutants isolated (Su et al., 1970; Kung et af., 1972; Greene et al., 1970, 1973) were fully decontrolled for methionine production. The non-B, ,-dependent transmethylation step which is one of the two convergent routes for methionine biosynthesis (Fig. l), synthesizing the methyl group from serine, was also found to be under the same repressor control (Smith, 1971). The regulatory roles of MetJ protein and AdoMet in the expression of the 5,lO- methylenetetrahydrofolate reductase gene (Shoeman et al., 1985a) and of AdoMet in the synthesis of serine hydroxymethyltransferase have been demonstrated (Greene & Radovich, 1975, Dev & Harvey, 1984). In this paper, we report a reinvestigation of the methionine control system by studying spontaneously occurring Abbreviations : AdoEt, S-adenosyl-DL-ethionine ; AdoMet, S-adeno- syl-L-methionine; Et, DL-ethionine; MM, a-methyl-DL-methionine; NL, DL-norleucine. methionine-analogue-resistant mutants of E. coli K 12, with the hope that mutants thus isolated might have a single locus altered among the large numbers of loci involved in the overall control of methionine biosynthe- sis and might thereby yield a better understanding of the control mechanism. Methods Bacterial strains and growth conditions. Escherichia coli K 12 strains (see Table 1) and phage P1 were obtained from the E. coli Genetic Stock Center, Yale University School of Medicine, New Haven, CT, USA. Minimal medium was that of Davis & Mingioli (1950) with 1 % (w/v) glucose. Cells were grown in shake flasks at 37 "C. Chemicals. N-Methyl-N'-nitro-N-nitrosoguanidine (NTG) was ob- tained from Fluka. DL-Ethionine (Et), DL-norleucine (NL), a-methyl- DL-methionine (M M), S-adenosyl-L-me t hionine ( AdoMet), S-adenosyl- DL-ethionine (AdoEt) and ATP (sodium salt) were purchased from Sigma. SP-Sephadex (C-25) was obtained from Pharmacia. [?3]Meth- ionine was obtained from the Bhabha Atomic Research Centre, Bombay, India. Determination of minimum inhibitory concentrations (MICs) of methion- ine analogues and selection of analogue-resistant mutants. MIC values were determined by dilution in minimal medium. To obtain analogue- resistant mutants in liquid medium, cells from the mid-exponential phase were harvested, washed and suitably diluted with saline, and inoculated into analogue-containing minimal medium (AM) at an initial population of approximately lo5 cells ml-l, growth being permitted up to 72 h. For mutant isolation on solid medium, 0001-6095 O 1991 SGM