Fig. 6: ZorE can degrade phage and chromosomal DNA.

a ZorE-Strep titrated against a constant amount of supercoiled pSG483 plasmid DNA (6 nM). Samples were incubated at 37 °C for 60 min in the presence of 5 mM Mg ²⁺. b Densitometry quantification of nicking of pSG483 by ZorE as shown in panel (a). c ZorE (768 nM) was incubated with supercoiled plasmid pSG483 (6 nM) at 37 °C for 0 to 60 min with 5 mM Mg^2+. d Densitometry quantification of nicking of pSG483 by ZorE as shown in panel (c). e ZorE (768 nM) was incubated with relaxed plasmid pSG483 (6 nM) for 0 to 60 min with 5 mM Mg²⁺ at 37 °C. f Densitometry quantification of nicking of pSG483 by ZorE as shown in panel (d). For panels a, c, and e, reactions were stopped by the addition of EDTA and SDS, and products were analyzed by gel electrophoresis in a 1× TAE, 1.4% agarose gel, post-stained with ethidium bromide. In all gels, control lanes represent forms of plasmid pSG483; R, relaxed (multiple topoisomers); N, nicked; L, linear; S, supercoiled. For panels b, d, and f densitometry was performed using ImageJ (version 1.54g) with background subtracted and band intensity measured in triplicate. The percentage of nicked, linear, and supercoiled pSG483 DNA of the total pSG483 DNA per lane was determined by calculating the average intensity (n = 3) of each lane’s nicked, linear, and supercoiled bands, respectively. as a percentage of the total average intensity of all bands per lane. Relative band intensity was determined by normalizing the average (n = 3) intensity of the “0 μM ZorE” lane to 100% and taking the average intensity of the subsequent lanes’ bands as a percentage of the “0 μM ZorE” lane. Error bars represent the standard error of the mean of triplicate data. g E. coli MT56 harboring empty vector (VC, pGM39) or the same plasmid encoding Zorya I, ZorAB I, ZorCD, Zorya II, ZorAB II, or ZorE were grown in LB supplemented with 0.2% l-Rhamnose for 2 h. Following incubation, cells were stained with DAPI and imaged by fluorescence microscopy. Scale bar 5 µm. h E. coli MT56 harboring empty vector (VC, pGM39) or the same plasmid encoding Zorya I, ZorAB I, or ZorCD were grown as in (g) and total genomic DNA (gDNA) was extracted. Neutral and alkaline treatment of gDNA, followed by electrophoretic analysis (Methods) was used to assess for DNA breaks. i E. coli MT56 harboring empty vector (VC, pGM39) or plasmids expressing Zorya II, ZorAB II or ZorE were induced as in (g) and total gDNA was isolated as in (h). Genomic DNA was subjected to neutral and alkaline treatment, as described in “Methods”, and subjected to electrophoretic analysis. j E. coli MT56 carrying the empty vector (VC, pSUPROM) or the same plasmid harboring Zorya II, ZorAB II, or ZorE under the control of their native promoter were grown in the presence and absence of ɸphAvM (MOI 0.1) until first burst event. Total gDNA was extracted and subjected to neutral and alkaline treatment as in panels (h–i). k E. coli MT56 carrying the empty vector (VC, pSUPROM) or the same plasmid harboring Zorya II, ZorAB II, or ZorE under the control of their native promoter were grown in the presence and absence of ɸphAvM (MOI 0.1). Strains were fractionated to produce a soluble cytoplasmic fraction (CP) and a total membrane fraction (TM). Samples were analyzed by immunoblot with antibodies to the His₆ tag (for detection of ZorE) and Strep-tag (for detection of ZorB). OmpC was used as a membrane control and CsrA as cytoplasmic control. For panels g–k, gels are representative of three independent experiments.