Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes

In Listeria monocytogenes, the full details of how stress signals are integrated into the sigma(B) regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (sigma(B)). These mutants were tested for acid tolerance, a trait that is known to be under sigma(B) regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking sigma(B) (Delta sigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for sigma(B) activation, independently of the transposon location. Reduced sigma(B) activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixedculture experiments at 42 degrees C demonstrated that the loss of sigma(B) activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting sigma(B) activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype. IMPORTANCE In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced sigma(B) activity and were found to have a growth advantage under conditions of mild heat stress (42 degrees C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating sigma(B) activity. Together, these findings suggest that the occurrence of mutations that attenuate sigma(B) activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.