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

Abstract

Plasmids, when transferred by conjugation, 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, but not from E. coli to E. coli. Surprisingly, 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. Thus, ArdC has antirestriction activity against HsdRMS system, and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and in the presence of Mn 2+ . ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity is not needed for antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggers 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. ResultsardC is required for R388 conjugation from E. coli to P. putida R388 plasmid is composed by three functional sectors ( Fig EV1). One for general maintenance (modules of replication, stable inheritance and establishment) located in the leading region, a sector for Ab R and integration and a third one for conjugation (modules of DNA transfer replication and mating pore formation) [11]. We expected the stable inheritance and establishment region to be required in interspecies conjugation. pSU2007, a Kn R R388 derivative, is transferred with different efficiencies from E. coli BW27783-Nx R to other bacteria ( Fig EV2). All conjugation experiments were done at the recipient cells optimal growing temperature except for P. putida KT2440 to which we observed that the conjugation was more effective at 37 °C. The transfer of pIC10 (R388∆kfrA-orf14), an R388 derivative without the stability and maintenance region, is similar to pSU2007 in all species but in P. putida KT2440 where the conjugation frequencies dropped around 1000 times. We have performed the conjugation experiments at both the optimal E. coli growing temperature (37 °C) or P. putida growing temperature (30 °C). In the stability and maintenance gene region deleted in pIC10 there are 13 genes that code for proteins homologous to some with predicted function of: fertility inhibition in a different system (kfrA, nuc1, nuc2 and osa) [12], hypothetical proteins of unknown function (orf7, orf8, orf9, orf12 and orf14), transcriptional regulators (ardk and klcB), ssDNA binding

Comparative whole-genome analysis of novel marine Vibrio campbellii from pure and non-axenic cultures reveals a unique plasmid

“…In contrast, the ardC genes observed in our plasmid have anti-restriction activity against the type I R-M system [93], which enables the plasmid to overpass the R-M systems of the recipient cell once they are transferred by conjugation. In that way, plasmids broaden their host range [94].…”

Genome archaeology of two laboratory Salmonella enterica enterica sv Typhimurium

“…Bacteria express variable surface features to thwart entry (e. g. flagellae, outer membrane proteins, O-antigen sugar chains) and internal limitation mechanisms to abort establishment (e. g. restriction systems and abortive-infection suicide factors) (Azam and Tanji 2019;Doron et al 2018). Phages and plasmids may defeat host restriction-modification (RM) systems with antirestriction proteins, which frequently block type I DNA restriction-modification enzymes (e. g. (Balabanov et al 2012;González-Montes et al 2020;Piya et al 2017;Roberts et al 2012;Serfiotis-Mitsa et al 2010;Zavilgelsky and Rastorguev 2009). Low-specificity DNA modification enzymes expressed only for the entering molecule are also found in prophages (Murray et al 2018) and plasmids (Fomenkov et al 2020).…”