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Mutagenesis is responsive to many environmental factors. Evolution therefore depends on the environment not only for selection but also in determining the variation available in a population. One such environmental dependency is the inverse relationship between mutation rates and population density in many microbial species. Here, we determine the mechanism responsible for this mutation rate plasticity. Using dynamical computational modelling and in culture mutation rate estimation, we show that the negative relationship between mutation rate and population density arises from the collective ability of microbial populations to control concentrations of hydrogen peroxide. We demonstrate a loss of this density-associated mutation rate plasticity (DAMP) when Escherichia coli populations are deficient in the degradation of hydrogen peroxide. We further show that the reduction in mutation rate in denser populations is restored in peroxide degradation-deficient cells by the presence of wild-type cells in a mixed population. Together, these model-guided experiments provide a mechanistic explanation for DAMP, applicable across all domains of life, and frames mutation rate as a dynamic trait shaped by microbial community composition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Green et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterized the spectrum of spontaneous mutations in Escherichia coli grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a Δ luxS deletant strain - a gene with roles in both quorum sensing and the recycling of methylation products used in E. coli 's DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive Δ luxS deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.

Fluctuation assays are widely used for estimating mutation rates in microbes growing in liquid environments. Many cultures are each inoculated with a few thousand cells, each sensitive to a selective marker that can be assayed phenotypically. These parallel cultures grow for many generations in the absence of the phenotypic marker. A subset of cultures is used to estimate the total number of cells at risk of mutations (i.e., the population size at the end of the growth period, or Nt). The remaining cultures are plated onto the selective agar. The distribution of observed resistant mutants among parallel cultures is then used to estimate the expected number of mutational events, m, using a mathematical model. Dividing m by Nt gives the estimate of the mutation rate per locus per generation. The assay has three critical aspects: the chosen phenotypic marker, the chosen volume of parallel cultures, and ensuring that the surface on the selective agar is completely dry before the incubation. The assay is relatively inexpensive and only needs standard laboratory equipment. It is also less laborious than alternative approaches, such as mutation accumulation and single-cell assays. The assay works on organisms that go through many generations rapidly and it depends on assumptions about the fitness effects of markers and cell death. However, recently developed tools and theoretical studies mean these issues can now be addressed analytically. The assay allows mutation rate estimation of different phenotypic markers in cells with different genotypes growing in isolation or in a community. By conducting multiple assays in parallel, assays can be used to study how an organism's environmental context affects spontaneous mutation rate, which is crucial for understanding antimicrobial resistance, carcinogenesis, aging, and evolution.

Evolution depends on mutations. For an individual genotype, the rate at which mutations arise is known to increase with various stressors (stress-induced mutagenesis-SIM) and decrease at high final population density (density-associated mutation-rate plasticity-DAMP). We hypothesised that these two forms of mutation-rate plasticity would have opposing effects across a nutrient gradient. Here we test this hypothesis, culturing Escherichia coli in increasingly rich media. We distinguish an increase in mutation rate with added nutrients through SIM (dependent on error-prone polymerases Pol IV and Pol V) and an opposing effect of DAMP (dependent on MutT, which removes oxidised G nucleotides). The combination of DAMP and SIM results in a mutation rate minimum at intermediate nutrient levels (which can support 7 × 10 8  cells ml -1 ). These findings demonstrate a strikingly close and nuanced relationship of ecological factors-stress and population density-with mutation, the fuel of all evolution.

Evolutionary rescue following environmental change requires mutations permitting population growth in the new environment. If change is severe enough to prevent most of the population reproducing, rescue becomes reliant on mutations already present. If change is sustained, the fitness effects in both environments, and how they are associated-termed 'environmental pleiotropy'-may determine which alleles are ultimately favoured. A population's demographic history-its size over time-influences the variation present. Although demographic history is known to affect the probability of evolutionary rescue, how it interacts with environmental pleiotropy during severe and sustained environmental change remains unexplored. Here, we demonstrate how these factors interact during antibiotic resistance evolution, a key example of evolutionary rescue fuelled by pre-existing mutations with pleiotropic fitness effects. We combine published data with novel simulations to characterise environmental pleiotropy and its effects on resistance evolution under different demographic histories. Comparisons among resistance alleles typically revealed no correlation for fitness-i.e., neutral pleiotropy-above and below the sensitive strain's minimum inhibitory concentration. Resistance allele frequency following experimental evolution showed opposing correlations with their fitness effects in the presence and absence of antibiotic. Simulations demonstrated that effects of environmental pleiotropy on allele frequencies depended on demographic history. At the population level, the major influence of environmental pleiotropy was on mean fitness, rather than the probability of evolutionary rescue or diversity. Our work suggests that determining both environmental pleiotropy and demographic history is critical for predicting resistance evolution, and we discuss the practicalities of this during in vivo evolution.

Variation of mutation rate at a particular site in a particular genotype, in other words mutation rate plasticity (MRP), can be caused by stress or ageing. However, mutation rate control by other factors is less well characterized. Here we show that in wild-type Escherichia coli (K-12 and B strains), the mutation rate to rifampicin resistance is plastic and inversely related to population density: lowering density can increase mutation rates at least threefold. This MRP is genetically switchable, dependent on the quorum-sensing gene luxS--specifically its role in the activated methyl cycle--and is socially mediated via cell-cell interactions. Although we identify an inverse association of mutation rate with fitness under some circumstances, we find no functional link with stress-induced mutagenesis. Our experimental manipulation of mutation rates via the social environment raises the possibility that such manipulation occurs in nature and could be exploited medically.

Understanding the connections among genotype, phenotype, and fitness through evolutionary time is a central goal of evolutionary genetics. Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations and show substantial morphological and fitness differences. Previous work identified genes contributing to the evolutionary success of WS, in particular the di-guanylate cyclase response regulator, WspR. Here we scrutinize the Wsp signal transduction pathway of which WspR is the primary output component. The pathway has the hallmarks of a chemosensory pathway and genetic analyses show that regulation and function of Wsp is analogous to the Che chemotaxis pathway from Escherichia coli. Of significance is the methyltransferase (WspC) and methylesterase (WspF) whose opposing activities form an integral feedback loop that controls the activity of the kinase (WspE). Deductions based on the regulatory model suggested that mutations within wspF were a likely cause of WS. Analyses of independent WS genotypes revealed numerous simple mutations in this single open reading frame. Remarkably, different mutations have different phenotypic and fitness effects. We suggest that the negative feedback loop inherent in Wsp regulation allows the pathway to be tuned by mutation in a rheostat-like manner. [ABSTRACT FROM AUTHOR]