ISOZYMES OF S-ADENOSYLMETHIONINE SYNTHETASE ARE ENCODED BY TANDEMLY DUPLICATED GENES IN ESCHERICHIA-COLI

The sole biosynthetic route to S-adenosylmethionine, the primary biolo gical alkylating agent, is catalysed by S-adenosylmethionine synthetas e (ATP: L-methionine S-adenosyltransferase). In Escherichia coli and S almonella typhimurium numerous studies have located a structural gene (metK) for this enzyme at 63 min on the chromosomal map. We have now i dentified a second structural gene for S-adenosylmethionine synthetase in E. coli by DNA hybridization experiments with metK as the probe; w e denote this gene as metX. The metX gene is located adjacent to metK with the gene order speA metK metX speC. The metK and metX genes are s eparated by approximately 0.8 kb. The metK and the metX genes are orie nted convergently as indicated by DNA hybridization experiments using sequences from the 5' and 3' ends of metK. The metK gene product is de tected immunochemically only in cells growing in minimal media, wherea s the metX gene product is detected immunochemically in cells grown in rich media at all growth phases and in stationary phase in minimal me dia. Mutants in metK or metX were obtained by insertion of a kanamycin resistance element into the coding region of the cloned metK gene (me tK=kan), followed by use of homologous recombination to disrupt the ch romosomal metK or metX gene. The metK=kan mutant thus prepared does no t grow on minimal media but does grow normally on rich media, while th e corresponding metX=kan mutant does not grow on rich media although i t grows normally on minimal media. These results indicate that metK ex pression is essential for growth of E. coli on minimal media and metX expression is essential for growth on rich media. Our results demonstr ate that AdoMet synthetase has an essential cellular and/or metabolic function. Furthermore, the growth phenotypes, as well as immunochemica l studies, demonstrate that the two genes that encode S-adenosylmethio nine synthetase isozymes are differentially regulated. The mutations i n metK and metX are highly unstable and readily yield kanamycin-resist ant cells in which the chromosomal location of the kanamycin-resistanc e element has changed.