THE FOLATE BRANCH OF THE METHIONINE BIOSYNTHESIS PATHWAY IN STREPTOMYCES-LIVIDANS - DISRUPTION OF THE 5,10-METHYLENETETRAHYDROFOLATE REDUCTASE GENE LEADS TO METHIONINE AUXOTROPHY

In enterobacteria, the methyl group of methionine is donated by 5-meth yltetrahydrofolate that is synthesized from N-5,N-10-methylenetetrahyd rofolate by the 5,10-methylenetetrahydrofolate reductase. The Streptom yces lividans metF gene, which encodes 5,10-methylenetetrahydrofolate reductase, has been cloned. It encodes a protein of 307 amino acids wi th a deduced molecular mass of 33,271 Da. S1 exonuclease mapping of th e transcription initiation site showed that the metF gene is expressed , forming a leaderless mRNA. A 13-bp tandem repeat located immediately upstream of the promoter region shows homology with the consensus Met R-binding sequence of Salmonella typhimurium. Expression of metF in mu lticopy plasmids in S. lividans resulted in accumulation of a 32-kDa p rotein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrop horesis. Disruption of the metF gene led to methionine auxotrophy. Int egration of the disrupting plasmid at the metF locus was confirmed by Southern hybridization in three randomly isolated transformants. The m ethionine auxotrophy was complemented by transformation of the auxotro phs with an undisrupted metF gene. These results indicate that the fol ate branch is essential for methionine biosynthesis in streptomycetes, as occurs in enterobacteria.