Abstract
In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F’-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10-12 CFU/recipient per hour.
Highlights
In prokaryotes, horizontal gene transfer (HGT; called lateral gene transfer) is a massive force of evolutionary change and adaptation
HGT is responsible for the global spread of antibiotic resistance [2,3], it shapes microbial communities like the human microbiome [4], and it is interconnected with biofilm formation [5], the CRISPR/cas immunity system [6], virulence [7,8], and other critical cellular functions
DNA transfer begins at oriT within the F and proceeds in one direction at about 40 kb/min [27]
Summary
Horizontal gene transfer (HGT; called lateral gene transfer) is a massive force of evolutionary change and adaptation. A Novel E. coli Recombination Mechanism commercial company that supplies reagents and strains for use in research, including some used in sequencing. The ICR consists of a variable region of migratory genes surrounded by conserved framework genes (yjiPRS and yjiAXY) [19,20] (S1 Fig) This region shows signatures of high rates of homologous genetic exchange [14,21], but underlying that signature is a structure suggesting that it is a likely target of site-specific replacement [22]. We created a conjugal system to facilitate the study of RecA-independent horizontal transfer of the ICR in E. coli and relatives, and qualified its properties in the model strain K-12 This conjugal system proved to be an effective tool for discovering and analyzing novel mechanisms of horizontal gene transfer. We identified the previously uncharacterized protein YjiP as an enhancer of recA-independent recombination
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