Crystal structure of Bacillus subtilis SPP1 phage gp23.1, a putative chaperone

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 Å 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.     

Binding-induced stabilization and assembly of the phage P22 tail accessory factor gp4

To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (Tm 34 degrees C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1)12:gp(4)6 assembly intermediate, which is stably populated at 30 degrees C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1)12:gp(4)12 complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.     

Bacteriophage P22 tail accessory factor GP26 is a long triple-stranded coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.     

The phage tail tape measure protein,an inner membrane protein and a periplasmic chaperone play connected roles in the genome injection process of E. coli phage HK97

Phages play critical roles in the spread of virulence factors and control of bacterial populations through their predation of bacteria. An essential step in the phage lifecycle is genome entry, where the infecting phage must productively interact with the components of the bacterial cell envelope in order to transmit its genome out of the viral particle and into the host cell cytoplasm. In this study, we characterize this process for the Escherichia coli phage HK97. We have discovered that HK97 genome injection requires the activities of the inner membrane glucose transporter protein, PtsG, and the periplasmic chaperone, FkpA. The requirements for PtsG and FkpA are determined by the sequence of the phage tape measure protein (TMP). We also identify a region of the TMP that mediates inhibition of phage genome injection by the HK97 superinfection exclusion protein, gp15. This region of the TMP also determines the PtsG requirement, and we show that gp15‐mediated inhibition requires PtsG. Based on these data, we present a model for the in vivo genome injection process of phage HK97 and postulate a mechanism by which the inhibitory action of gp15 is reliant upon PtsG.     

Smooth specific phage adsorption: endorhamnosidase activity of tail parts of P22

The tail parts of phage P22 as well as the phage particles cleave the O-antigen of its host bacterium, $\text{Salmonella typhimurium}$. The cleavage is caused by specific breakage of α-rhamnosyl 1–3 galactose linkages. Thus the tail parts of this phage consist of an enzyme, endorhamnosidase. The enzyme was not detected in nonpermissible strain infected with an amber gene 9 mutant of P22. Head without tail parts gains infectivity only after incubation with the tail parts which carry this enzymatic activity.     

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.     

Absence of a protein constituent and occurrence of an oversized DNA genome in a high density mutant particle of phage P22

Summary Mutants of P22 phage with abnormal density in CsCl solution (P22ndc phage) were analyzed in detail for this report. Two dimensional polyacrylamide gel electrophoresis revealed that wild-type P22ndc ⁺ phage virions contained a new protein (gpU) in addition to nine already identified proteins, while P22ndc lacked gpU. The molecular weight of gpU was essentially the same as that of gp5 (45 500), and one mature virion of phage P22ndc ¹ contained as many as 30–50 molecules of gpU. As P22ndc is a plaque-forming phage, gpU cannot be essential for the growth and assembly of P22 phage. Both genetical and biochemical analysis of the phage DNA in the virion revealed that P22ndc phage contained 2%–4% longer DNA than wild type P22ndc ⁺. A model is presented to account for the formation of P22ndc phage.     

A touch of glue to complete bacteriophage assembly: the tail‐to‐head joining protein (THJP) family

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.     

The flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi‐like protein tail fibre

The discovery of a Salmonella‐targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi‐like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi‐like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.     

Function of bacteriophage G7C esterase tailspike in host cell adsorption

Bacteriophages recognize and bind to their hosts with the help of receptor‐binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C – tailspikes gp63.1 and gp66 – are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N‐terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1‐gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1‐gp66 interaction is widespread among all three families of tailed bacteriophages.     

Structural plasticity of the phage P22 tail needle gp26 probed with xenon gas

The tail needle, gp26, is a highly stable homo‐trimeric fiber found in the tail apparatus of bacteriophage P22. In the mature virion, gp26 is responsible for plugging the DNA exit channel, and likely plays an important role in penetrating the host cell envelope. In this article, we have determined the 1.98 Å resolution crystal structure of gp26 bound to xenon gas. The structure led us to identify a calcium and a chloride ion intimately bound at the interior of α‐helical core, as well as seven small cavities occupied by xenon atoms. The two ions engage in buried polar interactions with gp26 side chains that provide specificity and register to gp26 helical core, thus enhancing its stability. Conversely, the distribution of xenon accessible cavities correlates well with the flexibility of the fiber observed in solution and in the crystal structure. We suggest that small internal cavities in gp26 between the helical core and the C‐terminal tip allow for flexible swinging of the latter, without affecting the overall stability of the protein. The C‐terminal tip may be important in scanning the bacterial surface in search of a cell‐envelope penetration site, or for recognition of a yet unidentified receptor on the surface of the host.     

