Indigenous and environmental modulation of frequencies of mutation in Lactobacillus plantarum.

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 stresses. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution for survival under stress conditions. However, this may lead to accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effects of specific fermentation or processing conditions on the mutation frequency is an important step toward 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 (N-methyl-N′-nitro-N-nitrosoguanidine) and by controlled expression of endogenous candidate mutator genes (e.g., a truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, under low-pH conditions, at high salt concentrations, or under starvation conditions did not have a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide resulted in a 100-fold increase in the mutation frequency compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42°C prior to treatment with sublethal levels of hydrogen peroxide, there was a 10-fold increase in survival after peroxide treatment, and there was a concomitant 50-fold decrease in 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 obtained by pretreatment of cultures with other, nonmutagenic stress conditions.