Measuring the rate of conjugal plasmid transfer in a bacterial population using quantitative PCR.
Measuring the rate of conjugal plasmid transfer in a bacterial population using quantitative PCR.
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DOI：
10.1016/j.bpj.2011.04.054
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发表时间：
2011-07
影响因子：
3.4
通讯作者：
Zhenmao Wan;J. Varshavsky;Sushma Teegala;Jamille McLawrence;N. Goddard
中科院分区：
文献类型：
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作者：
Zhenmao Wan;J. Varshavsky;Sushma Teegala;Jamille McLawrence;N. Goddard
物种间基因水平转移是细菌基因组进化的重要机制。在大肠杆菌中，偶联是通过细胞间接触从供体（F+）细胞转移到受体（F−）细胞。我们证明了我们认为是一种新的qPCR方法，通过枚举质粒和染色体特有的遗传位点的相对丰度来定量F质粒在群体中的转移动力学。这种方法使我们能够查询质粒转移率，而无需选择性培养，具有前所未有的单位点分辨率。我们将结果拟合到质量作用模型中，其中质粒生长速率包括新形成的F+转偶联体的滞后时间和F+供体连续偶联事件之间的恢复时间。通过测定在恒定接种密度下基因典型相同的供体和受体细胞的确定混合物，我们提取了F质粒转移率为5 × 10−10（细胞/mL·min）−1。我们确认在整个批生长过程中，质粒/染色体比例为1:1。令人惊讶的是，在一些混合实验中，我们观察到在早期饱和阶段过量的F质粒平衡到每条染色体一个质粒的最终比例。
Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. InEscherichia coli, conjugation is the transfer from a donor (F+) to a recipient (F−) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F+transconjugants and the recovery time between successive conjugation events of the F+donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10−10(cells/mL · min)−1. We confirm a plasmid/chromosome ratio of 1:1 in homogenous F+populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.