Lactococcin A overexpression in a Lactococcus lactis subsp. lactis transformant containing a Tn 5 insertion in the lcnD gene.

Lactococcin A production in lactococci has recently been linked to a signal-sequence-independent secretory system consisting of a four-gene cluster. Lactococcus lactis subsp. lactis LLM23L-A1 has been obtained after Tn 5 mutagenesis of pLLM23, a plasmid containing the gene cluster responsible for lactococcin A production. In contrast to other Tn 5-generated mutants, strain LLM23L-A1 exhibited a 12-fold increase in lactococcin A production. Overproduction of lactococcin A was not linked to an increased pLLM23 copy number. Restriction-enzyme analysis indicated the site of Tn 5 insertion to be at the 3′ end of lcnD and upstream of the lcnA structural gene. From DNA sequencing, the Tn 5 insertion was located −79 bp upstream of the transcription start site of the lcnA and lciA genes, eliminating eight amino acids from the C-terminal end of lactococcin D. Northern blots revealed overproduction of a 500-base transcript in strain LLM23L-A1, which corresponded to that predicted from the positions of the lactococcin A operon transcriptional start site and the termination structures. This result suggests that the overproduction of lactococcin A in strain LLM23L-A1 is at the transcriptional level and provides further impetus for elucidating the complete regulatory mechanism for lactococcin A expression. [ABSTRACT FROM AUTHOR]