CONSTRUCTION AND CHARACTERIZATION OF PROMOTER-PROBE VECTORS FOR CORYNEBACTERIA USING THE KANAMYCIN-RESISTANCE REPORTER GENE

Several multicopy promoter-probe plasmid vectors have been constructed that replicate in Brevibacterium lactofermentum and related coryneform amino acid-producing bacteria. Transcriptional activity is detected by the expression of a promoterless aminoglycoside phosphotransferase gene (kan) derived from transposon Tn5; expression of this gene confers kanamycin resistance in B. lactofermentum. An efficient transcriptional terminator from the B. lactofermentum trp operon has been inserted upstream of the kan coding region to prevent significant transcriptional readthrough from vector promoters. The cat gene from Streptomyces acrimycini or the hygromycin-resistance gene from S. hygroscopicus are used as primary selection markers in the promoter-probe plasmid vectors. Using the promoter-probe vectors described in this paper, we have cloned several transcriptionally active fragments from the endogenous plasmid pBL1 of B. lactofermentum into Escherichia coli and/or B. lactofermentum.