Data from: Linking system-wide impacts of RNA polymerase mutations to the fitness cost of rifampin resistance in Pseudomonas aeruginosa

Fitness costs play a key role in the evolutionary dynamics of antibiotic resistance in bacteria by generating selection against resistance in the absence of antibiotics. Although the genetic basis of antibiotic resistance is well understood, the precise molecular mechanisms linking the genetic basis of resistance to its fitness cost remain poorly characterized. Here, we examine how the system-wide impacts of mutations in the RNA polymerase (RNAP) gene rpoB shape the fitness cost of rifampin resistance in Pseudomonas aeruginosa. Rifampin resistance mutations reduce transcriptional efficiency, and this explains 76% of the variation in fitness among rpoB mutants. The pleiotropic consequence of rpoB mutations is that mutants show altered relative transcript levels of essential genes. We find no evidence that global transcriptional responses have an impact on the fitness cost of rifampin resistance as revealed by transcriptome sequencing (RNA-Seq). Global changes in the transcriptional profiles of rpoB mutants compared to the transcriptional profile of the rifampin-sensitive ancestral strain are subtle, demonstrating that the transcriptional regulatory network of P. aeruginosa is robust to the decreased transcriptional efficiency associated with rpoB mutations. On a smaller scale, we find that rifampin resistance mutations increase the expression of RNAP due to decreased termination at an attenuator upstream from rpoB, and we argue that this helps to minimize the cost of rifampin resistance by buffering against reduced RNAP activity. In summary, our study shows that it is possible to dissect the molecular mechanisms underpinning variation in the cost of rifampin resistance and highlights the importance of genome-wide buffering of relative transcript levels in providing robustness against resistance mutations.

IMPORTANCE Antibiotic resistance mutations carry fitness costs. Relative to the characteristics of their antibiotic-sensitive ancestors, resistant mutants show reduced growth rates and competitive abilities. Fitness cost plays an important role in the evolution of antibiotic resistance in the absence of antibiotics; however, the molecular mechanisms underlying these fitness costs is not well understood. We applied a systems-level approach to dissect the molecular underpinnings of the fitness costs associated with rifampin resistance in P. aeruginosa and showed that most of the variation in fitness cost can be explained by the direct effect of resistance mutations on the enzymatic activity of the mutated gene. Pleiotropic changes in transcriptional profiles are subtle at a genome-wide scale, suggesting that the gene regulatory network of P. aeruginosa is robust in the face of the direct effects of resistance mutations.

适应性成本在细菌抗生素抗性的进化动态中发挥关键作用，通过在无抗生素环境下产生对抗抗性的选择压力。尽管抗生素抗性的遗传基础已被充分阐明，但将抗性遗传基础与其适应性成本关联起来的精确分子机制仍未得到充分表征。
在此，我们探究了RNA聚合酶（RNA polymerase, RNAP）基因rpoB的突变所产生的全系统效应，如何塑造铜绿假单胞菌（Pseudomonas aeruginosa）利福平（rifampin）抗性的适应性成本。利福平抗性突变会降低转录效率，该效应可解释rpoB突变体中76%的适应性变异。rpoB突变具有多效性，会导致突变体的必需基因相对转录水平发生改变。我们通过转录组测序（transcriptome sequencing, RNA-Seq）未发现全局转录反应对利福平抗性适应性成本存在影响的证据。相较于利福平敏感的祖先菌株，rpoB突变体的转录组全局变化较为微弱，这表明铜绿假单胞菌的转录调控网络对rpoB突变引发的转录效率降低具有鲁棒性。在更精细的尺度下，我们发现利福平抗性突变通过降低rpoB上游衰减子的转录终止效率，提升了RNA聚合酶的表达量；我们认为，这一机制可通过缓冲RNA聚合酶活性的降低，帮助最小化利福平抗性的适应性成本。综上，本研究证实，我们可以解析驱动利福平抗性适应性成本变异的分子机制，并强调了全基因组相对转录水平缓冲在为抗性突变提供鲁棒性方面的重要性。

研究意义
抗生素抗性突变会带来适应性成本。相较于其抗生素敏感的祖先菌株，抗性突变体的生长速率与竞争能力均有所下降。适应性成本在无抗生素环境下的抗生素抗性进化中发挥关键作用；然而，这类适应性成本背后的分子机制仍未得到充分阐明。我们采用系统级研究方法，解析了铜绿假单胞菌利福平抗性相关适应性成本的分子基础，并证实绝大多数适应性成本变异可通过抗性突变对突变基因酶活性的直接效应得到解释。转录组的多效性变化在全基因组尺度上较为微弱，这表明铜绿假单胞菌的基因调控网络可抵御抗性突变带来的直接效应。