Background

Homologous recombination can occur between two strands of DNA that share a sufficient amount of sequence identity. Homologous recombination can result in either reciprocal exchanges (for example, during meiosis in diploid eukaryotes, when genetic information is exchanged between two homologous parental chromosomes to create novel gametic chromosomes), or non-reciprocal exchanges (such as during mating-type switching in Saccharomyces cerevisiae, when genetic information is copied from one location on a chromosome to another homologous location on the same chromosome, resulting in gene conversion). In reciprocal recombination no genetic information is lost, it is simply exchanged between two homologous chromosomes. In contrast, in a non-reciprocal recombination event, genetic information from one location is copied into another homologous location, and replaces the original information, resulting in gene conversion. In haploid bacteria there is no meiosis, but homologous recombination can take place between the bacterial chromosome and a foreign fragment of DNA, leading to a horizontal gene transfer event (Thomas and Nielsen, 2005) or between two homologous locations within the same chromosome, which can lead to gene conversion (Anderson and Roth, 1977; Abdulkarim and Hughes, 1996; Hughes, 2000).

To measure homologous recombination rates within bacterial chromosomes requires the ability to select for successful recombinant cells. Traditionally, this was achieved by the use of selectable markers. These were inserted in a mutated, inactive state into two locations of the chromosome and could recombine to form an active, selectable gene (Segall and Roth, 1989). The drawback of this method is that recombination occurs between the two inserted sequences and does not provide any information about the homologous recombination rates between native sites within the chromosome. We have developed a method that overcomes this drawback by the use of a counter-selectable marker (Brandis et al., 2018). A selectable/counter-selectable cat-sacB-yfp cassette (cat: selectable for chloramphenicol resistance, sacB: counter-selectable on sucrose (Gay et al., 1985); yfp: yellow fluorescence protein for screening) is inserted into a chromosomal location (selecting for chloramphenicol resistance) (Figures 1A and 1B) and homologous recombination rates are measured as a function of recombinational removal of the inserted cassette (selecting for loss of sacB, growth in the presence of sucrose) (Figures 1C and 1D).

This method enables the measurement of recombination rates between any native homologous sites within the bacterial chromosome.


Figure 1. Principle of the assay. A. The assay measures recombination rates between two homologous locations within the chromosome. B. Initially, a selectable/counter-selectable marker is inserted into one of the homologous locations (not part of this protocol). C and D. Spontaneous homologous recombination between the two locations (C) can lead to the removal of the inserted cassette (gene conversion) and the resulting strains (D) can be isolated due to the loss of the counter-selectable marker gene.