CLONING AND MOLECULAR ANALYSIS OF THE DIHYDROFOLATE-REDUCTASE GENE FROM LACTOCOCCUS-LACTIS

The Lactococcus lactis gene encoding trimethoprim resistance has been cloned and expressed in Escherichia coli and Bacillus subtilis. Severa l lines of evidence indicate that the cloned gene encodes dihydrofolat e reductase (DHFR). (i) It fully complements the fol ''null'' mutation in E. coli. (ii) Nucleotide sequencing of the cloned fragment reveale d the presence of one open reading frame encoding a protein that share s homology with the family of bacterial DHFR enzymes. (iii) Overexpres sion of this open reading frame in E. coli resulted in the appearance in cell extracts of a protein of the expected size as well as in a dra matic increase of DHFR activity. In cell extracts, the DHFR activity w as not inhibited by low trimethoprim concentration. By Northern (RNA) blotting and primer extension analyses, the size and the start point o f the dhfr transcript, respectively, have been determined. Results of these experiments indicate that in L. lactis the dhfr gene represents part of a larger transcription unit.