Daniel Muñoz-Espín

Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication

The transcription factor Spo0A is a master regulator for entry into sporulation in Bacillus subtilis and also regulates expression of the virulent B. subtilis phage ϕ29. Here, we describe a novel function for Spo0A, being an inhibitor of DNA replication of both, the ϕ29 genome and the B. subtilis chromosome. Binding of Spo0A near the ϕ29 DNA ends, constituting the two origins of replication of the linear ϕ29 genome, prevents formation of ϕ29 protein p6‐nucleoprotein initiation complex resulting in inhibition of ϕ29 DNA replication. At the B. subtilis oriC, binding of Spo0A to specific sequences, which mostly coincide with DnaA‐binding sites, prevents open complex formation. Thus, by binding to the origins of replication, Spo0A prevents the initiation step of DNA replication of either genome. The implications of this novel role of Spo0A for phage ϕ29 development and the bacterial chromosome replication during the onset of sporulation are discussed.

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

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.

Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages

A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of Φ29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of Φ29 TP led us to identify a bona fide NLS within residues 1–37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of Φ29 TP attached to the 5′ DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes.

The integral membrane protein p16.7 organizes in vivo φ29 DNA replication through interaction with both the terminal protein and ssDNA

Remarkably little is known about the in vivo organization of membrane‐associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage φ29 as a model system. Previously, we demonstrated that the φ29‐encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane‐associated φ29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the φ29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C‐terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA‐binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo φ29 DNA replication is presented.

Phage Φ29 DNA replication organizer membrane protein p16.7 contains a coiled coil and a dimeric, homeodomain-related, functional domain

The Bacillus subtilis phage φ29-encoded membrane protein p16.7 is one of the few proteins known to be involved in prokaryotic membrane-associated DNA replication. Protein p16.7 contains an N-terminal transmembrane domain responsible for membrane localization. A soluble variant lacking the N-terminal membrane anchor, p16.7A, forms dimers in solution, binds to DNA, and has affinity for the φ29 terminal protein. Here we show that the soluble N-terminal half of p16.7A can form a dimeric coiled coil. However, a second domain, located in the C-terminal half of the protein, has been characterized as being the main domain responsible for p16.7 dimerization. This 70-residue C-terminal domain, named p16.7C, also constitutes the functional part of the protein as it binds to DNA and terminal protein. Sequence alignments, secondary structure predictions, and spectroscopic analyses suggest that p16.7C is evolutionarily related to DNA binding homeodomains, present in many eukaryotic transcriptional regulator proteins. Based on the results, a structural model of p16.7 is presented.

Phage phi 29 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 ϕ29 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 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 phage-encoded 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.

Structural Basis for Membrane Anchorage of Viral ϕ29 DNA during Replication

Prokaryotic DNA replication is compartmentalized at the cellular membrane. Functional and biochemical studies showed that the Bacillus subtilis phage ϕ29-encoded membrane protein p16.7 is directly involved in the organization of membrane-associated viral DNA replication. The structure of the functional domain of p16.7 in complex with DNA, presented here, reveals the multimerization mode of the protein and provides insights in the organization of the phage genome at the membrane of the infected cell.

New insights in the ϕ29 terminal protein DNA-binding and host nucleoid localization functions

Protein‐primed DNA replication constitutes a strategy to initiate viral DNA synthesis in a variety of prokaryotic and eukaryotic organisms. Although the main function of viral terminal proteins (TPs) is to provide a free hydroxyl group to start initiation of DNA replication, there are compelling evidences that TPs can also play other biological roles. In the case of Bacillus subtilis bacteriophage ϕ29, the N‐terminal domain of the TP organizes viral DNA replication at the bacterial nucleoid being essential for an efficient phage DNA replication, and it contains a nuclear localization signal (NLS) that is functional in eukaryotes. Here we provide information about the structural properties of the ϕ29 TP N‐terminal domain, which possesses sequence‐independent DNA‐binding capacity, and dissect the amino acid residues important for its biological function. By mutating all the basic residues of the TP N‐terminal domain we identify the amino acids responsible for its interaction with the B. subtilis genome, establishing a correlation between the capacity of DNA‐binding and nucleoid localization of the protein. Significantly, these residues are important to recruit the DNA polymerase at the bacterial nucleoid and, subsequently, for an efficient phage DNA replication.

Nuclear and nucleoid localization are independently conserved functions in bacteriophage terminal proteins

Bacteriophage terminal proteins (TPs) prime DNA replication and become covalently linked to the DNA 5′‐ends. In addition, they are DNA‐binding proteins that direct early organization of phage DNA replication at the bacterial nucleoid and, unexpectedly, contain nuclear localization signals (NLSs), which localize them to the nucleus when expressed in mammalian cells. In spite of the lack of sequence homology among the phage TPs, these three properties share some common features, suggesting a possible evolutionary common origin of TPs. We show here that NLSs of three different phage TPs, Φ29, PRD1 and Cp‐1, are mapped within the protein region required for nucleoid targeting in bacteria, in agreement with a previously proposed common origin of DNA‐binding domains and NLSs. Furthermore, previously reported point mutants of Φ29 TP with no nuclear localization still can target the bacterial nucleoid, and Cp‐1 TP contains two independent NLSs, only one of them required for nucleoid localization. Altogether, our results show that nucleoid and nucleus localization sequence requirements partially overlap, but they can be uncoupled, suggesting that conservation of both features could have a common origin but, at the same time, they have been independently conserved during evolution.