Characterization of the nisin gene as part of a polycistronic operon in the chromosome of Lactococcus lactis ATCC 11454

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Abstract
The location and organization of the nisin locus in Lactococcus lactis ATCC 11454 were studied. Primer extension of in vivo mRNA transcripts of the gene that encodes the nisin prepropeptide sequence indicated the presence of a promoter at least 4 kb upstream from the nisin gene and that the mRNA has several processing sites. Restriction fragment patterns using rare-cutting enzymes, orthogonal pulsed-field clamped homogeneous electric field (CHEF) agarose gel electrophoresis, and hybridization with nisin gene probes showed that the nisin prepropeptide gene was located on a megabase-size restriction fragment, which was taken as proof of a chromosomal location. This is contrary to earlier reports, which had indicated that genes for nisin production were located on plasmids. There was no evidence of more than one chromosomal location or more than one copy of the nisin gene. The restriction patterns indicated that the size of the L. lactis genome is about 2,500 kb. The previously observed (G. W. Buchman, S. Banerjee, and J. N. Hansen, J. Biol. Chem. 263: 16260-16266, 1988) downstream open reading frame (ORF) was fully sequenced to reveal an 851-amino-acid coding region, an upstream putative mRNA processing site, and a putative rho-independent terminator. The ORF was analyzed for secondary structural features, and the sequence data bases were searched for homologies. The ORF contained many amphipathic helices, a C-terminal transmembrane helix, and homologies to some membrane-associated proteins. It lacked an N-terminal membrane insertion sequence and accordingly appears to be associated with, and anchored to, the cytoplasmic side of the membrane. An additional ORF that possessed a ribosome-binding sequence and tandem promoters, indicating the beginning of a new operon, was identified still farther downstream. The results were consistent with the nisin gene being part of a polycistronic operon with a size greater than 8.5 kb.