FIG 4.
pBS32 produces a defective phage that protects plasmid DNA. (A) DNase protection assay. Strain DK1539 (Physpank-wkRBSzpdN) was induced to lyse by the addition of 1 mM IPTG, and the resulting supernatant was harvested. Left lane, extracellular DNA was phenol-chloroform extracted and used as a PCR template to amplify the chromosomal locus fliG and the plasmid loci zpaB, zpbI, and zpcJ (Fig. 1A). Middle lane, extracellular DNA was treated with DNase prior to extraction and PCR. Right lane, extracellular DNA was heat treated and then DNase treated prior to extraction and PCR. Panels were cropped from the same gel (see Fig. S5A in the supplemental material). (B) DNase protection assay. Strain DK4112 (ΔzpbH ΔSPβ ΔPBSX Physpank-wkRBSzpdN) was induced to lyse by the addition of 1 mM IPTG, and the resulting supernatant was harvested, phenol-chloroform extracted, and used as a PCR template to amplify the plasmid loci zpaB, zpbI, and zpcJ. Middle lane, extracellular DNA was treated with DNase prior to extraction and PCR. Right lane, extracellular DNA was heat treated and then DNase treated prior to extraction and PCR. Panels were cropped from the same gel (see Fig. S5B in the supplemental material). (C and D) EM images of PEG-precipitated supernatants of a hag mutant (DS1143) (C) and a hag ΔpBS32 mutant (DK3650) (D) induced with mitomycin C. Scale bar, 100 nm. Inset, enlarged images of PBSX particles. (E and F) EM images of PEG-precipitated supernatants of a hag ΔpBS32 ΔSPβ ΔPBSX mutant (DK3652) (E) and a hag ΔSPβ ΔPBSX mutant (DK3651) (F) induced with mitomycin C. Scale bar, 100 nm. Inset, enlarged images. (G) Silver-stained fraction taken from CsCl gradient resolved at the interface between 1.45 and 1.55 g/ml of PEG-precipitated supernatant of DK1634 (hag ΔSPβ ΔPBSX) induced with mitomycin C. The triangle indicates the location of a band that was excised and determined to be the ZpbH capsid protein by mass spectrometry analysis.