In vivo assembly of phage phi 29 replication protein p1 into membrane-associated multimeric structures.
The mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown. In the case of the Bacillus subtilis phage 29, the viral protein p1 enhances the rate of in vivo viral DNA replication. Previous work showed that p1 generates highly ordered structures in vitro. We now show that protein p1, like integral membrane proteins, has an amphiphilic nature. Furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location. By combining in vivo chemical cross-linking and cell fractionation techniques, we also demonstrate that p1 assembles in infected cells into multimeric structures that are associated with the bacterial membrane. These structures exist both during viral DNA replication and when 29 DNA synthesis is blocked due to the lack of viral replisome components. In addition, protein p1 encoded by plasmid generates membrane-associated multimers and supports DNA replication of a p1-lacking mutant phage, suggesting that the pre-assembled structures are functional. We propose that a phage structure assembled on the cell membrane provides a specific site for 29 DNA replication.
Serrano-Heras
Gemma
G
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Salas
Margarita
M
Bravo
Alicia
A
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000737:chemistry* / Q000378:metabolism*; D001412:Bacillus subtilis / Q000378:metabolism / Q000821:virology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005816:Genetic Complementation Test; D007700:Kinetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D003432:Cross-Linking Reagents; D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40