Abstract
The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the numbers of cell divisions are crucial but neglected parameters in the evolvability of a population, and we provide experimental and computational tools and methods to study evolvability under stress, leading to a reassessment of the magnitude and significance of the stress-induced mutagenesis paradigm.
Highlights
One of the most puzzling and controversial microbial evolution experiments of the 20th century may be the one performed by Cairns and colleagues [1,2] in which lac− cells are plated on lactose as the sole carbon source and cannot grow
An antimicrobial treatment is subinhibitory if the population grows
This discrepancy is due to both miscalculation of mutation rates and misconceptions about the link between mutation rate and evolvability in these classical papers
Summary
One of the most puzzling and controversial microbial evolution experiments of the 20th century may be the one performed by Cairns and colleagues [1,2] in which lac− cells are plated on lactose as the sole carbon source and cannot grow. Revertants toward the lac+ genotype continuously appear after plating at a rate and timing seemingly incompatible with the Darwinian hypothesis of selection of preexisting mutants. In the lac− construct, the lacZ coding sequence is present but nonfunctional, because it is out of frame with the start codon. The lac+ revertants are frameshift mutants in which this coding sequence is back in frame with the start codon. Many additional experiments quickly suggested that this phenomenon can be explained by more standard Darwinian mechanisms, in which genetic changes are not targeted but occur randomly and are selected or not. While two seemingly conflicting molecular explanations—the stressinduced mutagenesis model and the gene amplification model—emerged, both are conceptually very similar
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