Transfection of Escherichia coli spheroplasts with a bacteriophage Mu DNA-protein complex.
We disrupted bacteriophage Mu particles by freeze-thaw treatment and recovered the DNA by CsCl density gradient centrifugation. This CsCl-purified DNA had a buoyant density which was indistinguishable from that of phenol-extracted Mu DNA. It was, however, 10(3) times more infective than phenol-extracted DNA for spheroplasts of exoV endI Escherichia coli. Infectivity was destroyed by proteinase K as well as by pancreatic DNase, indicating that the infective form was a DNA-protein complex. The infective properties of the complex demonstrated that the protein protects. Mu DNA against degradation by exonuclease V and that it serves at least one other function in bacteriophage Mu infection. The infectivity of the CsCl-purified DNA was due to a small class of highly infective molecules which sedimented 1.2. times faster than phenol-extracted Mu DNA on neutral sucrose gradients. This change in sedimentation rate is best explained by the formation of protein-linked circular monomers or linear dimers of Mu DNA. In vitro labeling of the DNA-protein complex, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that the CsCl-purified DNA contained a noncovalently associated 65,000-dalton polypeptide. A 65,000-dalton protein was also found to be a minor component of the bacteriophage Mu particle. No protein was found in phenol-extracted Mu DNA. These results suggest that the 65,000-dalton protein is necessary for successful phage infection and is normally injected into the host cell with the Mu genome.
D010583:Bacteriophage mu / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D003852:Deoxyribonucleoproteins / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D009698:Nucleoproteins / Q000235:genetics*; D013104:Spheroplasts / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification
D004279:DNA, Viral; D003852:Deoxyribonucleoproteins; D009698:Nucleoproteins; D014764:Viral Proteins