ABSTRACT Relatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of the Sinorhizobium meliloti -infecting T4 superfamily phage ΦM9. This phage and its close relative Rhizobium phage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-like S. meliloti phages of the ΦM12 group. Structurally, ΦM9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the ΦM9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. ΦM9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, ΦM9 and S. meliloti phage ΦM12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide. IMPORTANCE Despite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil bacterium-phage dynamics are genetic, molecular, and structural characterizations of these groups of phages. The T4 superfamily phages are among the most complex phages; they have large genomes packaged within an icosahedral head and a long, contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study of S. meliloti -infecting T4 superfamily phage ΦM9 provides new insight into the diversity of this family. The comparison of structure-related genes in both ΦM9 and S. meliloti -infecting T4 superfamily phage ΦM12, which comes from a completely different lineage of these phages, allows the identification of host infection-related factors.
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Conformational dynamics control assembly of an extremely long bacteriophage tail tube
Tail tube assembly is an essential step in the lifecycle of long-tailed bacteriophages. Limited structural and biophysical information has impeded an understanding of assembly and stability of their long, flexible tail tubes. The hyperthermophilic phage P74-26 is particularly intriguing as it has the longest tail of any known virus (nearly 1 μm) and is the most thermostable known phage. Here, we use structures of the P74-26 tail tube along with an in vitro system for studying tube assembly kinetics to propose the first molecular model for the tail tube assembly of long-tailed phages. Our high-resolution cryo-EM structure provides insight into how the P74-26 phage assembles through flexible loops that fit into neighboring rings through tight "ball-and-socket"-like interactions. Guided by this structure, and in combination with mutational, light scattering, and molecular dynamics simulations data, we propose a model for the assembly of conserved tube-like structures across phage and other entities possessing tail tube-like proteins. We propose that formation of a full ring promotes the adoption of a tube elongation-competent conformation among the flexible loops and their corresponding sockets, which is further stabilized by an adjacent ring. Tail assembly is controlled by the cooperative interaction of dynamic intraring and interring contacts. Given the structural conservation among tail tube proteins and tail-like structures, our model can explain the mechanism of high-fidelity assembly of long, stable tubes.
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- Award ID(s):
- 1817338
- PAR ID:
- 10476542
- Publisher / Repository:
- Journal of Biological Chemistry
- Date Published:
- Journal Name:
- Journal of Biological Chemistry
- Volume:
- 299
- Issue:
- 3
- ISSN:
- 0021-9258
- Page Range / eLocation ID:
- 103021
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Cingolani, Gino (Ed.)Background: Genome flow is a fundamental aspect of all biological systems. In viruses, it involves movement of nucleic acid genomes into and out of a proteinaceous capsid. Viruses must recover their newly replicated genomes into a protective capsid shell (packaging) and then safely re-introduce them into a new host (ejection) to initiate infection. While the mechanisms of DNA genome packaging in large icosahedral bacteriophages (phages) and viruses have been extensively investigated, the post-packaging mechanisms involving retention, positioning, and ejection of packaged genome are poorly understood. Aims: Using the tailed phage T4 as a model, we delineated the structural and assembly intermediates involved in transitioning a DNA-full head into an infectious virion particle, and then into a genome delivering supramolecular machine. These include intermediates of neck attachment, virion assembly, and genome release into E. coli. Methods: Various intermediates produced either by mutant phage infection or recombinant protein expression have been purified and biochemically characterized. Molecular genetic approaches were used to analyze the functional significance of amino acids involved in assembly. Structures of the purified particles were determined to near atomic resolution by cryo-electron microscopy and cryo-electron tomography. Results: Following termination of headful packaging, the pressurized T4 capsid containing tightly packed genome is sealed by the assembly of neck proteins gp13 and gp14. A dramatic conformational change in the portal dodecamer is evident, which expels the packaging motor while opening sites in portal’s “clip” domain exposed outside the capsid for binding the gp13 neck protein. Unexpectedly, we discovered that a host protein Hfq, a nucleic acid binding protein, plugs the neck structure. Hfq apparently helps to further stabilize the sealed head as it awaits tail attachment. After tail attachment, a genome end, likely the last packaged DNA, descends into the tail tube and precisely positions through interaction with an N-terminal DNA-binding motif of the tape measure protein (TMP) gp29. Six coiled-coil strands of TMP form the innermost tube of phage T4 tail, connected at the top end with DNA and at the bottom end with gp48 tube and baseplate. When the tail sheath contracts and the baseplate transform from hexagon to star shape, TMP pilots the genome to the tip of the tail tube, poised for delivery. Then, when the baseplate plug is opened fully, TMP is expelled by DNA pressure and remodels into a transmembrane channel and guides the genome to flow smoothly through the E. coli membrane envelope into the cytosol. Conclusion: Our studies describe the structural transitions of a complex and large myophage T4 in unprecedented detail. The mechanisms involve symmetry matches and mismatches, morphing, conformational transitions, and molecular remodeling that lead to genome retention, genome positioning, and genome release, precisely and efficiently.more » « less
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Abstract A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of
Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism. -
Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerS P76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerS P76-26 revealed that it forms a ring with a wide central pore and radially arrayed helix–turn–helix domains. The structure further showed that these helix–turn–helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerS P76-26 can bind DNA. Finally, the TerS P76-26 structure lacked the conserved C-terminal β-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal β-barrel domain is not required for TerS P76-26 to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerS P76-26 , revealing key differences between this thermophilic phage and its mesophilic counterparts.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.jbc.2023.103021
