Phage 29 phi protein p1 promotes replication by associating with the FtsZ ring of the divisome in Bacillus subtilis.

During evolution, viruses have optimized the interaction with host factors to increase the efficiency of fundamental processes such as DNA replication. Bacteriophage 29 protein p1 is a membrane-associated protein that forms large protofilament sheets that resemble eukaryotic tubulin and bacterial filamenting temperature-sensitive mutant Z protein (FtsZ) polymers. In the absence of protein p1, phage 29 DNA replication is impaired. Here we show that a functional fusion of protein p1 to YFP localizes at the medial region of Bacillus subtilis cells independently of other phage-encoded proteins. We also show that 29 protein p1 colocalizes with the B. subtilis cell division protein FtsZ and provide evidence that FtsZ and protein p1 are associated. Importantly, the midcell localization of YFP-p1 was disrupted in a strain that does not express FtsZ, and the fluorescent signal was distributed all over the cell. Depletion of penicillin-binding protein 2B (PBP2B) in B. subtilis cells did not affect the subcellular localization of YFP-p1, indicating that its distribution does not depend on septal wall synthesis. Interestingly, when 29 protein p1 was expressed, B. subtilis cells were about 1.5-fold longer than control cells, and the accumulation of 29 DNA was higher in mutant B. subtilis cells with increased length. We discuss the biological role of p1 and FtsZ in the 29 growth cycle.

Disclosing the in vivo organization of a viral histone-like protein in Bacillus subtilis mediated by its capacity to recognize the viral genome.

Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage 29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the 29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA, which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria.

Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid.

The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.