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Cloning and characterization of the genes for the two homocysteine transmethylases of Escherichia coli

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We have cloned the genes for the two homocysteine transmethylases of Escherichia coli K12. The vitamin B12-independent enzyme is encoded by the metE gene while the metH gene codes for the vitamin B12-requiring enzyme. Overexpression of the gene products and Tn1000 mutagenesis have enabled the metE and metH gene products to be identified as 99 kDa and 130 kDa polypeptides, respectively. The truncated polypeptides generated by Tn1000 insertion were used to determine the direction of transcription of the metE and metH genes. Negative complementation suggests that the MetH enzyme exists as an oligomer. Investigation of the expression of the chromosomal- and plasmid-encoded gene products confirms that metE is subject to negative control by vitamin B12 and methionine, and that metH is under positive control by the cofactor and negative control by methionine. For vitamin B12 and methionine to act as regulatory effectors in metE control, functional metH and metJ genes are required, respectively. The use of stable Tn1000-generated fragments of the metE product as electrophoretic markers for the plasmid-encoded metE gene product demonstrated that the two regulatory proteins involved in negative control of metE are present in excess. Under conditions whereby both forms of negative metE control are non-functional, the metE gene product represented about 90% of the total protein, and cell growth was severely impaired.

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References

  • Adams JM, Capecci MR (1966) N-formylmethionyl-s-RNA as the initiator of protein synthesis. Proc Natl Acad Sci USA 55:147–155

    Google Scholar 

  • Ahmed A (1973) Mechanism of repression of methionine biosynthesis in E. coli. I. The role of methionine, S-adenosylmethionine and methionyl-transfer ribonucleic acid in repression. Mol Gen Genet 123:299–324

    Google Scholar 

  • Appleyard RK, McGregor JF, Baird KM (1956) Mutations to extended host range and occurrence of phenotype mixing in temperate coliphage. Virology 2:565–574

    Google Scholar 

  • Bachman BJ (1983) Linkage map of Escherichia coli K-12. Edition 7. Microbiol Rev 47:180–230

    Google Scholar 

  • Belfaiza J, Parsot C, Martel A, de la Tour CB, Margarita D, Cohen GN, Saint-Girons I (1986) Evolution in biosynthetic pathways: Two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region. Proc Natl Acad Sci USA 83:867–871

    Google Scholar 

  • Belfaiza J, Guillou Y, Margarita D, Perrin D, Saint-Girons I (1987) Operator-constitutive mutations of the Escherichia coli metF gene. J Bacteriol 169:670–674

    Google Scholar 

  • Birge EA, Low KB (1974) Detection of transcribable recombination products following conjugation in Rec+, RecB−, and RecC− strains of Escherichia coli K12. J Mol Biol 83:447–457

    Google Scholar 

  • Blattner FR, Williams BG, Blechl AE, Denniston-Thompson K, Faber HE, Furlong L, Gunwald DG, Kiefer DO, Moore DD, Schum JM, Scheldon ER, Smithies O (1977) Charon phages: safe derivatives of bacteriophage lambda for DNA cloning. Science 194:161–169

    Google Scholar 

  • Boothroyd CM, Malet RM, Nene V, Glass RE (1983) Genetic studies on the β subunit of Escherichia coli RNA polymerase III. Analysis of low level resistance mutants. Mol Gen Genet 190:523–526

    Google Scholar 

  • Bullas LR, Ryu J-I (1983) Salmonella typhimurium LT2 strains which are r− m+ for all three chromosomally located systems of DNA restriction and modification. J Bacteriol 156:471–474

    Google Scholar 

  • Childs JD, Smith DA (1969) New methionine structural gene in Salmonella typhimurium. J Bacteriol 100:377–382

    Google Scholar 

  • Chu J, Shoeman R, Hart J, Coleman T, Mazaitis A, Kelker N, Brot N, Weissbach R (1985) Cloning and expression of the metE gene in E. coli. Arch Biochem Biophys 239:467–474

    Google Scholar 

  • Dawes J, Foster MA (1971) Vitamin B12 and methionine synthesis in Escherichia coli. Biochim Biophys Acta 237:455–464

    Google Scholar 

  • Duchange N, Zakin MM, Ferrara P, Saint-Girons I, Park I, Tran SV, Py M-C, Cohen GN (1983) Structure of the metJBLF cluster in Escherichia coli K12. Sequence of the metB structural gene and of the 5′- and 3′-flanking regions of the metBL operon. J Biol Chem 258:14868–14871

    Google Scholar 

  • Enquist L, Sternberg N (1979) In vitro packaging of Dam vectors and their use in cloning DNA fragments. Methods Enzymol 68:281–309

