ArdC, a ssDNA-binding protein with a metalloprotease domain, overpasses the recipient hsdRMS restriction system broadening conjugation host range.

Lorena González-Montes,Fernando De La Cruz,Irene Del Campo,M Pilar Garcillán-Barcia,Gabriel Moncalián,Diarmaid Hughes

doi:10.1371/journal.pgen.1008750

Lorena González-Montes, Fernando De La Cruz + Show 4 more

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https://doi.org/10.1371/journal.pgen.1008750

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Abstract

Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.

Highlights

Horizontal gene transfer (HGT) is the transmission of genetic material between organisms that are not in a parent–progeny relationship [1]
Horizontal gene transfer is the main mechanism by which bacteria acquire and disseminate new traits, such as antibiotic resistance genes, that allow adaptation and evolution
We have identified transcriptional changes associated with ardC-mediated conjugation. These results show that ArdC is involved in broadening the R388 plasmid host range

Summary

Introduction

Horizontal gene transfer (HGT) is the transmission of genetic material between organisms that are not in a parent–progeny relationship [1]. The clinical relevance of the HGT process lies in the acquisition and dissemination of genes involved in conferring bacterial resistance to antibiotics (AbR) between unrelated pathogens. When bacteria face selective pressures, as those exerted by antibiotics, horizontal acquisition of AbR allows the diversification of the genomes, increasing survival opportunities [2]. Conjugation is the main HGT process that allows the transfer of genes encoded in autonomous plasmids. This process requires the machinery to build a direct contact between a donor and a recipient cell [1]. Conjugation can be modulated by environmental factors or bacterial strategies based on genetic approaches that are coded in the chromosome (host barriers) or plasmid DNA (plasmid barriers). Host barriers can be mediated through SOS response modulation [5,6], CRISPR-Cas systems [7] or restriction and modification (R-M) systems

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