In Listeria monocytogenes the full details of how stress signals are integrated into the σB 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 (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). 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 σB activation, independently of the transposon location. Reduced σB 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. Mixed culture experiments at 42°C demonstrated that loss of σB activity allowed mutants to take-over a population of parental bacteria. Together, our results suggest that mutations affecting σB 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 food-borne pathogen and emphasise the need for whole genome sequence analysis of newly constructed strains to confirm the expected genotype.ImportanceIn the present study we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures these mutants outcompeted the wildtype 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 σB activity. Together the findings suggest that the occurrence of mutations that attenuate σB activity can be favoured under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.