Rates of random, spontaneous mutation can vary plastically, dependent upon the environment. Such plasticity affects evolutionary trajectories and may be adaptive. We recently identified an inverse plastic association between mutation rate and population density at 1 locus in 1 species of bacterium. It is unknown how widespread this association is, whether it varies among organisms, and what molecular mechanisms of mutagenesis or repair are required for this mutation-rate plasticity. Here, we address all 3 questions. We identify a strong negative association between mutation rate and population density across 70 years of published literature, comprising hundreds of mutation rates estimated using phenotypic markers of mutation (fluctuation tests) from all domains of life and viruses. We test this relationship experimentally, determining that there is indeed density-associated mutation-rate plasticity (DAMP) at multiple loci in both eukaryotes and bacteria, with up to 23-fold lower mutation rates at higher population densities. We find that the degree of plasticity varies, even among closely related organisms. Nonetheless, in each domain tested, DAMP requires proteins scavenging the mutagenic oxidised nucleotide 8-oxo-dGTP. This implies that phenotypic markers give a more precise view of mutation rate than previously believed: having accounted for other known factors affecting mutation rate, controlling for population density can reduce variation in mutation-rate estimates by 93%. Widespread DAMP, which we manipulate genetically in disparate organisms, also provides a novel trait to use in the fight against the evolution of antimicrobial resistance. Such a prevalent environmental association and conserved mechanism suggest that mutation has varied plastically with population density since the early origins of life.

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 × 108 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.

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 N t). 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 N t 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. Video Link The video component of this article can be found at https://www.jove.com/video/60406/. Twenty-six years later, when Hermann Joseph Muller discovered the mutagenic action of X-rays 2 , mutations were already perceived as one of the driving forces of evolution. However, the nature of mutations was not clear. To answer the fundamental question of whether mutations emerge spontaneously (i.e., a spontaneous mutation) or in response to selection (i.e., an induced mutation), a method was needed to observe mutational events. Such a method would measure the expected number of mutations per cell division or what was already known as a mutation rate 3,4 .

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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 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 bacteria in increasingly rich media. We distinguish an increase in mutation rate with added nutrients through SIM (dependent on errorprone 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 result in a mutation rate minimum at intermediate nutrient levels ( ). These findings demonstrate a strikingly close and nuanced relationship of ecological factors -stress and population density -with mutation, the fuel of all evolution.