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

Abstract

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.

Phase variation of a signal transduction system controlsClostridioides difficilecolony morphology, motility, and virulence

“…For six of the switches, no correlation between morphology and sequence orientation was observed (Figure 3B). However, the orientation of the invertible element Cdi6, which is upstream of the operon CDR20291_3128-3126, showed a strong correlation with colony morphology (28, 30). Each of four independently isolated rough populations contained the sequence predominantly in the “on” orientation previously determined to favor gene expression, while each of the smooth populations contained the sequence predominantly in the inverse “off” orientation (28).…”

“…However, the orientation of the invertible element Cdi6, which is upstream of the operon CDR20291_3128-3126, showed a strong correlation with colony morphology (28, 30). Each of four independently isolated rough populations contained the sequence predominantly in the “on” orientation previously determined to favor gene expression, while each of the smooth populations contained the sequence predominantly in the inverse “off” orientation (28). CDR20291_3128-3126 encodes a putative phosphorelay system consisting of two predicted response regulators and a predicted histidine kinase (28).…”

“…Because colony morphology development exhibited characteristics suggestive of phase variation, we sought to identify the underlying mechanism. To date, the only known phase variation mechanism in C. difficile involves site-specific DNA recombination resulting in a reversible DNA inversion that is known or predicted to impact gene expression in cis (28). We postulated that one of the seven known invertible sequences regulates the expression of genes involved in colony morphology development, which would be evident as a correlation between the orientation of the invertible sequence and colony morphology (Figure 3A).…”

“…Each of four independently isolated rough populations contained the sequence predominantly in the “on” orientation previously determined to favor gene expression, while each of the smooth populations contained the sequence predominantly in the inverse “off” orientation (28). CDR20291_3128-3126 encodes a putative phosphorelay system consisting of two predicted response regulators and a predicted histidine kinase (28). Accordingly, we named the operon c olony m orphology regulators RST, where cmrR and cmrT encode the response regulators and cmrS encodes the histidine kinase, and refer to the Cdi6 invertible element as the “ cmr switch”.…”

“…C. difficile encodes multiple factors that are regulated through phase variation. In C. difficile , seven invertible DNA sequences, or “switches”, have been identified (28). Two have been characterized: one controlling phase variation of the cell wall protein CwpV and the other controlling flagellar phase variation (19, 29–31).…”