<i>In Vitro</i> Evolution of Listeria monocytogenes Reveals Selective Pressure for Loss of SigB and AgrA Function at Different Incubation Temperatures

Abstract

The alternative sigma factor B (σ^B) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σ^B loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed <i>in vitro</i> evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of <i>sigB</i> operon mutations at 42°C. However, at 30°C, independent alleles in the <i>agr</i> locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both <i>sigB</i> mutants and <i>agr</i> mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. <i>sigB</i> mutant strains outcompeted the parental strain at 42°C but not at 30°C, while <i>agr</i> mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the <i>sigB</i> and <i>agrCA</i> loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying <i>agrCA</i> alleles. Our results suggest that the loss of σ^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. <b>IMPORTANCE</b> To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σ^B). However, σ^B becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σ^B loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σ^B loss-of-function alleles in the <i>sigB</i> operon. In contrast, lower temperatures of 30°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 σ^B in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.