In vitro evolution of Listeria monocytogenes reveals selective pressure for loss of SigB and AgrA function at different incubation temperatures.

The alternative sigma factor B ({sigma}B) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the General Stress Response. We previously showed that {sigma}B loss-of-function mutations arise frequently in strains of L. monocytogenes, and suggested that mild stresses might favour the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42{degrees}C) or lower (30{degrees}C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42{degrees}C. However, at 30{degrees}C independent alleles in the agr locus arose, resulting in the inactivation of the Agr quorum sensing. Colonies of both sigB- and agr- strains exhibited a greyer colouration on 7-days-old agar plates compared with the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB- strains outcompeted the parental strain at 42{degrees}C, but not at 30{degrees}C, whilst agr- strains showed a small increase in competitive fitness at 30{degrees}C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of {sigma}B or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. ImportanceTo withstand environmental aggressions L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B ({sigma}B). However, {sigma}B becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to {sigma}B loss-of-function alleles. Temperatures of 42{degrees}C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of {sigma}B loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30{degrees}C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of {sigma}B in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.