Data from: Effective polyploidy causes phenotypic delay and influences bacterial evolvability

Whether mutations in bacteria exhibit a noticeable delay before expressing their corresponding mutant phenotype was discussed intensively in the 1940s to 1950s, but the discussion eventually waned for lack of supportive evidence and perceived incompatibility with observed mutant distributions in fluctuation tests. Phenotypic delay in bacteria is widely assumed to be negligible, despite the lack of direct evidence. Here, we revisited the question using recombineering to introduce antibiotic resistance mutations into E. coli at defined time points and then tracking expression of the corresponding mutant phenotype over time. Contrary to previous assumptions, we found a substantial median phenotypic delay of three to four generations. We provided evidence that the primary source of this delay is multifork replication causing cells to be effectively polyploid, whereby wild-type gene copies transiently mask the phenotype of recessive mutant gene copies in the same cell. Using modeling and simulation methods, we explored the consequences of effective polyploidy for mutation rate estimation by fluctuation tests and sequencing-based methods. For recessive mutations, despite the substantial phenotypic delay, the per-copy or per-genome mutation rate is accurately estimated. However, the per-cell rate cannot be estimated by existing methods. Finally, with a mathematical model, we showed that effective polyploidy increases the frequency of costly recessive mutations in the standing genetic variation (SGV), and thus their potential contribution to evolutionary adaptation, while drastically reducing the chance that de novo recessive mutations can rescue populations facing a harsh environmental change such as antibiotic treatment. Overall, we have identified phenotypic delay and effective polyploidy as previously overlooked but essential components in bacterial evolvability, including antibiotic resistance evolution.

20世纪40至50年代，学界曾围绕细菌突变体在表达对应突变表型前是否存在显著延迟展开过激烈讨论，但由于缺乏支持性证据，且该假说与波动试验中观测到的突变体分布存在兼容性争议，相关讨论最终偃旗息鼓。尽管缺乏直接实验证据，学界目前普遍认为细菌的表型延迟可忽略不计。本研究通过重组工程（recombineering）技术，在特定时间点将抗生素抗性突变引入大肠杆菌（E. coli）基因组，随后实时追踪对应突变表型的表达过程，重新探讨了这一科学问题。与此前的普遍假设相悖，本研究观测到了长达3~4个世代的显著中位表型延迟。本研究证实，该延迟的主要来源为多叉复制（multifork replication）导致的细胞有效多倍体状态：同一细胞内的野生型基因拷贝会暂时性掩盖隐性突变基因拷贝的表型。本研究通过建模与仿真方法，分析了有效多倍体状态对波动试验及测序法估算突变率的影响。对于隐性突变而言，即便存在显著表型延迟，基于单拷贝或全基因组的突变率仍可被精准估算，但现有方法无法准确估算单细胞突变率。最后，本研究通过数学模型证实：有效多倍体状态会提升固有遗传变异库（standing genetic variation, SGV）中有害隐性突变的频率，进而增强其对进化适应的潜在贡献；但与此同时，该状态会大幅降低新发隐性突变拯救遭遇抗生素治疗等严苛环境变化的种群的概率。综上，本研究明确了表型延迟与有效多倍体状态是此前被忽视但对细菌演化能力（包括抗生素抗性演化）至关重要的关键因素。