Modified DNA substrate selectivity by GmrSD-family Type IV restriction enzyme BrxU

Abstract

Bacteriophages (phages), viral predators of bacteria, generate selection pressure that causes bacteria to evolve defence systems. Type I, II and III restriction enzymes cleave incoming non-modified phage DNAs. Phages have evolved to defend against these restriction systems by modifying their DNA so that they are no longer suitable substrates. Type IV restriction enzymes have evolved to recognize and cleave modified DNA. We have recently characterized and solved the first structure for the Type IV GmrSD-family enzymes, using the BrxU homologue from Escherichia fergusonii. Though promiscuous in target modifications, little is known about BrxU substrate preference. We used modified DNAs in in vitro assays to characterize the substrate preferences of BrxU and investigate the impact of the GmrSD-inhibitor IPI* on BrxU activity. These data extend our knowledge of phage-host interactions and inform mechanistic studies on the reaction cycle of BrxU and GmrSD homologues.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.

Keywords: BrxU; DNA modification; GmrSD; bacteriophage; phage defence; restriction enzyme.

Figure 1.

BrxU demonstrates preference between differing cytosine modifications. (A) Titrations of His₆-BrxU were incubated with 100 ng of pUC19 incorporated with dC, 5hmdC, 5mdC or glc-5mdC for 10 min. The reactions were performed in triplicate, and representative gels are shown. The presence of ATP in the reaction is denoted with a + or − sign, and ATPγS is denoted by γS. (B) Timecourse of His₆-BrxU (300 nM) mixed with 100 ng of pUC19 incorporated with dC, 5hmC, 5mC or glc-5hmC and incubated at 37 °C for from 1 to 30 min. The reactions were performed in triplicate, and representative gels are shown. The presence of ATP in the reaction is denoted with a + or − sign, and ATPγS is denoted by γS. (C) The average percentage of DNA remaining in His₆-BrxU titration reactions with ATP compared with reactions without ATP, calculated from triplicate gels. Error bars represent standard deviation. (D) The average percentage DNA remaining in His₆-BrxU timecourse reactions with ATP compared with reactions without ATP, calculated from triplicate gels. Error bars represent standard deviation.

Figure 2.

BrxU phage defence can be inhibited by T4 IPI* counter-defence. Phages T4 WT and T4Δip1 were plated on E. coli DH5α pBAD30-his₆-brxU or DH5α pBAD30-brxU and compared against DH5α pBAD30. (A) EOP values for each phage are shown; values are mean EOPs from triplicate data with standard deviation. (B) Example images of plaques are provided.

Figure 3.

In vitro inhibition of BrxU by IPI*. (A) His₆-BrxU and BrxU samples incubated with IPI* or BSA for 30 min were further incubated with 100 ng glc-5mdC pUC19 and 1 mM ATP for 10 min. The products were resolved on 1% agarose gels. Reactions were performed in triplicate, and representative gels are shown. (B) The mean average percentage DNA remaining in reactions of His₆-BrxU and BrxU with IPI* or BSA, calculated from triplicate gels. Error bars represent standard deviation.