Lemon-shaped halo archaeal virus His1 with uniform tail but variable capsid structure

Lemon-shaped viruses are common in nature but so far have been observed to infect only archaea. Due to their unusual shape, the structures of these viruses are challenging to study and therefore poorly characterized. Here, we have studied haloarchaeal virus His1 using cryo-electron tomography as well as biochemical dissociation. The virions have different sizes, but prove to be extremely stable under various biochemical treatments. Subtomogram averaging of the computationally extracted virions resolved a tail-like structure with a central tail hub density and six tail spikes. Inside the tail there are two cavities and a plug density that separates the tail hub from the interior genome. His1 most likely uses the tail spikes to anchor to host cells and the tail hub to eject the genome, analogous to classic tailed bacteriophages. Upon biochemical treatment that releases the genome, the lemon-shaped virion transforms into an empty tube. Such a dramatic transformation demonstrates that the capsid proteins are capable of undergoing substantial quaternary structural changes, which may occur at different stages of the virus life cycle.There seems to be only a limited number of different virus particle architectures (virion-based structural lineages) due to the limited protein-fold space (1–3). Examples of the uncommon architectures are spindle-, bottle-, and droplet-shaped virions, so far found only in archaeal viruses (4). Archaea, organisms forming the third domain of cellular life, are known to thrive in both moderate and extreme environments (5–7). Interestingly, archaeal viruses are morphologically diverse, resembling eukaryotic viruses in this respect (4). Of these morphotypes, the spindle-shaped (also known as lemon-shaped) viruses are the most common ones in archaea-dominated habitats (8–15). It appears that this architecture is unique. However, deeper structural and biochemical analyses are needed to confirm this claim.Lemon-shaped virions are wider in the middle and narrow toward the ends. Three types of such viruses have been described based on the virion appearance: (i) those with one very short tail, (ii) those with one long tail, and (iii) those having two long tails (16–18). However, based on the comparison of the structural proteins, it was recently proposed that all known lemon-shaped viruses could be classified into two evolutionary lineages or viral families: Fuselloviridae, containing type i viruses, and Bicaudaviridae, containing type ii and iii viruses (19). Most of the isolated lemon-shaped viruses belong to the family Fuselloviridae, whose type species is the Sulfolobus spindle-shaped virus 1 (SSV1). Fuselloviruses infect hyperthermophilic crenarchaea and have one short tail with tail fibers (4, 20). The physical properties of the studied spindle-shaped viruses have recently been summarized (21). However, 3D structural understanding of lemon-shaped viruses remains limited.His1 is the only high-salinity lemon-shaped virus isolate, and it infects an extremely halophilic euryarchaeon, Haloarcula hispanica, and morphologically resembles fuselloviruses (22). However, unlike fuselloviruses, which have a circular double-stranded DNA (dsDNA) genome, His1 has a linear dsDNA genome encoding a putative type-B DNA polymerase; consequently, His1 has been classified in the floating genus Salterprovirus (20, 23). The 14,462-bp genome of His1 is predicted to have 35 ORFs, and 4 of these have been shown to encode structural proteins of the virion (21, 23). His1 virion contains one major capsid protein (MCP), VP21. In addition, a few minor ones have been detected: VP11, VP26, and VP27. Interestingly, VP21 exists in two forms. One form is lipid modified, although there is no detectable lipid bilayer in the His1 virion (21). The His1 MCP is 47% similar to the MCP of SSV1, indicating that His1 and fuselloviruses may share a common ancestor, and it has been proposed that His1 could be classified into the family Fuselloviridae (19, 21). In addition to the DNA polymerase, the His1 genome is predicted to encode an ATPase and a glycosyltransferase (21, 23). His1, like the other lemon-shaped viruses, is nonlytic, and many of them also encode an integrase (16, 17, 21, 24–26). Despite its typically hypersaline environment, His1 tolerates a variety of salinities, from 50 mM up to 4 M NaCl (21).Few attempts have been made to determine the structures of nonicosahedral viruses, mainly due to the fact that their pleomorphic nature introduces great challenges in particle structure classification and averaging. Recent advances in image-processing methods in cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) have been demonstrated to be a useful tool in the structural studies of nonicosahedral viruses, such as Tula hantavirus (27) and the immature capsid in HIV-1 virus (28), and some archaeal viruses, such as pleomorphic, two-tailed spindle-shaped, and linear viruses (17, 21, 29) and bottle-shaped and filamentous archaeal viruses (30–32). Recent structural studies of the spindle-shaped virus SSV1 revealed its 3D structure with a spindle body and a short tail at one end (33).Here, we used cryo-ET and symmetry-free and model-free subtomogram averaging to reveal the previously unrecognized tail organization of the lemon-shaped virus His1, with a central tail hub and six surrounding tail spikes. Further analysis of a larger population of subtomograms showed variable dimensions of the lemon particles but a constant structure of the tail. Biochemical analysis of the virion under different chemical conditions revealed unexpected biochemical properties of the His1 virion, which may be relevant to the life cycle of this virus.     

