Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria

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.应激诱导突变（stress-induced mutagenesis）假说提出，细菌在受到应激刺激时会提升其全基因组突变率，进而提高子代更好耐受该应激的概率。这一假说对抗生素治疗具有启示意义：已有研究表明，暴露于亚抑制剂量抗生素会提升细菌的突变率，进而可能加快耐药突变的出现速率，最终导致治疗失败。更广泛而言，该假说认为应激可提升进化能力（evolvability，即种群产生适应性遗传多样性的能力），从而加速进化进程。然而，在应激条件下测定突变率存在难题，因为现有方法均假设不存在细胞死亡。但亚抑制应激水平下可能引发显著的细胞死亡事件。为达到既定种群规模，死亡事件需要通过额外的细胞分裂来弥补，这也就为突变的产生提供了更多机会。本研究表明，忽略细胞死亡会导致应激条件下突变率的系统性高估。我们开发了一套基于质粒分离（plasmid segregation）的实验系统，可同时测定细菌种群的死亡速率与分裂速率。利用该系统，我们发现，在此前被报道可提升突变率的亚抑制抗生素浓度下，确实存在显著的细胞死亡速率。将该死亡速率纳入考量后，应激诱导突变的信号会被削弱，有时甚至会完全消失。此外，即便抗生素提升了细菌的突变率，我们的研究也表明，亚抑制剂量的抗生素处理并不会提升遗传多样性与进化能力，这同样是因为抗生素对种群动态的影响。我们由此得出结论：由于细胞死亡的影响，抗生素诱导的突变率被高估了；而要理解应激条件下的进化能力，既需要考量应激对突变率的影响，也需要考量其对种群动态的影响。本研究的目标具有双重性：其一，我们证明了种群动态，尤其是细胞分裂次数，是影响种群进化能力的关键却被忽视的参数；其二，我们提供了可用于研究应激条件下进化能力的实验与计算工具及方法，从而实现对“应激诱导突变范式”的影响程度与重要性的重新评估。