    Google Scholar 

  • Flavin M (1975) Methionine biosynthesis. In: Greenburg DM (ed) Metabolism of sulphur compounds. Metabolic Pathways 3rd Edition. Academic Press. London, pp 457–503

    Google Scholar 

  • Fletcher AJP, Pugsley AP, Glass RE (1986) Investigation of the regulation of the Escherichia coli btuB gene using operon fusions. J Gen Microbiol 132:2643–2646

    Google Scholar 

  • Foster MA, Tejerina G, Woods DD (1961a) Two enzymatic mechanisms for the methylation of homocysteine by Escherichia coli. Biochem J 81:1

    Google Scholar 

  • Foster MA, Jones KM, Woods DD (1961b) The purification and properties of a fraction containing vitamin B12 concerned in the synthesis of methionine by Escherichia coli. Biochem J 80:519–531

    Google Scholar 

  • Foster MA, Tejerina G, Guest JR, Woods DD (1964) Two enzymatic mechanisms for the methylation of homocysteine by extracts of Escherichia coli. Biochem J 92:476–488

    Google Scholar 

  • Fuji K, Huennekens FM (1974) Activation of methionine synthetase by a reduced triphosphopyridine nucleotide-dependent flavoprotein system. J Biol Chem 249:6745–6753

    Google Scholar 

  • Galivan J, Huennekens FM (1970) Resolution of the methionine synthetase system from E. coli K-12. Biochem Biophys Res Commun 38:46–51

    Google Scholar 

  • Greene RC, Smith AA (1984) Insertion mutagenesis of the metJBLF gene cluster of Escherichia coli K12: Evidence for a metBL operon. J Bacteriol 159:767–769

    Google Scholar 

  • Greene RC, Williams RD, Kung H-S, Spears C, Weissbach H (1973) Effects of methionine and vitamin B12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12. Arch Biochem Biophys 158:249–256

    Google Scholar 

  • Guest JR, Friedman S, Woods DD, Smith EL (1962) A methyl analogue of cobamide coenzyme in relation to methionine synthesis by bacteria. Nature 195:340–342

    Google Scholar 

  • Guest JR, Friedman S, Foster MA, Tejerina G, Woods DD (1964a) Transfer of the methyl group from N5-methyltetrahydrofolate to homocysteine in Escherichia coli. Biochem J 92:497–504

    Google Scholar 

  • Guest JR, Foster MA, Woods DD (1964b) Methyl derivatives of folic acid intermediates in the methylation of homocysteine by Escherichia coli. Biochem J 92:48–496

    Google Scholar 

  • Guyer MS (1978) The γδ sequence of F is an insertion sequence. J Mol Biol 126:347–365

    Google Scholar 

  • Hobson AC, Smith DA (1973) S-adenosylmethionine synthetase in methionine regulatory mutants of Salmonella typhimurium. Mol Gen Genet 126:7–18

    Google Scholar 

  • Hunter MG, Glass RE (1982) Analysis of btuB receptor function by use of nonsense suppression. J Bacteriol 151:1591–1594

    Google Scholar 

  • Hunter JSV, Greene RC, Su C-H (1975) Genetic characterization of the metK locus in Escherichia coli K-12. J Bacteriol 122:1144–1152

    Google Scholar 

  • Kadner RJ, Liggins GL (1973) Transport of vitamin B12 in Escherichia coli: Genetic studies. J Bacteriol 115:514–521

    Google Scholar 

  • Kung H, Spears C, Greene R, Weissbach H (1972) Regulation of the terminal reactions in methionine biosynthesis by vitamin B12 and methionine. Arch Biochem Biophys 150:23–31

    Google Scholar 

  • Lederberg EM, Cohen SN (1974) Transformation of S. typhimurium by plasmid deoxyribonucleic acid. J Bacteriol 119:1072–1074

    Google Scholar 

  • Loenen WAM, Brammar WJ (1980) A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene 20:249–259

    Google Scholar 

  • Lundrigan MD, DeVeaux LC, Mann BJ, Kadner RJ (1987) Separate regulatory systems for the repression of metE and btuB by vitamin B12 in Escherichia coli. Mol Gen Genet 206:401–407

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Marcker K, Sanger F (1964) N-formylmethionyl-S-RNA. J Mol Biol 8:835–840

    Google Scholar 

  • Mazaitis AJ, Palchaudhuri S, Glansdorff N, Maas WK (1976) Isolation and characterization of λdargECBH transducing phages and heteroduplex analysis of the argECBH cluster. Mol Gen Genet 143:185–196

    Google Scholar 

  • Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122

    Google Scholar 

  • Michaeli S, Ron EZ (1984) Expression of the metA gene of Escherichia coli K12 in recombinant plasmids. FEMS Microbiol Lett 23:125–129