Structural rearrangements in the phage head-to-tail interface during assembly and infection

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal–phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein–DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein–protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere.The dsDNA bacterial viruses (phages or bacteriophages) and herpes viruses keep their genetic information packed at high pressure inside an icosahedral protein capsid. During virus particle assembly the genome is translocated into a prebuilt procapsid through a specialized portal vertex of the capsid (1, 2). Termination of the DNA packaging reaction is coordinated with closure of the portal system to avoid leakage of the viral genome. The outflow of DNA is prevented by conformational changes in the portal protein and binding of head completion proteins building the viral genome gatekeeper (3). In bacteriophages, the resultant complex [connector (4)] provides the connection point for the tail. The head-to-tail interface (HTI), or neck, is composed of the connector and of the tail-completion protein(s) found between the connector and the helical tail tube (Fig. S1A) (3, 5). Phage tails are responsible for host cell recognition and delivery of the viral genome to the host cytoplasm (6). At the beginning of viral infection the phage adsorption apparatus, located at the tail end distal from the capsid, binds to the host receptor, generating a signal that triggers opening of the neck (7). DNA then moves through the tail tube to enter the host cell. That tailed bacteriophages are the most abundant biological entities on Earth indicates the evolutionary advantage of this strategy for infecting bacterial cells. Infection by these viruses plays a central role in microbial ecosystems dynamics and in the horizontal transmission of genetic information within the bacterial world (8).Bacillus subtilis tailed bacteriophage SPP1 is a paradigm for viruses with a portal system (9). The viral particle is composed of an isometric icosahedral capsid ∼60 nm in diameter, shielding the 45.9-kbp-long viral chromosome (10). The portal protein gp6 (57.3 kDa subunit mass) is incorporated at a single vertex of the procapsid as a circular oligomer with a central channel that serves as a conduit for DNA passage (11). The portal vertex acts as a platform for the assembly of the viral DNA-translocating motor (12). Termination of DNA packaging is coordinated with disassembly of the motor and binding of gp15 subunits (11.6 kDa) to gp6, extending the portal channel that is closed underneath by the gp16 protein (12.5 kDa) (Fig. S1A) (13). The assembled complex represents the 180-Å-high connector that consists of three stacked cyclical homo-oligomers, each composed of 12 subunits of the portal protein gp6, of the adaptor gp15, and of the stopper gp16 (4, 13). Gp16 operates as a docking platform for the SPP1 preassembled tail tapered by the tail-to-head joining protein gp17 (15 kDa) (14, 15). Binding of the flexible 1,600-Å-long helical tail to the connector completes the formation of the HTI (7, 16). The capsid-distal region of the tail features an adsorption apparatus. Binding of this apparatus to the host cell receptor YueB (17, 18) triggers a domino-like cascade of conformational changes within the gp17.1/gp17.1* tail tube (7, 16, 19), signaling for opening of the gp16 stopper to initiate delivery of the SPP1 genome to the host cell.We report here subnanometer structures of the SPP1 HTI before and after DNA release obtained by cryoEM and single-particle analysis. The EM structures were used for flexible docking of X-ray and NMR atomic models of protein components of the HTI, allowing the uncovering the network of protein–protein and protein–DNA interactions in the complete HTI. The follow-up structure-driven functional analysis unraveled the allosteric mechanism by which the gatekeeper system assembles to lock DNA inside the virion after the genome-packaging reaction. It also provided experimental evidence supporting a model in which reversible diaphragm-like motion is the mechanism that controls viral genome release from the HTI for delivery to the host cell.     

