Transfection of Escherichia coli spheroplasts with a bacteriophage Mu DNA-protein complex.

Abstract

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.

• Authors: C D Chase, R H Benzinger
• Journal: Journal of virology (J Virol) Vol. 42 Issue 1 Pg. 176-85 (Apr 1982) ISSN: 0022-538X [Print] United States
• PMID: 6211551 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA, Viral
• Deoxyribonucleoproteins
• Nucleoproteins
• Viral Proteins

Topics

• Bacteriophage mu (genetics)
• DNA, Viral (genetics)
• Deoxyribonucleoproteins (genetics, isolation & purification)
• Escherichia coli (genetics)
• Genes, Viral
• Nucleoproteins (genetics)
• Spheroplasts (metabolism)
• Transfection
• Viral Proteins (genetics, isolation & purification)