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Bacillus subtilis bacteriophage SPP1 is a lytic siphovirus first described 50 years ago [1]. Its complete DNA sequence was reported in 1997 [2]. Here we present an updated annotation of the 44,016 bp SPP1 genome and its correlation to different steps of the viral multiplication process. Five early polycistronic transcriptional units encode phage DNA replication proteins and lysis functions together with less characterized, mostly non- essential, functions. Late transcription drives synthesis of proteins necessary for SPP1 viral particles assembly and for cell lysis, together with a short set of proteins of unknown function. The extensive genetic, biochemical and structural biology studies on the molecular mechanisms of SPP1 DNA replication and phage particle assembly rendered it a model system for tailed phages research. We propose SPP1 as the reference species for a new SPP1-like viruses genus of the Siphoviridae family., Competing Interests: The authors declare no conflict of interest.

Unlabelled: All viruses are obligate intracellular parasites and depend on certain host cell functions for multiplication. However, the extent of such dependence and the exact nature of the functions provided by the host cell remain poorly understood. Here, we investigated if nonessential Bacillus subtilis genes are necessary for multiplication of bacteriophage SPP1. Screening of a collection of 2,514 single-gene knockouts of nonessential B. subtilis genes yielded only a few genes necessary for efficient SPP1 propagation. Among these were genes belonging to the yuk operon, which codes for the Esat-6-like secretion system, including the SPP1 receptor protein YueB. In addition, we found that SPP1 multiplication was negatively affected by the absence of two other genes, putB and efp. The gene efp encodes elongation factor P, which enhances ribosome activity by alleviating translational stalling during the synthesis of polyproline-containing proteins. PutB is an enzyme involved in the proline degradation pathway that is required for infection in the post-exponential growth phase of B. subtilis, when the bacterium undergoes a complex genetic reprogramming. The putB knockout shortens significantly the window of opportunity for SPP1 infection during the host cell life cycle. This window is a critical parameter for competitive phage multiplication in the soil environment, where B. subtilis rarely meets conditions for exponential growth. Our results in combination with those reported for other virus-host systems suggest that bacterial viruses have evolved toward limited dependence on nonessential host functions., Importance: A successful viral infection largely depends on the ability of the virus to hijack cellular machineries and to redirect the flow of building blocks and energy resources toward viral progeny production. However, the specific virus-host interactions underlying this fundamental transformation are poorly understood. Here, we report on the first systematic analysis of virus-host cross talk during bacteriophage infection in Gram-positive bacteria. We show that lytic bacteriophage SPP1 is remarkably independent of nonessential genes of its host, Bacillus subtilis, with only a few cellular genes being necessary for efficient phage propagation. We hypothesize that such limited dependence of the virus on its host results from a constant "evolutionary arms race" and might be much more widespread than currently thought., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Icosahedral capsids of viruses are lattices of defined geometry and homogeneous size. The (quasi-)equivalent organization of their protein building blocks provides, in numerous systems, the binding sites to assemble arrays of viral polypeptides organized with nanometer precision that protrude from the capsid surface. The capsid of bacterial virus (bacteriophage) SPP1 exposes, at its surface, the 6.6-kDa viral polypeptide gp12 that binds to the center of hexamers of the major capsid protein. Gp12 forms an elongated trimer with collagen-like properties. This is consistent with the fold of eight internal GXY repeats of gp12 to build a stable intersubunit triple helix in a prokaryotic setting. The trimer dissociates and unfolds at near physiological temperatures, as reported for eukaryotic collagen. Its structural organization is reacquired within seconds upon cooling. Interaction with the SPP1 capsid hexamers strongly stabilizes gp12, increasing its Tm to 54 °C. Above this temperature, gp12 dissociates from its binding sites and unfolds reversibly. Multivalent binding of gp12 trimers to the capsid is highly cooperative. The capsid lattice also provides a platform to assist folding and association of unfolded gp12 polypeptides. The original physicochemical properties of gp12 offer a thermoswitchable system for multivalent binding of the polypeptide to the SPP1 capsid surface., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bacteriophage SPP1 is a nanomachine built to infect the bacterium Bacillus subtilis. The phage particle is composed of an icosahedric capsid, which contains the viral DNA, and a long non-contractile tail. Capsids and tails are produced in infected cells by two distinct morphogenetic pathways. Characterization of the suppressor-sensitive mutant SPP1sus82 showed that it produces DNA-filled capsids and tails but is unable to assemble complete virions. Its purified tails have a normal length but lack a narrow ring that tapers the tail end found at the tail-to-head interface. The mutant is defective in production of gp17. The gp17 ring is exposed in free tails competent for viral assembly but becomes shielded in the final virion structure. Recombinant gp17 is active in an in vitro assay to stick together capsids and tails present in extracts of SPP1sus82-infected cells, leading to formation of infectious particles. Gp17 thus plays a fundamental role in the tail-to-head joining reaction, the ultimate step of virus particle assembly. This is the conserved function of gp17 and its structurally related proteins like lambda gpU. This family of proteins can also provide fidelity to termination of the tail tube elongation reaction in a subset of phages including coliphage lambda., (© 2014 John Wiley & Sons Ltd.)

