The EcoKI Type I Restriction-Modification System in Escherichia coli Affects but Is Not an Absolute Barrier for Conjugation

Abstract

The rapid evolution of bacteria is crucial to their survival and is caused by exchange, transfer, and uptake of DNA, among other things. Conjugation is one of the main mechanisms by which bacteria share their DNA, and it is thought to be controlled by varied bacterial immune systems. Contradictory results about restriction-modification systems based on phenotypic studies have been presented as reasons for a barrier to conjugation with and other means of uptake of exogenous DNA. In this study, we show that inactivation of the R.EcoKI restriction enzyme in strain Escherichia coli K-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly, the results were not absolute, and uptake of unmethylated pOLA52 was still observed in the wild-type strain (with an intact hsdR gene) but at a reduction of 85% compared to the uptake of the mutant recipient with a disrupted hsdR gene. This leads to the conclusion that EcoKI restriction-modification affects the uptake of DNA by conjugation but is not a major barrier to plasmid transfer. The exchange of chromosomal and/or extrachromosomal DNA, such as plasmids, viruses, and transposons, is crucial for the evolution of bacteria and their ability to adapt to new environments. Exchange of genetic material occurs among both related and unrelated species of bacteria and is driven by the three horizontal gene transfer (HGT) mechanisms: conjugation, transformation, and transduction (1-3). Restriction-modification (RM) systems are described as major barriers to HGT (4-6) and comprise restriction endonucleases with a cognate methyltransferase. These recognize and cleave DNA not modified by the methyltransferase, thereby making the bacterium able to distinguish between its own (methylated) DNA and incoming nonmethylated DNA.Based on their protein-complex subunit composition and functionality, RM systems can be divided into four types; this study focuses on type I. Type I systems require products of the three genes hsdR (restriction), hsdM (methylation), and hsdS (sequence specificity) and cleave randomly at a remote distance from the recognition sequence. Restriction occurs only when a protein complex of all three gene products (R 2 M 2 S) is formed, whereas methylation of the DNA requires formation of a complex of only the HsdM and HsdS proteins (M 2 S) (7).Some studies have indicated that transfer by conjugation is unaffected by RM systems but that unmodified phage or free DNA in transformation is readily degraded (8-11). This has led to the view that the conjugational transfer of plasmids through a singlestranded DNA intermediate is immune to restriction by RM systems, as the great majority of these recognize only nonmethylated double-stranded DNA (12-17). Other studies have, however, contradicted this. In 1964, Arber and Morse (18) proposed that host specificity (RM systems) might play a role in the acceptance or rejection of DNA transferred by conjugation in Escherichia coli. In many studies from the 1960s, the t...

ArdC protein overpasses the recipient hsdRMS restriction system broadening conjugation host range

“…Type I R-M systems attack dsDNA and thus, are not expected to degrade ssDNA during bacterial conjugation. However, it has been reported that the EcoKI R-M system affects the uptake of DNA by conjugation [26] and, ArdC is not expressed until plasmid DNA is in dsDNA shape. Thus, Type I R-M system could be attacking late, once ArdC is generated from the dsDNA plasmid after DNA entrance and establishment in recipient cells.…”

Plasmid transfer is biased towards close kin in bacteria from natural populations

“…We identified restriction-modification as a likely mechanism contributing to discrimination in transfer, showing that recipients with inactivated RM receive R1 plasmid at higher rates from field isolates. Restriction was previously shown to limit plasmid conjugation rates with relatively low efficiency (Roer et al, 2015), likely because the first transconjugants that escaped restriction are then protected from further restriction when transferring to kin. RM systems are tremendously variable and mobile among E. coli isolates (Murray, 2002;Barcus et al, 1995).…”

“…The restriction to clone-mates of high transfer rates suggests that barriers to transfer are not simply a function of genetic similarity. One barrier could be variation in restrictionmodification (RM) systems, which can lead to decrease in transfer rates (Roer et al, 2015):…”

The Impact of Restriction-Modification Systems on Mating inHaloferax volcanii

“…The ability of these systems to restrict foreign DNA could allow them to limit genetic exchange between species, thus potentially driving the diversification of microbial populations (Erwin et al, 2008;Budroni et al, 2011). Studies have demonstrated that RM systems can limit conjugal transfer of plasmids (Roer et al, 2015), and that they can limit the size of recombinant DNA fragments that are obtained via natural transformation (Lin et al, 2009).…”