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.Bacillus subtilis | phage ø29 | DNA polymerase | DNA-binding | bacterial cytoskeleton P rotein-primed DNA replication is a mechanism used to initiate DNA synthesis in a variety of prokaryotic and eukaryotic organisms (1). Phages such as ϕ29 (infecting Bacillus subtilis) and PRD1 (infecting Escherichia coli) (1, 2), animal viruses such as adenoviruses (1, 3), bacterial species from the Streptomyces genus (1, 4), and viruses infecting Archaea (5-7) possess replication origins at their linear chromosomes constituted by inverted terminal repetitions with a terminal protein (TP) linked to both 5′ genome ends. TPs also have been reported in linear plasmids isolated from bacteria, yeast, fungi, and higher plants (1) and in animal and plant RNA viruses (1).The development of an in vitro replication system with purified proteins and DNA from the B. subtilis phage ϕ29 laid the foundations for investigating the protein-primed mechanism of DNA replication and makes ϕ29 a paradigm for studying this process (1, 2). A schematic overview of the in vitro ϕ29 DNA replication mechanism is shown in Fig. S1. The ϕ29 genome consists of a 19,285-bp linear dsDNA, with a TP of 31 kDa (the parental TP) covalently linked to each 5′ end. DNA replication starts with the recognition of the TP-containing DNA ends by a heterodimer formed by the ϕ29 DNA polymerase and a free TP molecule (the primer TP) (8). The DNA polymerase then catalyzes the formation of a covalent bond between deoxyAMP and the hydroxyl group Ser 232 of the primer TP. Replication is coupled to strand displacement, and continuous elongation of the DNA polymerase from both DNA ends generates replication intermediates that finally converge in the complete duplication of the parental strands (reviewed in ref.2). Phage ϕ29 DNA transcription is divided into early and late stages (9). Fig. S1 shows a genetic and a transcriptional map. Genes 2 and 3, encoding phage DNA polymerase and TP, respectively, are located i...

During evolution, viruses have optimized the interaction with host factors to increase the efficiency of fundamental processes such as DNA replication. Bacteriophage ϕ29 protein p1 is a membraneassociated protein that forms large protofilament sheets that resemble eukaryotic tubulin and bacterial filamenting temperaturesensitive mutant Z protein (FtsZ) polymers. In the absence of protein p1, phage ϕ29 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 phageencoded proteins. We also show that ϕ29 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 ϕ29 protein p1 was expressed, B. subtilis cells were about 1.5-fold longer than control cells, and the accumulation of ϕ29 DNA was higher in mutant B. subtilis cells with increased length. We discuss the biological role of p1 and FtsZ in the ϕ29 growth cycle.bacterial enlargement | cytokinesis | viral replication | Z ring D NA replication of bacterial viruses seems to occur at specific intracellular locations, similar to the replication of DNA from viruses infecting eukaryotic cells. The use of organizing structures of the host constitutes a general viral mechanism to enhance the efficiency of the replication process. Over the last 40 years, different lines of evidence have indicated that the bacterial membrane provides a framework to support replication of distant viral genomes (1-3), thus compartmentalizing this fundamental biological process. In this sense, it has been shown that DNA replication of bacteriophage ϕ29 occurs in association with the Bacillus subtilis membrane (4-6).The B. subtilis phage ϕ29 is one of the best-characterized phages and serves as a model for the study of DNA replication both in vitro and in vivo. The genome of ϕ29 consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked at each 5′ end, and is replicated by a protein-primed mechanism (7, 8) (see Fig. S1 for details). In vitro ϕ29 DNA replication starts with the formation of a heterodimer between the ϕ29 DNA polymerase and a free TP molecule (primer TP) that recognizes the replication origins located at both ends of the viral genome. The DNA polymerase then catalyzes the formation of a covalent linkage between dAMP and the hydroxyl group of serine232 of the primer TP. Replication is coupled to strand displacement, and continuous elongation by the DNA polymerase from both DNA ends gives rise to the formation of replication intermediates that finally converge in the generation of two full-...

Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage ϕ29 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 ϕ29 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.