TRANSFER OF NISIN GENE CLUSTER FROM Lactococcus lactis INTO THE CHROMOSOME OF Bacillus subtilis 168

Abstract

The posttranslationally modified antimicrobial peptide nisin belongs to the family of lantibiotics, which constitute a group of small peptides that contain a high proportion of unusual amino acids. These unusual residues confer unique properties that are essential for its biological activity. Nisin is produced by Lactococcus lactis, and has a broad spectrum of activity against gram-positive bacteria including food-spoilage bacteria and pathogens.

The aim of this work was to express nisin from Bacillus subtilis 168 by transferring the nisin gene cluster from its natural host into the chromosome of B. subtilis 168.This would allow the exploration of the use of genetic engineering techniques to create structural analogs of nisin and also enable a more facile study of the post-translational modification machinery. A variety of strategies to achieve this transfer were explored. In one strategy, a l genomic library of L. lactis 11454 was constructed. The clones containing the 16.5 kb nisin gene cluster and immediate neighboring genes was then subcloned into a cassette vector, pLPVcat, and transformed into B. subtilis ermDsunA, which was then integrated into the B. subtilis chromosome by a double recombination. This recombinant was tested for its ability to express mature nisin into the culture supernatant by using mass spectrometry. However, no nisin or nisin like peptide could be detected when the culture supernatant was analyzed. Integration of a constitutive promoter in front of the nisin structural gene in an attempt to insure that the nisin gene was expressed did not solve the problem. There could be several explanations for this result. One possibility is that transcription of the nisin gene cluster was inadequate despite the presence of the integrated promoter. Another possibility is that transcription was adequate, but there was a lesion in the posttranslational processing pathway. RT-PCR was employed to establish that all of the nis ABTCIPRK genes were transcriptionally active, but the possibility that the level of transcription was too low to give detectable levels of nisin could not be ruled out. It is noted that nisin biosynthesis is a complex process, and that one or more of the posttranslational events could be inefficient in the B. subtilis 168 host.

An important achievement in this work was the construction of a B. subtilis 168 strain that contains the entire nisin gene cluster integrated into the chromosome. This will greatly facilitate future work on expression of nisin in a heterologous strain that is more suitable for laboratory studies than is the natural producer strain.