Role of gene 10 protein in the hierarchical assembly of the bacteriophage P22 portal vertex structure

The portal vertex structure of the phage P22 is a 2.8 MDa molecular machine that mediates attachment and injection of the viral genome into the host Salmonella enterica serovar Typhimurium. Five proteins form this molecular machine: the portal protein, gp1; the tail-spike, gp9; the tail-needle, gp26, and the tail accessory factors, gp4 and gp10. In order to understand the assembly of the portal vertex structure, we have isolated the gene encoding tail accessory factor gp10 and defined its structural composition and assembly within the portal vertex structure. In solution, monomeric gp10 is a beta-sheet-rich protein with a stable conformational structure, which spontaneously assembles into hexamers, likely via a dimeric intermediate. This oligomerization enhances the structural stability of the protein, which then becomes competent for assembly to a preformed portal protein:gp4 complex, and acts as a structural adaptor bridging the nascent phage tail to gp26 and gp9. Notably, in vitro purified tail accessory factors gp4, gp10, and gp26 do not significantly interact with each other in solution, but their assembly takes place efficiently when these factors are added sequentially onto an immobilized portal protein. This suggests that the assembly of the P22 tail is a highly sequential and cooperative process, likely mediated by structural rearrangements in the assembly components. The assembled portal vertex structure represents both a membrane-binding and penetrating device as well as a plug that retains the pressurized phage DNA inside the capsid.     

A novel nucleotide kinase encoded by gene 1.7 of bacteriophage T7

Gene 1.7 of bacteriophage T7 confers sensitivity of both phage T7 and its host Escherichia coli to dideoxythymidine (ddT). We have purified the product of gene 1.7, gp1.7. It exists in two forms of molecular weight 22 181 and 17 782. Only the C‐terminal half of the protein is required to confer ddT sensitivity. We show that gp1.7 catalyses the phosphorylation of dGMP and dTMP to dGDP and dTDP, respectively, by using either GTP, dGTP or dTTP as the phosphate donor. Either form of gp1.7 exhibit identical kinase activity as compared with wild‐type gp1.7 that contains a mixture of both forms. The K_m of 70 µM and Kcat of 4.3 s^?1 for dTMP are similar to those found for E. coli thymidylate kinase. However, unlike the host enzyme, gp1.7 efficiently catalyses the conversion of the chain‐terminating dideoxythymidylate (ddTMP) to ddTDP. This finding explains the sensitivity of phage T7 but not E. coli to exogenous ddT. Gp1.7 is unusual in that it has no sequence homology to any known nucleotide kinase, it has no identifiable nucleotide‐binding motif and its activity is independent of added metal ions. When coupled with nucleoside diphosphate kinase, gp1.7 exponentially converts dTMP to dTTP.     

Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

Bacteriophages engage in complex dynamic interactions with their bacterial hosts and with each other. Bacteria have numerous mechanisms to resist phage infection, and phages must co‐evolve by overcoming bacterial resistance or by choosing an alternative host. Phages also compete with each other, both during lysogeny by prophage‐mediated defense against viral attack and by superinfection exclusion during lytic replication. Phages are enormously diverse genetically and are replete with small genes of unknown function, many of which are not required for lytic growth, but which may modulate these bacteria–phage and phage–phage dynamics. Using cellular toxicity of phage gene overexpression as an assay, we identified the 93‐residue protein gp52 encoded by Cluster F mycobacteriophage Fruitloop. The toxicity of Fruitloop gp52 overexpression results from interaction with and inactivation of Wag31 (DivIVA), an essential Mycobacterium smegmatis protein organizing cell wall biosynthesis at the growing cellular poles. Fruitloop gene 52 is expressed early in lytic growth and is not required for normal Fruitloop lytic replication but interferes with Subcluster B2 phages such as Hedgerow and Rosebush. We conclude that Hedgerow and Rosebush are Wag31‐dependent phages and that Fruitloop gp52 confers heterotypic superinfection exclusion by inactivating Wag31.     

