Restriction lift date: 2031-09-30
Natural transformation in lactic acid bacteria
O'Connell, Laura Michelle
University College Cork
Fermentation has been commercially exploited to produce food products for millennia. The most intensely exploited microorganisms used in modern industry to commercially produce fermented products are yeasts and lactic acid bacteria (LAB). LAB are most widely employed in dairy processing, where they contribute to the biochemical changes that convert milk to cheese and yoghurt. To ensure consistency, large-scale, industrial fermentations use starter cultures of LAB, meaning a known strain or a sometimes-undefined combination of multiple strains with well-defined characteristics. Hypothetically, through natural genetic modification, which exploits DNA transfer systems occurring in nature, desirable traits could be introduced to strains to improve the efficacy of the starter culture. The focus of this thesis is natural competence. Competence refers to the specialised physiological state in which bacteria are able to undergo transformation through the internalisation of exogenous DNA. Recently an early com system was described for the industrially relevant LAB, Streptococcus thermophilus and a late com system homologous to that of S. pneumoniae was described in another LAB widely employed in cheese production, Lactococcus lactis. This thesis aimed to explore the functionality of the proven and predicted natural com systems encoded these LAB. In the case of S. thermophilus, this was achieved by defining the nutritional requirements of competence development, which involved defining a minimal cultivation medium. This medium was used in competence assays involving a model strain, S. thermophilus LMD-9, to confirm the functionality of the competence system in the minimal medium. The optimised medium and assay were then applied to a panel of industrial strains to determine the transferability of the transformation protocol. While it was observed that the minimal medium could support the growth of the industrially relevant strains, no transformation was detected in any industrial strains. A different approach was taken to study competence in L. lactis, as the system is far less defined compared to that of S. thermophilus. For this reason, an investigation of the gene activity of the lactococcal competence components was conducted, as opposed to competence assays. Specifically, the activity of the com-promoters was assessed via blue/white selection, using a promoter-probe vector constructed in this study. Several conditions were trialled to identify those optimal for competence development. This included the nisin-induction of the master competence regulator, ComX, which was the only condition that proved capable of activating the com-promoters. Chemical mutagenesis was employed in an attempt to generate random mutants that did not require comX-induction; however, this method did not prove effective, and no such mutants were isolated and the mechanism of lactococcal competence remains elusive.
DNA transfer , Lactic acid bacteria , Chemically defined medium , Blue/white selection , Beta-glucuronidase , Streptococcus thermophilus , Lactococcus lactis , Natural transformation , Competence
O'Connell, L. M. 2020. Natural transformation in lactic acid bacteria. MRes Thesis, University College Cork.