Elucidation of regulatory mechanisms effecting nisin biosynthesis in Lactococcus lactis ATCC 11454

The bacteriocin nisin is an anti-microbial peptide produced by several strains of Lactococcal lactis. The actual biosynthetic mechanism has been well described but knowledge on the regulatory mechanisms is incomplete. To date, a well-established model supports regulation at the level of transcription wherein nisin is believed to auto-regulate its own synthesis. The investigations presented in this thesis identify a novel induction mechanism and three repressive mechanisms that impact nisin transcription thereby affecting nisin biosynthesis. The results indicate that three different regulatory mechanisms are in effect in each of the repressive effects presented. It appears that repression of nisin biosynthesis in glycerol possibly involves a repressor model, which is inactivated in the presence of cAMP. In the case of growth at an elevated temperature (40°C), the nisRK system seems to be affected and addition of extragenous nisin helps overcome the repression resulting in a restoration of both nisin transcription and nisin production. In the third scenario, both nisin production and nisin transcription were abolished upon the electroporation of plasmids but could be restored to a very low-level by the addition of exogenous nisin. This restoration probably occurs in a nisRK independent fashion, as indicated by a lack of a dose dependence response to the inducer molecule. In addition, the novel nisin independent galactose-mediated induction was further investigated by first determining the nisA start site during growth in galactose. The region involved in the galactose-mediated induction was identified by directed deletion analysis of a 200 by region upstream of the nisA promoter in the transcription fusion, pDOC99. The induction of the deletion derivatives by galactose and nisin were compared phenotypically and the regions necessary for the induction determined by sequence analysis. These findings reveal the complexity of nisin regulation in L. lactis that includes but is not limited to the nisin auto-regulatory process. Future work in this area may help unravel these intriguing mechanisms and possibly provide a deeper insight into the complex processes that constitute the regulation of nisin biosynthesis. This knowledge may then be extrapolated to understand the regulation of other lantibiotics.