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

Ognjen Sekulovic,Rita Tamayo,Andrew Camilli,Elizabeth Mathias Garrett,Aimee Shen,Jacob Bourgeois,Ivan Matic

doi:10.1371/journal.pgen.1007332

Ognjen Sekulovic, Rita Tamayo + Show 5 more

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https://doi.org/10.1371/journal.pgen.1007332

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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.

Highlights

Clonal microbial populations often exhibit phenotypic and behavioral heterogeneity
We developed a method for genome-wide detection of conservative site-specific recombination in bacteria using paired-end high-throughput sequencing
We demonstrate the utility of our method by detecting and experimentally confirming known and novel invertible sites in the opportunistic pathogen C. difficile

Summary

Introduction

Clonal microbial populations often exhibit phenotypic and behavioral heterogeneity This feature is widely observed in the microscopic world–from viruses to protozoa–and reflects the common need to adapt to the ever-changing environmental milieu. A common bet-hedging strategy adopted by bacteria is to alter the expression of specific cellular components (e.g. flagella or pili) in a dichotomous on-off mode This process, termed phase variation, is heritable, reversible, and occurs at frequencies well-above typical mutation rates [1]. Phase variation can quickly become the major source of heterogeneity with a profound impact on the survival or fitness capacity of the population as a whole This is best exemplified in pathogens that need to constantly change the expression of surface antigens as a consequence of the pressure from the host immune system [2,3,4]

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