Lactococcin A overexpression in a Lactococcus lactis subsp. lactis transformant containing a Tn5 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 Tn5 mutagenesis of pLLM23, a plasmid containing the gene cluster responsible for lactococcin A production. In contrast to other Tn5-generated mutants, strain LLM23L-Al 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 Tn5 insertion to be at the 3' end of lcnD, and upstream of the lcnA structural gene. From DNA sequencing, the Tn5 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.
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Department of Food Science and Nutrition, University of Minnesota, St. Paul 55108, USA.
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Stoddard
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McKay
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D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001430:Bacteriocins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D001430:Bacteriocins; D002352:Carrier Proteins; C098554:LcnD protein, Lactococcus lactis; D008565:Membrane Proteins; C069325:lactococcin A