Quantitative characterization of quantum dot‐labeled lambda phage for Escherichia coli detection

We characterize CdSe/ZnS quantum dot (QD) binding to genetically modified bacteriophage as a model for bacterial detection. Interactions among QDs, lambda (λ) phage, and Escherichia coli are examined by several cross‐validated methods. Flow and image‐based cytometry clarify fluorescent labeling of bacteria, with image‐based cytometry additionally reporting the number of decorated phage bound to cells. Transmission electron microscopy, image‐based cytometry, and electrospray differential mobility analysis allow quantization of QDs attached to each phage (4–17 QDs) and show that λ phage used in this study exhibits enhanced QD binding to the capsid by nearly a factor of four compared to bacteriophage T7. Additionally, the characterization methodology presented can be applied to the quantitative characterization of other fluorescent nanocrystal‐biological conjugates. Biotechnol. Bioeng. 2009;104: 1059–1067. Published 2009 Wiley Periodicals, Inc.     

Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity

A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1–150] and the lysozyme (aa 401–624) domains but not the linker domain (aa 151–400) caused efficient lysis of S . aureus . Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Kinetics of filamentous phage assembly

Filamentous phages release their progeny particles by a secretory process without lysing the bacterial cell. By this process about 6 viral particles per min are secreted from each cell. We show here that when the major coat protein (gp8) is provided from a plasmid we observe a phage progeny production rate depending on the induction of gp8 by IPTG. We also show that a transfection of Escherichia coli lacking F-pili is observed using a mutant of M13 that carries an ampicillin resistance gene, and phage particles are secreted in the absence of an F-plasmid. Extruding phage was visualized by atomic force microscopy (AFM) and by transmission electron microscopy (TEM) using gold-labeled antibodies to the major coat protein.     

An Evolutionarily Conserved Family of Virion Tail Needles Related to Bacteriophage P22 gp26: Correlation between Structural Stability and Length of the α-Helical Trimeric Coiled Coil

Bacteriophages of the Podoviridae family use short noncontractile tails to inject their genetic material into Gram-negative bacteria. In phage P22, the tail contains a thin needle, encoded by the phage gene 26, which is essential both for stabilization and for ejection of the packaged viral genome. Bioinformatic analysis of the N-terminal domain of gp26 (residues 1-60) led us to identify a family of genes encoding putative homologues of the tail needle gp26. To validate this idea experimentally and to explore their diversity, we cloned the gp26-like gene from phages HK620, Sf6 and HS1, and characterized these gene products in solution. All gp26-like factors contain an elongated α-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240 to 300 Å. gp26 tail needles display a high level of structural stability in solution, with T_m (temperature of melting) between 85 and 95 °C. To determine how the structural stability of these phage fibers correlates with the length of the α-helical core, we investigated the effect of insertions and deletions in the helical core. In the P22 tail needle, we identified an 85-residue-long helical domain, termed MiCRU (minimal coiled-coil repeat unit), that can be inserted in-frame inside the gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of the HS1 tail needle, minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases nonlinearly with the length of the α-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the α-helical core.     

The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein

The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified.     

Domain organization and polarity of tail needle GP26 in the portal vertex structure of bacteriophage P22

The attachment of tailed bacteriophages to the host cell wall as well as the penetration and injection of the viral genome into the host is mediated by the virion tail complex. In phage P22, a member of the Podoviridae family that infects Salmonella enterica, the tail contains an approximately 220 A elongated protein needle, previously identified as tail accessory factor gp26. Together with tail factors gp4 and gp10, gp26 is critical to close the portal protein channel and retain the viral DNA inside the capsid. By virtue of its topology and position in the virion, the tail needle gp26 is thought to function as a penetrating device to perforate the Salmonella cell wall. Here, we define the domain organization of gp26, characterize the structural determinants for its stability, and define the polarity of the gp26 assembly into the phage portal vertex structure. We have found that the N-terminal 27 residues of gp26 form a functional domain that, although not required for gp26 trimerization and overall stability, is essential for the correct attachment to gp10, which is thought to plug the portal vertex structure. The region downstream of domain I, domain II, folds into helical core, which exhibits four trimerization octad repeats with consensus Ile-xx-Leu-xxx-Val/Tyr. We demonstrate that in vitro, domain II represents the main self-assembling, highly stable trimerization core of gp26, which retains a folded conformation both in an anhydrous environment and in the presence of 10% SDS. The C terminus of gp26, immediately downstream of domain II, contains a beta-sheet-rich region, domain III, and a short coiled coil, domain IV, which, although not required for gp26 trimerization, enhance its thermodynamic stability. We propose that domains III and IV of the tail needle form the tip utilized by the phage to penetrate the host cell wall.