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Populations with large mutation supplies adapt via the "greedy" substitution of the fittest genotype available, leading to fast and repeatable short-term responses. At longer time scales, smaller mutation supplies may in theory lead to larger improvements when distant high-fitness genotypes more readily evolve from lower-fitness intermediates. Here we test for long-term adaptive benefits from small mutation supplies using in vitro evolution of an antibiotic-degrading enzyme in the presence of a novel antibiotic. Consistent with predictions, large mutant libraries cause rapid initial adaptation via the substitution of cohorts of mutations, but show later deceleration and convergence. Smaller libraries show on average smaller initial, but also more variable, improvements, with two lines yielding alleles with exceptionally high resistance levels. These two alleles share three mutations with the large-library alleles, which are known from previous work, but also have unique mutations. Replay evolution experiments and analyses of the adaptive landscape of the enzyme suggest that the benefit resulted from a combination of avoiding mutational cohorts leading to local peaks and chance. Our results demonstrate adaptive benefits from limited mutation supplies on a rugged fitness landscape, which has implications for artificial selection protocols in biotechnology and argues for a better understanding of mutation supplies in clinical settings.

Microbial population heterogeneity allows for a differential microbial response to environmental stresses and can lead to the selection of stress resistant variants. In this study, we have used two different stress resistant variants of Listeria monocytogenes LO28 with mutations in the rpsU gene encoding ribosomal protein S21, to elucidate features that can contribute to fitness, stress-tolerance and host interaction using a comparative gene profiling and phenotyping approach. Transcriptome analysis showed that 116 genes were upregulated and 114 genes were downregulated in both rpsU variants. Upregulated genes included a major contribution of SigB-controlled genes such as intracellular acid resistance-associated glutamate decarboxylase (GAD) (gad3), genes involved in compatible solute uptake (opuC), glycerol metabolism (glpF, glpK, glpD), and virulence (inlA, inlB). Downregulated genes in the two variants involved mainly genes involved in flagella synthesis and motility. Phenotyping results of the two rpsU variants matched the gene profiling data including enhanced freezing resistance conceivably linked to compatible solute accumulation, higher glycerol utilisation rates, and better adhesion to Caco 2 cells presumably linked to higher expression of internalins. Also, bright field and electron microscopy analysis confirmed reduced flagellation of the variants. The activation of SigB-mediated stress defence offers an explanation for the multiple-stress resistant phenotype in rpsU variants.

This review elucidates the state-of-the-art knowledge about pathogen population heterogeneity and describes the genotypic and phenotypic analyses of persister subpopulations and stress-resistant variants. The molecular mechanisms underlying the generation of persister phenotypes and genetic variants are identified. Zooming in on Listeria monocytogenes, a comparative whole-genome sequence analysis of wild types and variants that enabled the identification of mutations in variants obtained after a single exposure to lethal food-relevant stresses is described. Genotypic and phenotypic features are compared to those for persistent strains isolated from food processing environments. Inactivation kinetics, models used for fitting, and the concept of kinetic modeling-based schemes for detection of variants are presented. Furthermore, robustness and fitness parameters of L. monocytogenes wild type and variants are used to model their performance in food chains. Finally, the impact of stress-resistant variants and persistence in food processing environments on food safety is discussed.

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