MUTATIONAL ANALYSIS OF CAT-86 GENE-EXPRESSION CONTROLLED BY LACTOCOCCAL PROMOTERS IN LACTOCOCCUS-LACTIS SUBSP LACTIS AND ESCHERICHIA-COLI

Promoters were cloned from the chromosomal DNA of Lactococcus lactis s ubsp. lactis NP4510 by using promoter-probe vector pGKV210. N-Methyl-N '-nitro-N-nitrosoguanidine-induced mutagenesis of L. lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced exp ression. Subcloning and sequencing of the mutated plasmid designated p BV415 revealed that the mutation is located within the PstI-HindIII fr agment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine). A set of otherwi se identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by sit e-directed mutagenesis. These plasmids containing cat-86 derivatives d isplayed a significant variation in cat expression in L. lactis and E. coli. The data suggest that cat expression is dependent on the second ary structure of the cat mRNA. New cat-86 derivatives described here c an be used in lactococci, in which they provide additional flexibility for promoter cloning.