title: Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum.
abstract: Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite D,L-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of L-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.
authors.affiliation: Institute of Biotechnology, Swiss Federal Institute of Technology (ETH), Zurich 8093, Switzerland. mampel@micro.biol.ethz.ch
authors.forename: Jörg
authors.initials: J
authors.lastname: Mampel
authors.forename: Hartwig
authors.initials: H
authors.lastname: Schröder
authors.forename: Stefan
authors.initials: S
authors.lastname: Haefner
authors.forename: Uwe
authors.initials: U
authors.lastname: Sauer
mesh_terms: D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000502:physiology; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media; D003547:Cysteine Synthase / Q000235:genetics / Q000502:physiology; D004251:DNA Transposable Elements / Q000235:genetics; D005001:Ethionine / Q000378:metabolism; D017353:Gene Deletion; D005816:Genetic Complementation Test; D008715:Methionine / Q000096:biosynthesis*; D009154:Mutation; D016366:Open Reading Frames; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics / Q000502:physiology
chemical_list: D003470:Culture Media; D004251:DNA Transposable Elements; D008715:Methionine; D050862:Oxidoreductases Acting on Sulfur Group Donors; D003547:Cysteine Synthase; C018725:O-acetylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases; D005001:Ethionine