Recombinant transfer in the basic genome of Escherichia coli

An approximation to the ∼4-Mbp basic genome shared by 32 strains of Escherichia coli representing six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.The increasing availability of complete genome sequences of many different bacterial and archaeal species, as well as metagenomic sequencing of mixed populations from natural environments, has stimulated theoretical and computational approaches to understand mechanisms of speciation and how prokaryotic species should be defined (1–8). Much genome analysis and comparison has been at the level of gene content, identifying core genomes (the set of genes found in most or all genomes in a group) and the continually expanding pan-genome. Population genomics of Escherichia coli has been particularly well studied because of its long history in laboratory research and because many pathogenic strains have been isolated and completely sequenced (9–14). Proposed models of how related groups or species form and evolve include isolation by ecological niche (7–9, 11, 15), decreased homologous recombination as divergence between isolated populations increases (2–4, 8, 14, 16), and coevolving phage and bacterial populations (6).E. coli genomes are highly variable, containing an array of phage-related mobile elements integrated at many different sites (17), random insertions of multiple transposable elements (18), and idiosyncratic genome rearrangements that include inversions, translocations, duplications, and deletions. Although E. coli grows by binary cell division, genetic exchange by homologous recombination has come to be recognized as a significant factor in adaptation and genome evolution (9, 10, 19). Of particular interest has been the relative contribution to genome variability of random mutations (single base-pair differences referred to as SNPs) and replacement of genome regions by homologous recombination with fragments imported from other genomes (here referred to as recombinant transfers or transferred regions). Estimates of the rate, extent, and average lengths of recombinant transfers in the core genome vary widely, as do methods for detecting transferred regions and assessing their impact on phylogenetic relationships (12–14, 20, 21).In a previous comparison of complete genome sequences of the K-12 reference strain MG1655 and the reconstructed genome of the B strain of Delbrück and Luria referred to here as B-DL, we observed that SNPs are not randomly distributed among 3,620 perfectly matched pairs of coding sequences but rather have two distinct regimes: sharply decreasing numbers of genes having 0, 1, 2, or 3 SNPs, and an abrupt transition to a much broader exponential distribution in which decreasing numbers of genes contain increasing numbers of SNPs from 4 to 102 SNPs per gene (22). Genes in the two regimes of the distribution are interspersed in clusters of variable lengths throughout what we referred to as the basic genome, namely, the ∼4 Mbp shared by the two genomes after eliminating mobile elements. We speculated that genes having 0 to 3 SNPs may primarily have been inherited clonally from the last common ancestor, whereas genes comprising the exponential tail may primarily have been acquired by horizontal transfer from diverged members of the population.The current study was undertaken to extend these observations to a diverse set of 32 completely sequenced E. coli genomes and to analyze how SNP distributions in the basic genome change as a function of evolutionary divergence between the 496 pairs of strains in this set. We have taken a simpler approach than those of Touchon et al. (13), Didelot et al. (14), and McNally et al. (21), who previously analyzed multiple alignments of complete genomes of E. coli strains. The appreciably larger basic genome derived here is not restricted to protein-coding sequences and retains positional information.     

