Farr Andrew D, Vasileiou Christina, Lind Peter A, Rainey Paul B
Department of Microbial Population Biology, Max Planck Institute for Evolutionary Biology, Plön, Germany.
New Zealand Institute for Advanced Study, Massey University, Auckland, New Zealand.
PLoS Genet. 2025 Jan 31;21(1):e1011572. doi: 10.1371/journal.pgen.1011572. eCollection 2025 Jan.
Mutation rate varies within and between genomes. Within genomes, tracts of nucleotides, including short sequence repeats and palindromes, can cause localised elevation of mutation rate. Additional mechanisms remain poorly understood. Here we report an instance of extreme mutational bias in Pseudomonas fluorescens SBW25 associated with a single base-pair change in nlpD. These mutants frequently evolve in static microcosms, and have a cell-chaining (CC) phenotype. Analysis of 153 replicate populations revealed 137 independent instances of a C565T loss-of-function mutation at codon 189 (CAG to TAG (Q189*)). Fitness measures of alternative nlpD mutants did not explain the deterministic evolution of C565T mutants. Recognising that transcription can be mutagenic, and that codon 189 overlaps with a predicted promoter (rpoSp) for the adjacent stationary phase sigma factor, rpoS, transcription across this promoter region was measured. This confirmed rpoSp is induced in stationary phase and that C565T mutation caused significant elevation of transcription. The latter provided opportunity to determine the C565T mutation rate using a reporter-gene fused to rpoSp. Fluctuation assays estimate the C565T mutation rate to be ~5,000-fold higher than expected. In Pseudomonas, transcription of rpoS requires the positive activator PsrA, which we show also holds for SBW25. Fluctuation assays performed in a ∆psrA background showed a ~60-fold reduction in mutation rate confirming that the elevated rate of mutation at C565T mutation rate is dependent on induction of transcription. This hotspot suggests a generalisable phenomenon where the induction of transcription causes elevated mutation rates within defining regions of promoters.
突变率在基因组内部和基因组之间存在差异。在基因组内部，核苷酸序列，包括短序列重复和回文序列，可导致突变率局部升高。其他机制仍知之甚少。在这里，我们报告了荧光假单胞菌SBW25中一个极端突变偏向的实例，该实例与nlpD中的一个单碱基对变化有关。这些突变体在静态微观世界中频繁进化，并具有细胞连锁（CC）表型。对153个重复群体的分析揭示了密码子189（CAG突变为TAG（Q189*））处C565T功能丧失突变的137个独立实例。其他nlpD突变体的适应性测量并不能解释C565T突变体的确定性进化。认识到转录可能具有诱变作用，并且密码子189与相邻的静止期sigma因子rpoS的预测启动子（rpoSp）重叠，于是对该启动子区域的转录进行了测量。这证实了rpoSp在静止期被诱导，并且C565T突变导致转录显著升高。后者提供了使用与rpoSp融合的报告基因来确定C565T突变率的机会。波动试验估计C565T突变率比预期高约5000倍。在假单胞菌中，rpoS的转录需要正激活因子PsrA，我们发现SBW25也是如此。在∆psrA背景下进行的波动试验显示突变率降低了约60倍，证实了C565T突变率的升高取决于转录的诱导。这个热点表明了一种普遍现象，即转录的诱导会导致启动子定义区域内的突变率升高。
