Abstract
Microbial population heterogeneity contributes to differences in stress response between individual cells in a population, and can lead to the selection of genetically stable variants with increased stress resistance. We previously provided evidence that the multiple-stress resistant Listeria monocytogenes LO28 variant 15, carries a point mutation in the rpsU gene, resulting in an arginine-proline substitution in ribosomal protein RpsU (RpsU17Arg-Pro). Here, we investigated the trade-off between general stress sigma factor SigB-mediated stress resistance and fitness in variant 15 using experimental evolution. By selecting for higher fitness in two parallel evolving cultures, we identified two evolved variants: 15EV1 and 15EV2. Whole genome sequencing and SNP analysis showed that both parallel lines mutated in the same codon in rpsU as the original mutation resulting in RpsU17Pro-His (15EV1) and RpsU17Pro-Thr (15EV2). Using a combined phenotyping and proteomics approach, we assessed the resistance of the evolved variants to both heat and acid stress, and found that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity. Proteome analysis of L. monocytogenes LO28 WT, variant 15, 15EV1, and 15EV2 revealed high level expression of SigB regulon members only in variant 15, whereas protein profiles of both evolved variants were highly similar to that of the LO28 WT. Experiments with constructed RpsU17Arg-Pro mutants in L. monocytogenes LO28 and EGDe, and RpsU17Arg-His and RpsU17Arg-Thr in LO28, confirmed that single amino acid substitutions in RpsU enable switching between multiple-stress resistant and high fitness states in L. monocytogenes.
Highlights
Listeria monocytogenes is a foodborne pathogen that is ubiquitously present in the environment, and can cause the rare but severe disease listeriosis (Lecuit, 2007; Radoshevich and Cossart, 2018; VazquezBoland et al, 2001)
We addressed this issue and subjected L. monocytogenes LO28 variant 15, with its single point mutation in rpsU, resulting in RpsU17Arg-Pro, to an experimental evolution regime where we selected for higher fitness, defined as an increased maximum specific growth rate, when compared to the ancestor variant 15
For acid stress experiments (Fig. 2b), variant 15 again only showed a small (
Summary
Listeria monocytogenes is a foodborne pathogen that is ubiquitously present in the environment, and can cause the rare but severe disease listeriosis (Lecuit, 2007; Radoshevich and Cossart, 2018; VazquezBoland et al, 2001). Metselaar et al (2015) combined phenotypic clustering of a collection of stable stress resistant L. monocytogenes variants, based on reduced growth rate and increased resistance against acid, heat, high hydrostatic pressure (HHP), and benzalkonium chloride, with a whole genome sequencing and Structural Variation (SV) analysis. This analysis showed that 11 of the 23 selected variants with a shared phenotype had a mutation in the ribosomal rpsU gene locus encoding S30 ribosomal protein RpsU (small ribosomal protein 21) (Metselaar et al, 2015). An additional factor contributing to increased risk following the initial selection of multiple stress resistant variants (Abee et al, 2016) could be the subsequent se lection of other variants that originate from the ancestor variant and have increased fitness and loss of the stress resistant phenotype
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