Rapid cloning of metK encoding methionine adenosyltransferase from Corynebacterium glutamicum by screening a genomic library on a high density colony-array

The genes SAM1 and SAM2 encoding the two different methionine adenosyltrans ferases (EC 2.5.1.6) in Saccharomyces cerevisiae were used as templates to generate specific DNA-probes. This heterologous mixture of DNA-probes was h ybridized under low stringency hybridization conditions to a Corynebacteriu m glutamicum colony-array representing the complete genome. Subsequently, o ne genomic fragment was isolated which contained the C. glutamicum methioni ne adenosyltransferase gene metK (1.224 kb). When overproduced in Escherich ia coli, MetK (44.2 kDa) of C. glutamicum had methionine adenosyltransferas e activity. In addition, overexpression of metK in C. glutamicum led to an increased intracellular S-adenosylmethionine concentration. The metK transc ript was detected by reverse transcription PCR in C. glutamicum cells in th e exponential growth phase but not in the stationary phase. (C) 2000 Federa tion of European Microbiological Societies. Published by Elsevier Science B .V. All rights reserved.