Conjugation Mediated Bacterial Genome Mixing (CoMBacGeMi) to transfer and detect genes of interest in Escherichia coli and unravel the recombination patterns

Abstract

Conjugation Mediated Bacterial Genome Mixing (CoMBacGeMi) is a technique developed to enable the exchange of genetic material between bacterial strains, in a manner that simulates the process occurring in the wild. CoMBacGeMi aims to elucidate the molecular basis of functional diversity observed in medically significant bacterial species, such as Escherichia coli. The method employs a library of strains, each harboring a conjugative plasmid integrated at a random position in the genome. In this study, we employed CoMBacGeMi to investigate the effect of a mutant matP recipient on the bias in recombination frequency observed at the terminus. Analysis of the pools revealed that MatP does not significantly regulate the recombination pattern. To go beyond a single locus and to investigate the recombination pattern across the entire genome, we utilized the Hfr library and the recipient tse2 library. Our results revealed that the recombination frequencies across the genome were non-uniform, with regions such as the Ter macrodomain exhibiting low recombination frequencies. Additionally, we observed that the conjugation efficiencies of recipient clones were related to the position of the loci of selection in the genome. Lastly, we tested the ability of CoMBacGeMi to identify specific targets of selection. Analysis of the pools enabled us to identify, with precision, the three distinct insertion positions of tse2 in the three tested clones. Our results demonstrate that CoMBacGeMi is a simple, precise, and accurate method with the potential to identify key genetic loci and provide insight into the complexities of genetic traits in diverse bacterial populations.