HNH proteins are a widespread component of phage DNA packaging machines

The genome packaging reactions of tailed bacteriophages and herpes viruses require the activity of a terminase enzyme, which is comprised of large and small subunits. Phage genomes are replicated as linear concatemers composed of multiple copies of the genome joined end to end. As the terminase enzyme packages the genome into the phage capsid, it cleaves the DNA into single genome-length units. In this work, we show that the phage HK97 HNH protein, gp74, is required for the specific endonuclease activity of HK97 terminase and is essential for phage head morphogenesis. HNH proteins are a very common family of proteins generally associated with nuclease activity that are found in all kingdoms of life. We show that the activity of gp74 in terminase-mediated cleavage of the phage cos site relies on the presence of an HNH motif active-site residue, and that the large subunit of HK97 terminase physically interacts with gp74. Bioinformatic analysis reveals that the role of HNH proteins in terminase function is widespread among long-tailed phages and is uniquely required for the activity of the Terminase_1 family of large terminase proteins.Tailed bacteriophages and herpes viruses package their large double-stranded DNA genomes into a preformed protein shell, known as the “prohead,” using terminase enzymes. In both types of viruses, the genome is synthesized as concatemers composed of multiple copies of the genome joined end to end. This concatemeric DNA is packaged into the prohead and cleaved into genome-length units by terminase in an ATP-dependent reaction. Phage terminases are composed of two proteins: the large subunit harbors an endonuclease domain and an ATPase that powers the DNA packaging reaction, and the small subunit mediates specific DNA-binding required for recognition of packaging sites in the phage genome. A variety of elegant structural and biophysical studies have recently provided insight into the molecular mechanisms of terminase function (1, 2). However, the factors that affect the action of terminase enzymes in vivo have been less well characterized.Terminase enzymes perform several functions. They specifically recognize and bind the viral genome, interact with the prohead, then drive the DNA into the head through the narrow entry channel formed by the portal protein that is positioned at a single vertex of the head. During this process terminases also cleave the viral DNA, either nonspecifically upon head filling or at a specific site known as “cos.” The efficient packaging of a phage genome in vivo may require phage-encoded cofactors in addition to the terminase enzyme. For example, Escherichia coli phage λ gpFI facilitates interaction of the terminase–DNA complex with proheads (3–6). A wide variety of phages appear to encode proteins with a function similar to λ gpFI (7). Additionally, the activity of Bacillus subtilis phage phi29 terminase requires a phage-encoded RNA molecule bound to its portal protein (8), and in vivo packaging of the E. coli phage T4 genome can only be completed with the participation of the phage-encoded endonuclease, gp49 (9). The general prevalence and importance of terminase cofactors is difficult to evaluate because few studies have addressed this issue.We recently reported that phage genomes often encode proteins possessing an HNH motif near their terminase genes (10). The HNH motif is ∼35 aa long, and is characterized by the presence of two highly conserved His residues and one Asn residue. These HNH motifs, as defined by the large (∼7,400-member) HNH Pfam (11) protein sequence family (PF01844), are often found in proteins that possess endonuclease activity, such as site-specific homing endonucleases (12, 13), colicins (14, 15), S pyocins (16), and restriction enzymes (17–19). HNH motif-containing proteins comprised of primarily an HNH motif as found in E. coli colicins, usually possess nonspecific endonuclease activity. Conversely, HNH motif-containing proteins may contain DNA-recognition domains in addition to the HNH motif and thus possess high sequence specificity, as found in the homing endonucleases.The frequent juxtaposition of HNH and phage terminase genes (10, 20) suggests a unique role for HNH proteins in the endonuclease and/or packaging activities of the terminases. To address this issue, we investigated the function of E. coli phage HK97 gp74, a 119-residue protein containing an HNH motif. The gene encoding gp74 is located at the extreme 3′ end of the mature linear HK97 genome, adjacent to the cos site. In both the lysogen and replicative form of the HK97 genome gene 74 is immediately adjacent to genes 1 and 2, which encode the small and large subunits of terminase (TerS and TerL), respectively. Whereas gp74 was previously found to possess endonuclease activity (10), its role in the HK97 replication cycle remained uncharacterized. In this study we used functional and bioinformatic analyses to investigate its function.     

