Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA; Rutgers Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA; Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08540, USA.
Curr Biol. 2021 May 24;31(10):2039-2050.e7. doi: 10.1016/j.cub.2021.02.040. Epub 2021 Mar 11.
Genetic mutants have demonstrated the importance of homologous recombination (HR) to fluoroquinolone (FQ) persistence, which suggests that single-cell chromosome (Chr) abundance might be a phenotypic variable of importance to persisters. Here, we sorted stationary-phase E. coli based on ploidy and subjected the subpopulations to tolerance assays. Subpopulations sorted to contain diploid cells harbored up to ∼40-fold more FQ persisters than those sorted to contain monoploid cells. This association was observed with distinct FQs, in independent environmental conditions, and with more than one strain of E. coli (MG1655; uropathogenic CFT073) but was abolished in HR-deficient strains (ΔrecA and ΔrecB). It was observed that the persister level of monoploid subpopulations exceeded those of ΔrecA and ΔrecB by 10-fold or more, and subsequent high-purity sorting confirmed that observation. Those data suggested the existence of distinct FQ persister subtypes: those that are and are not proficient with HR. Time-lapse microscopy revealed significant differences in initial size and growth dynamics during the post-antibiotic recovery period for persisters from monoploid- and diploid-enriched subpopulations. In addition, non-persisters in monoploid-enriched subpopulations elongated minimally following FQ treatment, resembling previous observations of HR-deficient strains, whereas non-persisters in diploid-enriched subpopulations on average filamented extensively. Together, these results identify a phenotypic variable with a significant impact on FQ persistence, establish the existence of more than one type of persister to the same antibiotic in an isogenic culture, and reveal roles for RecA and RecB in FQ persistence, even in the absence of homologous chromosomes.
遗传突变体已经证明了同源重组（HR）对氟喹诺酮（FQ）持续存在的重要性，这表明单细胞染色体（Chr）丰度可能是与持续存在相关的重要表型变量。在这里，我们根据倍性对静止期大肠杆菌进行了分选，并对亚群进行了耐量测定。与分选以包含单倍体细胞的亚群相比，分选以包含二倍体细胞的亚群中含有多达约 40 倍的 FQ 持续存在体。这种关联在不同的 FQ 中观察到，在独立的环境条件下，在不止一个大肠杆菌菌株（MG1655；尿路致病性 CFT073）中观察到，但在 HR 缺陷菌株（ΔrecA 和 ΔrecB）中被消除。观察到单倍体亚群的持续存在体水平比 ΔrecA 和 ΔrecB 高出 10 倍或更多，随后的高纯度分选证实了这一观察结果。这些数据表明存在不同的 FQ 持续存在体亚型：那些具有 HR 能力的和那些没有 HR 能力的。延时显微镜观察到在抗生素后恢复期间，来自单倍体和二倍体富集亚群的持续存在体在初始大小和生长动力学方面存在显著差异。此外，在单倍体富集亚群中的非持续存在体在 FQ 处理后最小程度地伸长，类似于 HR 缺陷菌株的先前观察结果，而在二倍体富集亚群中的非持续存在体平均广泛地丝状化。这些结果共同确定了一个对 FQ 持续存在有重大影响的表型变量，在同一种抗生素的同基因培养物中建立了存在不止一种类型的持续存在体的存在，并揭示了 RecA 和 RecB 在 FQ 持续存在中的作用，即使在没有同源染色体的情况下也是如此。