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Moir PD, Hunter MG, Armstrong JT, Glass RE (1987) Studies on the gene for the multivalent vitamin B12 receptor of Escherichia coli. FEMS Microbiol Lett 41:103–108

    Google Scholar 

  • Mulligan JT, Margolin W, Kreuger JH, Walker GC (1982) Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions. J Bacteriol 151:609–619

    Google Scholar 

  • Paessens A Rudiger H (1980) Purification and chemical characterization of the vitamin B12-dependent 5-methyltetrahydrofolatehomocysteine methyltransferase from Escherichia coli B. Eur J Biochem 112:47–51

    Google Scholar 

  • Poston JM, Stadtman TC (1975) Cobamides as Cofactors: Methyl cobamides and the synthesis of methionine, methane and acetate. In: Babior BM (ed) Cobalamin: Biochemistry and Pathophysiology. John Wiley and Sons. New York, London, Sydney, Toronto, pp 111–140

    Google Scholar 

  • Roehrdanz RL, Dove WF (1971) Studies on the stimulation by helper of site-specific recombination in lytic crosses. Virology 79:32–40

    Google Scholar 

  • Rowbury RJ (1983) Methionine biosynthesis and its regulation. In: Herrmann KM, somerville RL (eds) Amino acids: biosynthesis and genetic regulation. Addison Wesley, London, pp 191–211

    Google Scholar 

  • Rudiger H, Jaenicke L (1970) Methionine synthetase. Existence and interconversion of two enzyme species. Eur J Biochem 16:92–95

    Google Scholar 

  • Rudiger H, Jaenicke L (1973) The biosynthesis of methionine. Mol Cell Biochem 1:157–168

    Google Scholar 

  • Saint-Girons I, Duchange N, Zakin MM, Park I, Margarita D, Ferrara P, Cohen GN (1983) Nucleotide sequence of metF, the E. coli structural gene for 5, 10-methylene tetrahydrofolate reductase and its control region. Nucleic Acids Res 11:6723–6732

    Google Scholar 

  • Saint-Girons I, Duchange N, Cohen GN, Zakin MM (1984) Structure and autoregulation of the metJ regulatory gene in Escherichia coli. J Biol Chem 259:14282–14285

    Google Scholar 

  • Saint-Girons I, Belfaiza J, Buillou Y, Perrin D, Guiso N, Barzu O, Cohen GN (1986) Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine. J Biol Chem 261:10936–10940

    Google Scholar 

  • Saint-Girons I, Parsot C, Zakin MM, Barzu O, Cohen G (1987) Methionine biosynthesis in Enterobacteriaceae: Biochemical, regulatory and evolutionary aspects. CRC Crit Rev Biochem (in press)

  • Schulte LL, Stauffer LT, Stauffer GV (1984) Cloning and characterization of the Salmonella typhimurium metE gene. J Bacteriol 158:928–933

    Google Scholar 

  • Shoeman R, Redfield B, Coleman T Greene RC, Smith AA, Brot N, Weissbach H (1985a) Regulation of methionine synthesis in Escherichia coli: Effect of metJ gene product and S-adenosylmethionine on the expression of the metF gene. Proc Natl Acad Sci USA 82:3601–3605

    Google Scholar 

  • Shoeman R, Coleman T, Redfield B, Greene RC, Smith AA, Saint-Girons I, Brot N, Weissbach H (1985b) Regulation of methionine synthesis in Escherichia coli: Effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL, and metJ genes. Biochem Biophys Res Commun 133:731–739

    Google Scholar 

  • Shoeman R, Redfield B, Coleman T, Brot N, Weissbach H, Greene RC, Smith AA, Saint-Girons I, Zakin MM, Cohen GN (1985c) Regulation of the methionine regulon in Escherichia coli. Bioessays 3:210–213

    Google Scholar 

  • Silhavy TJ, Berman ML, enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Smith DA (1971) S-Amino acid metabolism and its regulation in Escherichia coli and Salmonella typhimurium. Adv Genet 16:141–165

    Google Scholar 

  • Smith DA, Childs JD (1966) Methionine genes and enzymes of Salmonella typhimurium. Heredity 21:265–286

    Google Scholar 

  • Soberon X, Covarrubias L, Bolivar F (1980) Construction and characterizsation of new cloning vehicles IV. Deletion derivatives of pBR322 and pBR325. Gene 9:287–305

    Google Scholar 

  • Stavrianopoulos J, Jaenicke L (1967) Reaktionsschritte der Methionin-Synthese bei Escherichia coli. Eur J Biochem 3:95–106

    Google Scholar 

  • Sternberg N, Tiemeier D, Enquist L (1977) In vitro packaging of a Dam vector containing EcoRI DNA fragments of Escherichia coli and phage PI. Gene 1:255–280