Tailed bacteriophages and herpesviruses package DNA inside the viral capsid by a powerful molecular motor. This packaging machine is composed of the portal protein, which provides a gate for DNA entry, the large terminase subunit whose ATPase activity fuels DNA translocation, and most frequently, a small terminase subunit that recognizes the viral packaging site. Here we review the mechanisms how the virulent Bacillus subtilis phage SPP1 packages DNA into a preformed procapsid. Encapsidation of the SPP1 DNA follows a processive unidirectional headful mechanism that starts with the recognition and cleavage of a unique genomic sequence (pac) by the viral terminase. The viral genome is then translocated through the central channel of the portal protein found at a single vertex of the procapsid. Packaging is terminated by an endonucleolytic cleavage of the concatemeric DNA substrate, following by disassembly of the packaging motor and closure of the portal system by the gatekeepers preventing leakage of the viral genome. Recent advances are providing new molecular insights on the mechanisms that ensure precise coordination of these critical steps required to accomplish the packaging encapsidation cycle., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The mechanism of genome transfer from the virion to the host cytoplasm is critical to understand and control the beginning of viral infection. The initial steps of bacteriophage SPP1 infection of the Gram-positive bacterium Bacillus subtilis were monitored by following changes in permeability of the cytoplasmic membrane (CM). SPP1 leads to a distinctively faster CM depolarization than the one caused by podovirus ϕ29 or myovirus SP01 during B. subtilis infection. Depolarization requires interaction of SPP1 infective virion to its receptor protein YueB. The amplitude of depolarization depends on phage input and concentration of YueB at the cell surface. Sub-millimolar concentrations of Ca(2+) are necessary and sufficient for SPP1 reversible binding to the host envelope and thus to trigger depolarization while DNA delivery to the cytoplasm depends on millimolar concentrations of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bacteriophages recognize and bind specific receptors to infect suitable hosts. Bacteriophage SPP1 targets at least two receptors of the Bacillus subtilis cell envelope, the glucosylated wall teichoic acids and the membrane protein YueB. Here, we identify a key virion protein for YueB binding and for the trigger of DNA ejection. Extracts from B. subtilis-infected cells applied to a YueB affinity matrix led to preferential capturing of gp21 from SPP1. To assess the significance of this interaction, we isolated mutant phages specifically affected in YueB binding. The mutants exhibited a very low inactivation rate and a strong defect to eject DNA when challenged with YueB. The phenotype correlated with presence of a single amino acid substitution in the gp21 carboxyl terminus, defining a region involved in YueB binding. Immunoelectron microscopy located the gp21 N-terminus in the SPP1 cap and probably in the adjacent tail spike region whereas the gp21 C-terminus was mapped further down in the spike structure. Antibodies against this part of gp21 interfered with the interaction of YueB with SPP1 and triggered DNA ejection. The gp21 C-terminal region thus plays a central role in two early key events that commit the virus to deliver its genome into host cells., (© 2011 Blackwell Publishing Ltd.)

