The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum.
In order to isolate transcriptional regulatory proteins involved in L-methionine-dependent repression in Corynebacterium glutamicum, proteins binding to the putative promoter region upstream of the metY gene were isolated by DNA affinity chromatography. One of the isolated proteins was identified as a putative transcriptional repressor of the TetR-family by a mass spectrometry fingerprint technique based on the complete C. glutamicum genome sequence. The respective gene, designated mcbR, was deleted in the mutant strain C. glutamicum DR1. Using 2D-PAGE, the protein contents of the C. glutamicum wild type and the mutant strain DR1 grown in media with or without L-methionine supplementation were compared and a set of six proteins was identified. Their abundance was drastically enhanced in the mutant strain and no longer influenced by L-methionine added to the growth medium. The corresponding genes were identified by mass spectrometry fingerprint analysis. They included metY encoding O-acetyl-L-homoserine sulfhydrylase, metK encoding S-adenosyl-methionine synthethase, hom encoding homoserine dehydrogenase, cysK encoding L-cysteine synthase, cysI encoding an NADPH dependant sulfite reductase, and ssuD encoding an alkanesulfonate monooxygenase. Evidently, the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis of L-methionine in C. glutamicum. The C. glutamicum mcbR mutant can be considered to represent a first step in the construction of an L-methionine production strain.
Rey
Daniel Alexander
DA
Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstrasse 25, D-33501 Bielefeld, Germany.
Pühler
Alfred
A
Kalinowski
Jörn
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D003352:Corynebacterium / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004734:Energy Metabolism / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008715:Methionine / Q000096:biosynthesis* / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013457:Sulfur Compounds / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology
D000596:Amino Acids; C483725:McbR protein, C glutamicum; D012097:Repressor Proteins; D013457:Sulfur Compounds; C034811:tetracycline resistance-encoding transposon repressor protein; D008715:Methionine