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-methyltetrahydrofolate that is synthesized from N5,10-methylenetetrahydrofolate by the 5,10-methylenetetrahydrofolate reductase. The Streptomyces lividans metF gene, which encodes 5,10-methylenetetrahydrofolate reductase, has been cloned. It encodes a protein of 307 amino acids with a deduced molecular mass of 33,271 Da. S1 exonuclease mapping of the 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 MetR-binding sequence of Salmonella typhimurium. Expression of metF in multicopy plasmids in S. lividans resulted in accumulation of a 32-kDa protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Disruption of the metF gene led to methionine auxotrophy. Integration of the disrupting plasmid at the metF locus was confirmed by Southern hybridization in three randomly isolated transformants. The methionine auxotrophy was complemented by transformation of the auxotrophs with an undisrupted metF gene. These results indicate that the folate branch is essential for methionine biosynthesis in streptomycetes, as occurs in enterobacteria.
Blanco
J
J
Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of León, Spain.
Coque
J J
JJ
Martin
J F
JF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D008715:Methionine / Q000096:biosynthesis* / Q000378:metabolism*; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012486:Salmonella typhimurium / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C058169:MetR protein, Bacteria; D012333:RNA, Messenger; D013763:Tetrahydrofolates; D015534:Trans-Activators; D008715:Methionine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D015719:Single-Strand Specific DNA and RNA Endonucleases; C005984:5-methyltetrahydrofolate