Entry into the host bacterial cell is one of the least understood steps in the life cycle of bacteriophages. The different envelopes of Gram-negative and Gram-positive bacteria, with a fluid outer membrane and exposing a thick peptidoglycan wall to the environment respectively, impose distinct challenges for bacteriophage binding and (re)distribution on the bacterial surface. Here, infection of the Gram-positive rod-shaped bacterium Bacillus subtilis by bacteriophage SPP1 was monitored in space and time. We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB, which is encoded by a putative type VII secretion system gene cluster. YueB was found to concentrate at the cell poles and to display a punctate peripheral distribution along the sidewalls of B. subtilis cells. The kinetics of SPP1 DNA entry and replication were visualized during infection. Most of the infecting phages DNA entered and initiated replication near the cell poles. Altogether, our results reveal that the preferentially polar topology of SPP1 receptors on the surface of the host cell determines the site of phage DNA entry and subsequent replication, which occurs in discrete foci., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

SPP1 is a siphophage infecting the gram-positive bacterium Bacillus subtilis. The SPP1 tail electron microscopy (EM) reconstruction revealed that it is mainly constituted by conserved structural proteins such as the major tail proteins (gp17.1), the tape measure protein (gp18), the Distal tail protein (Dit, gp19.1), and the Tail associated lysin (gp21). A group of five small genes (22-24.1) follows in the genome but it remains to be elucidated whether their protein products belong or not to the tail. Noteworthy, an unassigned EM density accounting for ~245 kDa is present at the distal end of the SPP1 tail-tip. We report here the gp23.1 crystal structure at 1.6 A resolution, a protein that lacks sequence identity to any known protein. We found that gp23.1 forms a hexamer both in the crystal lattice and in solution as revealed by light scattering measurements. The gp23.1 hexamer does not fit well in the unassigned SPP1 tail-tip EM density and we hypothesize that this protein might act as a chaperone., (Copyright © 2010 The Protein Society.)

The majority of bacteriophages have a long non-contractile tail (Siphoviridae) that serves as a conduit for viral DNA traffic from the phage capsid to the host cell at the beginning of infection. The 160-nm-long tail tube of Bacillus subtilis bacteriophage SPP1 is shown to be composed of two major tail proteins (MTPs), gp17.1 and gp17.1*, at a ratio of about 3:1. They share a common amino-terminus, but the latter species has approximately 10 kDa more than gp17.1. A CCC.UAA sequence with overlapping proline codons at the 3' end of gene 17.1 drives a programmed translational frameshift to another open reading frame. The recoding event generates gp17.1*. Phages carrying exclusively gp17.1 or gp17.1* are viable, but tails are structurally distinct. gp17.1 and the carboxyl-terminus of gp17.1* have a distinct evolutionary history correlating with different functions: the polypeptide sequence identical in the two proteins is responsible for assembly of the tail tube while the additional module of gp17.1* shields the structure exterior exposed to the environment. The carboxyl-terminal extension is an elaboration present in some tailed bacteriophages. Different extensions were found to combine in a mosaic fashion with the MTP essential module in a subset of Siphoviridae genomes.

Tailed bacteriophage particles carry DNA highly pressurized inside the capsid. Challenge with their receptor promotes release of viral DNA. We show that addition of the osmolyte polyethylene glycol (PEG) has two distinct effects in bacteriophage SPP1 DNA ejection. One effect is to inhibit the trigger for DNA ejection. The other effect is to exert an osmotic pressure that controls the extent of DNA released in phages that initiate ejection. We carried out independent measurements of each effect, which is an essential requirement for their quantitative study. The fraction of phages that do not eject increased linearly with the external osmotic pressure. In the remaining phage particles ejection stopped after a defined amount of DNA was reached inside the capsid. Direct measurement of the size of non-ejected DNA by gel electrophoresis at different PEG concentrations in the latter sub-population allowed determination of the external osmotic pressure that balances the force powering DNA exit (47 atm for SPP1 wild-type). DNA exit stops when the ejection force mainly due to repulsion between DNA strands inside the SPP1 capsid equalizes the force resisting DNA insertion into the PEG solution. Considering the turgor pressure in the Bacillus subtilis cytoplasm the energy stored in the tight phage DNA packing is only sufficient to power entry of the first 17% of the SPP1 chromosome into the cell, the remaining 83% requiring application of additional force for internalization.

