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

• Published in: Applied and environmental microbiology Vol. 88; no. 11; p. e0033022
• Main Authors: Guerreiro, Duarte N, Wu, Jialun, McDermott, Emma, Garmyn, Dominique, Dockery, Peter, Boyd, Aoife, Piveteau, Pascal, O'Byrne, Conor P
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 14.06.2022
• Subjects: Alleles; Bacterial Proteins - metabolism; Codons; Colonies; Coloration; Evolution; Fitness; Foodborne pathogens; Gene Expression Regulation, Bacterial; Gene sequencing; Genetics and Molecular Biology; Genomes; Inactivation; Incubation; Life Sciences; Listeria; Listeria monocytogenes; Loci; Mutants; Mutation; Quorum sensing; Regulon; Reproductive fitness; Scanning electron microscopy; Sigma factor; Sigma Factor - genetics; Sigma Factor - metabolism; Spotlight Selection; Temperature; general stress response; in vitro evolution; quorum sensing; agr system; bacterial competition; Listeria monocytogenes; σB; sigma(B)
• ISSN: 0099-2240; 1098-5336; 1098-5336
• DOI: 10.1128/aem.00330-22

The alternative sigma factor B (σ ) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σ 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 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 operon mutations at 42°C. However, at 30°C, independent alleles in the locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both mutants and 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. mutant strains outcompeted the parental strain at 42°C but not at 30°C, while 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 and loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying alleles. Our results suggest that the loss of σ 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. To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σ ). However, σ becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σ 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 loss-of-function alleles in the 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 in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.