A mutant ofEscherichia colitemperature sensitive in the biosynthesis ofS-adenosylmethionine

Abstract

S-Adenosylmethionine (SAM) is synthesized in vitro by SAM synthetase, which is coded for by the metK gene of Escherichia coli. Since E. coli cells are impermeable to SAM it has been impossible to obtain mutants which require SAM for growth. The finding that SAM is required for regulating methionine biosynthesis was used to select metK mutants which have the phenotype of resistance to methionine analogues. These mutants show reduced SAM synthesis in vitro, and a lower level of intracellular SAM. However, as all these mutants can grow, without added SAM, the block in SAM synthesis must be incomplete. To try to block SAM synthesis more completely we combined temperature-sensitive metK mutation with a block in the synthesis of cystathionine (metA), a precursor that supports only slow growth because of slow entry. At the non-permissive temperature for metK these double mutants could grow on methionine but not on cystathionine, in correlation with the greatly reduced intracellular level of SAM. This result indicates that it is possible to reduced intracellular SAM concentrations to levels which are low enough to prevent growth of E. coli. Moreover, since the mutation responsible for the reduction in SAM level is in the metK gene, it is suggested that SAM synthetase is the enzyme which catalyses SAM biosynthesis in E. coli cells.