Table_6_Ribosomal mutations enable a switch between high fitness and high stress resistance in Listeria monocytogenes.XLSX

Multiple stress resistant variants of Listeria monocytogenes with mutations in rpsU encoding ribosomal protein RpsU have previously been isolated after a single exposure to acid stress. These variants, including L. monocytogenes LO28 variant V14 with a complete deletion of the rpsU gene, showed upregulation of the general stress sigma factor Sigma B-mediated stress resistance genes and had a lower maximum specific growth rate than the LO28 WT, signifying a trade-off between stress resistance and fitness. In the current work V14 has been subjected to an experimental evolution regime, selecting for higher fitness in two parallel evolving cultures. This resulted in two evolved variants with WT-like fitness: 14EV1 and 14EV2. Comparative analysis of growth performance, acid and heat stress resistance, in combination with proteomics and RNA-sequencing, indicated that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity, due to lack of Sigma B-activated stress defense. Notably, genotyping of 14EV1 and 14EV2 provided evidence for unique point-mutations in the ribosomal rpsB gene causing amino acid substitutions at the same position in RpsB, resulting in RpsB22Arg-His and RpsB22Arg-Ser, respectively. Combined with data obtained with constructed RpsB22Arg-His and RpsB22Arg-Ser mutants in the V14 background, we provide evidence that loss of function of RpsU resulting in the multiple stress resistant and reduced fitness phenotype, can be reversed by single point mutations in rpsB leading to arginine substitutions in RpsB at position 22 into histidine or serine, resulting in a WT-like high fitness and low stress resistance phenotype. This demonstrates the impact of genetic changes in L. monocytogenes’ ribosomes on fitness and stress resistance.

此前，经单次酸性胁迫暴露后，研究人员已分离得到携带rpsU基因突变的多重胁迫抗性单核细胞增生李斯特菌（Listeria monocytogenes）变异株，该rpsU基因编码核糖体蛋白RpsU（ribosomal protein RpsU）。此类变异株——包括rpsU基因完全缺失的单核细胞增生李斯特菌LO28变异株V14——均表现出一般胁迫西格玛因子Sigma B介导的胁迫抗性基因的上调，且其最大比生长速率低于LO28野生型（Wild Type, WT）菌株，这意味着胁迫抗性与生存适合度之间存在权衡关系。本研究中，研究人员对变异株V14实施了实验进化培养方案，在两个平行进化培养体系中筛选更高适合度的菌株，最终获得两株具备野生型菌株相似适合度的进化变异株：14EV1与14EV2。通过对生长性能、酸性与热胁迫抗性的比较分析，结合蛋白质组学与RNA测序（RNA-sequencing）数据，研究发现两株进化株的适合度恢复至野生型水平的同时，其胁迫敏感性也回归野生型状态，这归因于Sigma B激活的胁迫防御通路的缺失。值得注意的是，对14EV1与14EV2的基因分型结果显示，核糖体rpsB基因存在独特的点突变（point-mutation），该突变导致RpsB蛋白的同一位置发生氨基酸替换，分别对应RpsB22Arg-His与RpsB22Arg-Ser变异。结合在V14遗传背景下构建的RpsB22Arg-His与RpsB22Arg-Ser突变株的实验数据，本研究证实：原本因RpsU功能丧失而表现出多重胁迫抗性与适合度降低的表型，可通过rpsB基因的单点突变实现逆转——该突变使RpsB蛋白第22位的精氨酸替换为组氨酸或丝氨酸，最终使菌株恢复至野生型般的高适合度与低胁迫抗性表型。该研究结果证实了单核细胞增生李斯特菌核糖体的遗传改变对其适合度与胁迫抗性的影响。