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.
Author summary Bacteria in many ecological niches experience a common challenge in the form of unpredictable environmental fluctuations. Rapid adaptation to challenging conditions is important for bacterial survival and successful proliferation. Altering gene expression through DNA inversion is a common mechanism adopted by many bacterial species that allows quick generation of distinct subpopulations with altered fitness. The characterization of these systems beyond a few classical cases is lagging due to the difficulties to accurately detect such inversion on a population level. In this study, we implement an easy-to-use method for detecting small genomic inversions in bacterial genomes. We successfully applied our approach to detect known and novel inversion sites in C. difficile. We further show that all detected sites undergo inversion and exist at different frequencies in vitro. The inversion of several sites seems dependent on the master recombinase RecV. We expand our analysis to a large collection of bacterial and archaeal strains and show that our method can be globally applied for detection of small genomic inversions. Taken together, this study advances the ability to characterize this important phenomenon.