Indigenous and environmental modulation of mutation frequencies in Lactobacillus plantarum

The reliability of microbial (starter) strains in terms of quality, functional properties, growth performance and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing such as acid, osmotic, temperature, and oxidative stress. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution towards survival under these stress conditions. However, this may lead to the accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effect of specific fermentation or processing conditions on the mutation frequencies is an important step towards preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (NTG) and by controlled expression of endogenous candidate mutator genes (e.g. truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, in low pH conditions, at high salt concentrations or in starvation conditions did not result in a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide showed a 100-fold increase of the mutation frequency when compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42 degrees C, prior to the treatment with sublethal levels of hydrogen peroxide, this induced a 10-fold increase in peroxide treatment survival, with a concomitant 50-fold decrease of the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be achieved by pre-treatment of cultures with other, non-mutagenic stress conditions