    Google Scholar 

  • Stoker NG, Pratt JM, Holland IB (1984) In vivo gene expression systems in prokaryotes. In: Hames BD, Higgins SJ (eds) Transcription and translation, a practical approach. IRL Press, Oxford, pp 153–177

    Google Scholar 

  • Tabor H, Rosenthal SM, Tabor CW (1958) The biosynthesis of spermidine and spermine from putrescine and methionine. J Mol Biol 233:907–914

    Google Scholar 

  • Taylor RT (1970) Escherichia coli B 5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase: Resolution and reconstitution of holoenzyme. Arch Biochem Biophys 137:529–546

    Google Scholar 

  • Taylor RT (1971) Escherichia coli B N5-methyltetrahydrofolatehomocysteine cobalamin methyltransferase: Gel filtration behaviour of apoenzyme and holoenyzmes. Biochim Biophys Acta 242:355–364

    Google Scholar 

  • Taylor RT, Weissbach H (1967) N5-methyltetrahydrofolate-homocysteine transmethylase. Partial purification and properties. J Biol Chem 242:1502–1508

    Google Scholar 

  • Taylor RT, Weissbach H (1969a) Escherichia coli B N5-methyltetrahydrofolate-homocysteine methyltransferase: Sequential formation of bound methylcobalamin with S-adenosyl-L-methionine and N5-methyltetrahydrofolate. Arch Biochem Biophys 129:728–744

    Google Scholar 

  • Taylor RT, Weissbach H (1969b) Escherichia coli B N5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase. Activation with S-adenosyl-L-methionine and the mechanism for methyl group transfer. Arch Biochem Biophys 129:745–766

    Google Scholar 

  • Taylor RT, Weissbach H (1973) N5-methyltetrahydrofolate-homocysteine transmethylase. Enzymes 19:121–165

    Google Scholar 

  • Twigg AJ, Sherratt D (1980) Trans-complementable copy-number mutants of plasmid ColE1. Nature 283:216–218

    Google Scholar 

  • Urbanowski ML, Stauffer GV (1986) The metH gene from Salmonella typhimurium LT2: cloning and initial characterization. Gene 44:211–217

    Google Scholar 

  • Urbanowski ML, Plamann LS, Stauffer GV (1987a) Mutations affecting the regulation of the metB gene of Salmonella typhimurium LT2. J Bacteriol 169:126–130

    Google Scholar 

  • Urbanowski ML, Stauffer LT, Plamann LS, Stauffer GV (1987b) A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium. J Bacteriol 169:1391–1397

    Google Scholar 

  • Weissbach H, Taylor RT (1970) Role of vitamin B12 and folic acid in methionine synthesis. Vitam Horm 28:415–550

    Google Scholar 

  • Whalen WA, Berg CM (1984) Gratuitous repression of autA in Escherichia coli and Salmonella typhimurium. J Bacteriol 158:571–574

    Google Scholar 

  • Whitehouse JM, Smith DA (1973) Methionine and vitamin B12 repression and precursor induction in the regulation of homocysteine methylation in Salmonella typhimurium. Mol Gen Genet 120:341–353

    Google Scholar 

  • Whitfield CD, Steers EJ, Weissbach H (1970) Purification and properties of 5-methyltetrahydropteroyltriglutamate-homocysteine transmethylase. J Biol Chem 245:390–401

    Google Scholar 

  • Woods DD, Foster MA, Guest JR (1965) Cobalamin-dependent and-independent methyl transfer in methionine biosynthesis. In: Shapiro SK, Sclenk F (eds) Transmethylation and Methionine Biosynthesis. University of Chicago Press, Chicago, London, pp 138–154

    Google Scholar 

  • Yang RCA, Lis J, Wu R (1979) Elution of DNA from agarose gels after electrophoresis. Methods Enzymol 68:176–182

    Google Scholar 

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  1. Michael G. Hunter

    Present address: Searle Research and Development, Division of G.D. Searle and Co., 4901 Searle Parkway, 60077, Skokie, IL, USA

  2. D. T. R. Wilson

    Present address: Department of Medicine, City Hospital, Lincoln, UK

  3. Carolyn A. Weatherston

    Present address: Department of Undergraduate Medicine, University Hospital, Derby Road, NG7 2UH, Nottingham, UK

Authors and Affiliations

  1. Department of Biochemistry, Queens Medical Centre, Clifton Boulevard, NG7 2UH, Nottingham, UK

    Iain G. Old, Michael G. Hunter, D. T. R. Wilson, Susan M. Knight, Carolyn A. Weatherston & Robert E. Glass

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Old, I.G., Hunter, M.G., Wilson, D.T.R. et al. Cloning and characterization of the genes for the two homocysteine transmethylases of Escherichia coli . Mol Gen Genet 211, 78–87 (1988). https://doi.org/10.1007/BF00338396

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