Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12

Abstract

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, in the E. coli K12 context too. However, it did not encode a site-specific recombinase as mobile genomic island usually do. Then, it was deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

“…The genetic basis of the RecA-independent recombination is independent of RecBCD, RecET, RecF, RecG, RuvAB and other known recombination mechanisms in E. coli, but its mechanism has not been fully understood [18,19]. It seems to be difficult to find a suitable host to eliminate RecA-independent recombination.…”

“…For repeats larger than about 200 bp in length, deletion can occur by RecA-dependent homologous recombination [15]. For homologies less than about 200 bp in length, the plasmid rearrangement can occur in a RecAindependent manner [15][16][17][18]. Depending on plasmid recombination products and the inverting sequences between DRs, three mechanisms of RecA-independent rearrangements were presented: slipped misalignment, sister-chromosome slipped misalignment, and single-strand annealing [19].…”

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

“…RecA-dependent recombination produces intermolecular interactions and appears to require a threshold of minimal homology [16]. For homologies less than 200 bp in length, the plasmid rearrangement can occur in a RecA-independent manner [17][18][19][20][21]. This type of rearrangement is enhanced by replication errors that hinder the DNA replication process [22].…”

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.

“…RecA-dependent recombination produces intermolecular interactions and appears to require a threshold of minimal homology [16]. For homologies less than 200 bp in length, the plasmid rearrangement can occur in a RecA-independent manner [17][18][19][20][21]. This type of rearrangement is enhanced by replication errors that hinder the DNA replication process [22].…”

“…The RecA protein was shown to be essential in most types of homologous recombination event, while RecBCD, RecJ or RecQ protein could process the dsDNA to produce ssDNA suitable for RecA function [15,16,30] . Although the RecA-involved RecBCD pathway is the major pathway for recombination in wild type cells, several of plasmid or genomic recombination events introduced by short DRs also occurred in manners independent of RecA protein [17][18][19][20][21]. Both RecA-dependent and RecAindependent recombination are homology-driven, with the former relies on the length of DRs more than 200 bp [17].…”