Gp7 is a minor capsid protein of the Bacillus subtilis bacteriophage SPP1. Homologous proteins are found in numerous phages but their function remained unknown. Deletion of gene 7 from the SPP1 genome yielded a mutant phage (SPP1del7) with reduced burst-size. SPP1del7 infections led to normal assembly of virus particles whose morphology, DNA and protein composition was undistinguishable from wild-type virions. However, only approximately 25% of the viral particles that lack gp7 were infectious. SPP1del7 particles caused a reduced depolarization of the B. subtilis membrane in infection assays suggesting a defect in virus genome traffic to the host cell. A higher number of SPP1del7 DNA ejection events led to abortive release of DNA to the culture medium when compared with wild-type infections. DNA ejection in vitro showed that no detectable gp7 is co-ejected with the SPP1 genome and that its presence in the virion correlated with anchoring of released DNA to the phage particle. The release of DNA from wild-type phages was slower than that from SPP1del7 suggesting that gp7 controls DNA exit from the virion. This feature is proposed to play a central role in supporting correct routing of the phage genome from the virion to the cell cytoplasm.

The irreversible binding of bacteriophages to their receptor(s) in the host cell surface triggers release of the naked genome from the virion followed by transit of viral DNA to the host cell cytoplasm. We have purified, for the first time, a receptor from a Gram-positive bacterium that is active to trigger viral DNA ejection in vitro. This extracellular region ("ectodomain") of the Bacillus subtilis protein YueB (YueB780) was a 7 S elongated dimer forming a 36.5-nm-long fiber. YueB780 bound to the tail tip of bacteriophage SPP1. Although a stable receptor-phage interaction occurred between 0 and 37 degrees C, complete blocking of phage DNA release or partial ejection events were observed at temperatures below 15 degrees C. We also showed that the receptor was exposed to the B. subtilis surface. YueB differed structurally from phage receptors from Gram-negative bacteria. Its properties revealed a fiber spanning the full length of the 30-nm-thick peptidoglycan layer. The fiber is predicted to be anchored in the cell membrane through transmembrane segments. These features, highly suitable for a virus receptor in Gram-positive bacteria, are very likely shared by a large number of phage receptors.

A large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection. These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices. The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient. The portal protein is an essential component of this DNA packaging machine. Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action. Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated. Functional regions of gp6 necessary at each step are identified within its primary structure. The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses.

An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.

The icosahedral procapsid of tailed bacteriophages is composed of a large number of identical subunits and of minor proteins found in a few copies. Proteins present in a very low copy number are targeted to the viral procapsid by an unknown mechanism. Bacteriophage SPP1 procapsids and mature virions contain two copies of gp7 on average. Gp7 forms stable complexes with the SPP1 portal protein gp6. Deletion of the gp6 carboxyl-terminus and the mutation Y467-->C localized in the same region prevent gp6-gp7 complex formation. Gp7 binds double-stranded and single-stranded DNA. Gp6 competes for this interaction, and purified gp6-gp7 complexes do not bind DNA. Procapsid structures assembled in the absence of gp6 or carrying the mutant gp6 Y467-->C lack gp7. The gp6-gp7 interaction thus targets gp7 to the procapsid where the portal protein is localized asymmetrically at a single vertex of the icosahedral structure. The interaction between the two proteins is disrupted during viral assembly. Proteins homologous to gp6 and gp7 are coded by contiguous genes in a variety of phage genomes from Gram-positive bacteria, suggesting that the gp6-gp7 complex is widespread in this group of phages. Transient association with the portal protein, an essential component of tailed bacteriophages and herpes viruses, provides a novel strategy to target minor proteins to the virion structure that might be operative in a large number of viruses.

In tailed bacteriophages and herpes viruses, the viral DNA is packaged through the portal protein channel. Channel closure is essential to prevent DNA release after packaging. Here we present the connector structure from bacteriophage SPP1 using cryo-electron microscopy and single particle analysis. The multiprotein complex comprises the portal protein gp6 and the head completion proteins gp15 and gp16. Although we show that gp6 in the connector has a fold similar to that of the isolated portal protein, we observe conformational changes in the region of gp6 exposed to the DNA-packaging ATPase and to gp15. This reorganization does not cause closure of the channel. The connector channel traverses the full height of gp6 and gp15, but it is closed by gp16 at the bottom of the complex. Gp16 acts as a valve whose closure prevents DNA leakage, while its opening is required for DNA release upon interaction of the virus with its host.