Starvation and stress response in lactic acid bacteria

This thesis reports a study of aspects of the response of lactic acid bacteria to various stresses. Long term starvation (2 years) of L. lactis LL41-1 resulted in the appearance of variants with altered genotype, phenotype and colony morphology. Changes in chromosomal DNA were demonstrated using PFGE. Progressive plasmid loss and variation in protein production was observed. Three distinct colony morphology variants were evident. Eventually one slow growing variant predominated. All of the 4 original plasmids were lost but a 12 kb novel plasmid was present in all of these slow growing variants. The 12 kb plasmid was sequenced and shown to encode either complete or truncated genes for ori. repB, orfX, hsdR, hsdM, IS elements. mobA, mob and mobe. Expression of repB and orjX, hsdR and hsdM and mobA and mobB appear to be translationally coupled. The hsdM gene and IS elements appears to be truncated. The exact contribution that the genes encoded on this plasmid may have had to starvation survival is unclear at this time, however they are involved in DNA maintenance, transfer, modification and expression.A similar starvation study was conducted on L. lactis M176 in which plasmid presence and plasmid encoded phenotypes were monitored for 365 days. Plasmid loss was frequently associated with phenotype loss as would be expected. However, some phenotypes were retained but the relevant plasmid was not visible. In addition, in some cases the phenotype was lost but the relevant plasmid was still visible.Exposure of L. lactis LL41-1 to severe stresses for short periods resulted in a survivor population that were able to grow normally in M17GSstd medium, after differing lag phase periods. These regrown survivor cells had increased resistance when exposed to the same stress and often showed cross-resistance when exposed to a different stress.An attempt was made to PCR amplify rpoS (primary stress regulator) from one strain each of Laclocaccus, Lactobacillus and Btfidobacleria. Only one product was obtained and that was from the Bifidobacterial culture. When sequenced it was in fact rpoD. If rpoS does exist in the tested cultures then the primers used did not allow its amplification