Genome-wide detection of conservative site-specific recombination in bacteria

The ability of clonal bacterial populations to generate genomic and phenotypic heterogeneity is thought to be of great importance for many commensal and pathogenic bacteria. One common mechanism contributing to diversity formation relies on the inversion of small genomic DNA segments in a process commonly referred to as conservative site-specific recombination. This phenomenon is known to occur in several bacterial lineages, however it remains notoriously difficult to identify due to the lack of conserved features. Here, we report an easy-to-implement method based on high-throughput paired-end sequencing for genome-wide detection of conservative site-specific recombination on a single-nucleotide level. We demonstrate the effectiveness of the method by successfully detecting several novel inversion sites in an epidemic isolate of the enteric pathogen Clostridium difficile. Using an experimental approach, we validate the inversion potential of all detected sites in C. difficile and quantify their prevalence during exponential and stationary growth in vitro. In addition, we demonstrate that the master recombinase RecV is responsible for the inversion of some but not all invertible sites. Using a fluorescent gene-reporter system, we show that at least one gene from a two-component system located next to an invertible site is expressed in an on-off mode reminiscent of phase variation. We further demonstrate the applicability of our method by mining 209 publicly available sequencing datasets and show that conservative site-specific recombination is common in the bacterial realm but appears to be absent in some lineages. Finally, we show that the gene content associated with the inversion sites is diverse and goes beyond traditionally described surface components. Overall, our method provides a robust platform for detection of conservative site-specific recombination in bacteria and opens a new avenue for global exploration of this important phenomenon.

克隆细菌种群产生基因组与表型异质性的能力，被认为对众多共生与致病细菌而言至关重要。促成多样性形成的一类常见机制，依赖于小型基因组DNA片段的倒位，该过程通常被称为保守位点特异性重组（conservative site-specific recombination）。目前已知该现象存在于多个细菌类群中，但由于缺乏保守特征，其识别难度始终较高。本研究报道了一种基于高通量双端测序的简易实现方法，可在全基因组范围内以单核苷酸水平检测保守位点特异性重组。我们通过在肠道致病菌艰难梭菌（Clostridium difficile）的一株流行分离株中成功检测到多个新型倒位位点，验证了该方法的有效性。借助实验手段，我们验证了艰难梭菌中所有检测到的位点的倒位潜能，并定量了其在体外指数生长期与稳定生长期的出现频率。此外，我们证实主要重组酶RecV可介导部分而非全部倒位位点的重组事件。利用荧光基因报告系统，我们发现位于倒位位点旁的一套双组分系统中的至少一个基因，以类似相变异的开-关模式进行表达。我们进一步通过挖掘209个公开可用的测序数据集，验证了该方法的适用性，结果显示保守位点特异性重组在细菌界中普遍存在，但部分类群中似乎并未出现该现象。最后，我们发现与倒位位点相关的基因内容具有多样性，且超出了传统认知的表面组分范畴。总体而言，我们的方法为细菌中保守位点特异性重组的检测提供了一套稳健的研究平台，为这一重要现象的全局探索开辟了新途径。