Broadness and specificity: ArdB, ArdA, and Ocr against various restriction-modification systems

Abstract

ArdB, ArdA, and Ocr proteins inhibit the endonuclease activity of the type I restriction-modification enzymes (RMI). In this study, we evaluated the ability of ArdB, ArdA, and Ocr to inhibit different subtypes of Escherichia coli RMI systems (IA, IB, and IC) as well as two Bacillus licheniformis RMI systems. Furthermore we explored, the antirestriction activity of ArdA, ArdB, and Ocr against a type III restriction-modification system (RMIII) EcoPI and BREX. We found that DNA-mimic proteins, ArdA and Ocr exhibit different inhibition activity, depending on which RM system tested. This effect might be linked to the DNA mimicry nature of these proteins. In theory, DNA-mimic might competitively inhibit any DNA-binding proteins; however, the efficiency of inhibition depend on the ability to imitate the recognition site in DNA or its preferred conformation. In contrast, ArdB protein with an undescribed mechanism of action, demonstrated greater versatility against various RMI systems and provided similar antirestriction efficiency regardless of the recognition site. However, ArdB protein could not affect restriction systems that are radically different from the RMI such as BREX or RMIII. Thus, we assume that the structure of DNA-mimic proteins allows for selective inhibition of any DNA-binding proteins depending on the recognition site. In contrast, ArdB-like proteins inhibit RMI systems independently of the DNA recognition site.

Keywords: ArdA; ArdB; RMI; antirestriction; conjugative plasmid.

Figure 1

The efficiency of defense provided by RMI systems. Results of the λ₀ phage plaquing (EOP) on a lawn of E. coli cells containing genes of various RMI systems of gram-negative bacteria. (A) EOP represents a ratio of a phage titer obtained on the experimental lawn relative to the TG1 lawn, which is sensitive to phage infection. Columns: pACYC184 – empty vector TG1pACYC184, EcoKI – TG1pACYCEcoKI, EcoAI – TG1pAM35, and EcoR124II – TG1pKF650. (B) Antirestriction activity of ArdB, ArdA, and Ocr. The residual activity of endonuclease (REA) represents the activity of endonuclease in the presence of ArdB, ArdA, or Ocr. Columns: EcoKI+ArdA – TG1pACYCEcoKIpUCArdA; EcoKI+ArdB – TG1pACYCEcoKIpArdBRam; EcoKI+Ocr – TG1pACYCEcoKIpBADOcr; EcoAI+ArdA – TG1pAM35pUCArdA; EcoAI+ArdB – TG1pAM35pArdBRam; EcoR124II+ArdB – TG1pKF650pArdBRam; EcoR124II+Ocr – TG1pKF650pBADOcr.

Figure 2

Influence of ArdB presence on the endonuclease activity of IB and IC RMI-systems EcoR124I and EcoAI in vitro. (A) 1% agarose gel electrophoresis: lane 1-−1kb ladder, lane 2—linear plasmid pARK, lane 3—circular plasmid pARK, lanes 4–11—incubation time (min) of the mix containing 10 nM pARK, 10nM EcoR124I, or EcoAI endonuclease; lanes 12–19—incubation time (min) of the mix containing 10 nM pARK, 10nM EcoR124I, or EcoAI endonuclease with 300 nM ArdB). (B) The percentage of linear plasmid at total plasmid band density (L band density, Lbd) measured with TotalLab Quant software. A typical experiment is presented. The ratios Lbd(EcoR124I)/Lbd(EcoR124I+ArdB) and Lbd(EcoAI24I)/Lbd(EcoAI24I+ArdB) were measured for three independent experiments at 5 min and equaled 1,35±0,1 and 3,38±0,3, respectively.

Figure 3

The efficiency of defense provided by RMI systems. (A) Results of the λ₀ phage plaquing (EOP) on a lawn of E. coli cells containing genes of various RMI systems of gram-positive bacteria compared to the EcoKI system. Columns: EcoKI – TG1pVMC3, BlihIA – TG1pIRal-2_RM-Ia, and BlihIB – TG1pIRal-2_RM-Ib. (B) Antirestriction activity of ArdA, ArdB, and Ocr proteins. Residual endonuclease activity (REA) represents the activity of endonuclease in the presence of antirestriction proteins. Columns: EcoKI+ArdA – TG1pVMC3p15araArdA, EcoKI+ArdB – TG1pVMC3p15araArdB, EcoKI+Ocr – TG1pVMC3p15araOcr, BlihIA+ArdA – TG1pIRal-2_RM-Ia-p15araArdA, BlihIA+ArdB – TG1pIRal-2_RM-Ia – p15araArdB, BlihIA+Ocr – TG1pIRal-2_RM-Ia – p15araOcr, BlihIB+ArdA – TG1pIRal-2_RM-Ib – p15araArdA, BlihIB+ArdB – TG1pIRal-2_RM-Ib – p15araArdB, and BlihIB+Ocr – TG1pIRal-2_RM – Ib – p15araOcr.

Figure 4

The efficiency of defense provided by BREX and RMIII (EcoPI) systems. (A) Results of the λ₀ phage plaquing (EOP) on a lawn of E. coli cells containing BREX or RMIII (EcoPI) defense systems of gram-negative bacteria. Columns: EcoPI – TG1pBTB_EcoPI and BREX – TG1pBREX. (B) Antirestriction activity of ArdA, ArdB, and Ocr proteins. Residual endonuclease activity (REA) represents the activity of endonuclease in the presence of antirestriction proteins. Columns: EcoPI+ArdA – TG1pBTB_EcoPIpUCArdA(pSR3), EcoPI+ArdB(prha) – TG1pBTB_EcoPIpArdBRham_AmpR, EcoKI – TG1pACYCEcoKI, BREX + ArdA – TG1pBREXpUCArdA(pSR3), BREX + ArdB(prha) – TG1pBREXpArdBRam, EcoPI – TG1pBTB_EcoPI, EcoPI + Ocr – TG1pBTB_EcoPIp15ara:ocr, and BREX+Ocr – TG1pBREXp15ara:ocr.