Department of Biological Sciences, University of Idaho, Moscow, Idaho, USA.
mBio. 2012 Jul 3;3(4):e00077-12. doi: 10.1128/mBio.00077-12. Print 2012.
Promiscuous plasmids replicate in a wide range of bacteria and therefore play a key role in the dissemination of various host-beneficial traits, including antibiotic resistance. Despite the medical relevance, little is known about the evolutionary dynamics through which drug resistance plasmids adapt to new hosts and thereby persist in the absence of antibiotics. We previously showed that the incompatibility group P-1 (IncP-1) minireplicon pMS0506 drastically improved its stability in novel host Shewanella oneidensis MR-1 after 1,000 generations under antibiotic selection for the plasmid. The only mutations found were those affecting the N terminus of the plasmid replication initiation protein TrfA1. Our aim in this study was to gain insight into the dynamics of plasmid evolution. Changes in stability and genotype frequencies of pMS0506 were monitored in evolving populations of MR-1 (pMS0506). Genotypes were determined by sequencing trfA1 amplicons from individual clones and by 454 pyrosequencing of whole plasmids from entire populations. Stability of pMS0506 drastically improved by generation 200. Many evolved plasmid genotypes with point mutations as well as in-frame and frameshift deletions and duplications in trfA1 were observed in all lineages with both sequencing methods. Strikingly, multiple genotypes were simultaneously present at high frequencies (>10%) in each population. Their relative abundances changed over time, but after 1,000 generations only one or two genotypes dominated the populations. This suggests that hosts with different plasmid genotypes were competing with each other, thus affecting the evolutionary trajectory. Plasmids can thus rapidly improve their stability, and clonal interference plays a significant role in plasmid-host adaptation dynamics.
Promiscuous plasmids play an important role in the spread of antibiotic resistance and many other traits between closely and distantly related bacteria. However, little is known about the dynamics by which these broad-host-range antibiotic resistance plasmids adapt to novel bacteria and thereby become more persistent, even in the absence of antibiotics. In this study, we show that after no more than 200 generations of growth in the presence of antibiotics, a plasmid that was initially poorly maintained in a novel bacterial host evolved to become drastically more persistent in the absence of antibiotics. In each of the evolving populations, an unexpectedly large number of bacterial variants arose with distinct mutations in the plasmid's replication initiation protein. Our results suggest that clonal interference, characterized by competition between variant clones in a population, plays a major role in the evolution of the persistence of drug resistance.
易变质粒在广泛的细菌中复制，因此在传播各种宿主有益特性方面发挥着关键作用，包括抗生素抗性。尽管具有医学相关性，但对于药物抗性质粒如何适应新宿主并因此在没有抗生素的情况下持续存在的进化动态，我们知之甚少。我们之前曾表明，不相容群 P-1（IncP-1）小型复制子 pMS0506 在抗生素选择下经过 1000 代后，在新宿主希瓦氏菌属（Shewanella oneidensis MR-1）中，其稳定性大大提高。唯一发现的突变是那些影响质粒复制起始蛋白 TrfA1 N 端的突变。我们在这项研究中的目标是深入了解质粒进化的动态。在 MR-1（pMS0506）的进化群体中，监测了 pMS0506 的稳定性和基因型频率的变化。通过对来自单个克隆的 trfA1 扩增子进行测序以及对整个群体的全质粒进行 454 焦磷酸测序来确定基因型。pMS0506 的稳定性在 200 代时大大提高。两种测序方法均观察到 pMS0506 的许多进化基因型具有点突变以及 trfA1 中的框内和移框缺失和重复。引人注目的是，在每个群体中，多种基因型同时以高频率（>10%）存在。它们的相对丰度随时间变化，但经过 1000 代后，只有一种或两种基因型占主导地位。这表明具有不同质粒基因型的宿主相互竞争，从而影响进化轨迹。因此，质粒可以迅速提高其稳定性，克隆干扰在质粒-宿主适应动态中起着重要作用。
易变质粒在密切和远距离相关细菌之间传播抗生素抗性和许多其他特性方面起着重要作用。然而，对于这些广谱抗生素抗性质粒如何适应新细菌并因此变得更具持久性，甚至在没有抗生素的情况下，人们知之甚少。在这项研究中，我们表明，在抗生素存在的情况下生长不超过 200 代后，最初在新型细菌宿主中保持不佳的质粒在没有抗生素的情况下进化为更持久。在每个进化群体中，都出现了大量具有不同突变的细菌变体，这些突变发生在质粒的复制起始蛋白中。我们的研究结果表明，克隆干扰，其特征是群体中变异克隆之间的竞争，在耐药性持久性的进化中起着主要作用。
