Phage ϕ29 protein p1 promotes replication by associating with the FtsZ ring of the divisome in Bacillus subtilis

Abstract

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-...

Splitsville: structural and functional insights into the dynamic bacterial Z ring

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“…In contrast to inhibiting FtsZ ring formation, a recent report described the role of B. subtilis Φ29 p1 in promoting phage replication through association with assembled FtsZ [72] . This strategy allows Φ29 to utilize an existing host cell scaffold to organize and optimize viral DNA production, but how a phage would benefit from perturbing FtsZ ring formation and inhibiting cell division is less clear.…”