From the Cover: Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans

The abundance, genetic diversity, and crucial ecological and evolutionary roles of marine phages have prompted a large number of metagenomic studies. However, obtaining a thorough understanding of marine phages has been hampered by the low number of phage isolates infecting major bacterial groups other than cyanophages and pelagiphages. Therefore, there is an urgent requirement for the isolation of phages that infect abundant marine bacterial groups. In this study, we isolated and characterized HMO-2011, a phage infecting a bacterium of the SAR116 clade, one of the most abundant marine bacterial lineages. HMO-2011, which infects “Candidatus Puniceispirillum marinum” strain IMCC1322, has an ∼55-kb dsDNA genome that harbors many genes with novel features rarely found in cultured organisms, including genes encoding a DNA polymerase with a partial DnaJ central domain and an atypical methanesulfonate monooxygenase. Furthermore, homologs of nearly all HMO-2011 genes were predominantly found in marine metagenomes rather than cultured organisms, suggesting the novelty of HMO-2011 and the prevalence of this phage type in the oceans. A significant number of the viral metagenome sequences obtained from the ocean surface were best assigned to the HMO-2011 genome. The number of reads assigned to HMO-2011 accounted for 10.3%–25.3% of the total reads assigned to viruses in seven viromes from the Pacific and Indian Oceans, making the HMO-2011 genome the most or second-most frequently assigned viral genome. Given its ability to infect the abundant SAR116 clade and its widespread distribution, Puniceispirillum phage HMO-2011 could be an important resource for marine virus research.Viruses are the most abundant biological entities in diverse marine environments, as revealed by electron microscopy, epifluorescence microscopy, and flow cytometry studies (1–3). The average number of virus-like particles in surface seawater is ∼10⁷ per mL, and it typically exceeds the number of prokaryotes by an order of magnitude. Marine viruses play an important role in nutrient cycles by mediating a significant proportion of bacterial mortality. This so-called “viral shunt” diverts organic matter from particulate forms to dissolved forms, influencing the overall biogeochemistry of various elements (4). Viruses affect the community composition and genetic diversity of marine organisms by selectively infecting susceptible hosts (5, 6), which also increases the genetic diversity of the viruses themselves through mechanisms such as antagonistic coevolution (6, 7).Recent metagenomic studies of the oceans have revealed the numerous novel genetic repertoires of marine viromes (8–14). However, most genome fragments in these viromes cannot be categorized into any known viral group (8, 9, 13, 14). Because most marine viruses are believed to be phages (15), a more thorough understanding of the ecological and evolutionary roles of marine viruses and a better interpretation of the rapidly increasing virome sequences require the isolation and genomic analysis of individual viruses, especially phages infecting diverse marine bacterioplankton.The importance of phage isolation is exemplified by studies on marine cyanophages and pelagiphages. The isolation and characterization of cyanophages have shown that they have auxiliary metabolic genes (16), including photosynthesis-related genes that are expressed during infection and modulate host metabolism toward successful infection (17–21). Cyanophages are important in the diversification of hosts and horizontal gene transfer and are extensively used to interpret marine metagenome sequences (6, 22, 23). Very recently, four pelagiphages, viruses infecting the SAR11 clade, were isolated (24). Pelagiphage genome sequences have been found to be crucial for the interpretation of viromes from the Pacific Ocean (24). However, there are many abundant marine bacterial groups, including SAR86, SAR116, and Bacteroidetes, for which few phages have been isolated or characterized. Given the poor assignment of virome sequences into specific viral groups and the presence of difficult-to-cultivate or unculturable bacterial groups in the ocean, cultivating these bacteria and isolating the phages infecting them are prerequisites for understanding phage diversity.The SAR116 clade is one of the most abundant groups of heterotrophic bacteria inhabiting the surface of the ocean. Since its initial discovery through the cloning of 16S rRNA genes from the Sargasso Sea (25), many culture-independent studies have shown that the SAR116 clade contributes significantly—more than 10% in some cases—to the bacterial assemblages of the marine euphotic zone (26–33). The genome sequences of two bacteria in this clade, HIMB100 and “Candidatus Puniceispirillum marinum” IMCC1322, have recently been reported (34, 35). Both strains have genes for proteorhodopsin-based photoheterotrophy, carbon monoxide dehydrogenase, and dimethylsulfoniopropionate demethylase, suggesting the diverse metabolic potential and biogeochemical importance of the SAR116 clade. Considering the widespread distribution and ecologically meaningful genome characteristics of this clade, their phages are likely to be abundant in marine environments and may provide useful references for functional and phylogenetic annotation of marine viromes.Here, we report the isolation and genomic characterization of HMO-2011, a phage that infects strain IMCC1322 of the SAR116 clade. The HMO-2011 genome harbors many novel genes, such as a gene encoding a DNA polymerase with a DnaJ central domain. The genome of HMO-2011 was a notable resource for the identification of unknown marine viral gene fragments, as HMO-2011 accounted for 10.3%–25.3% of viral metagenome reads assigned to viruses.     

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.DNA packaging is both a critical step in viral assembly and a unique model for understanding the physics of polymers under strong confinement. Before packaging, the DNA (∼6–60 µm long) forms a loose random coil of diameter ∼1–3 µm. After translocation into the viral prohead (∼50–100 nm in diameter), a ∼10,000-fold volume compaction is achieved. Packaging is driven by a powerful molecular motor that must work against the large forces resisting confinement arising from DNA bending, repulsion between DNA segments, and entropy loss (1–8).DNA packaging in bacteriophages phi29, lambda, and T4 has been directly measured via single-molecule manipulation with optical tweezers and the packaging motors have been shown to generate forces of >60 pN, among the highest known for biomotors, while translocating DNA at rates ranging from ∼100 bp (for phage phi29, which packages a 19.3-kbp genome into a 42 × 54-nm prohead shell) up to as high as ∼2,000 bp/s (for phage T4, which packages a 171-kbp genome into a 120 × 86-nm prohead) (9–15). The force resisting packaging rises steeply with prohead filling and has been proposed to play an important role in driving viral DNA ejection (16).Recently, a variety of theoretical models for viral DNA packaging have been proposed (3–5, 17–21). The simplest treat DNA as an elastic rod with repulsive self-interactions and assume that packaging is a quasistatic thermodynamic process, i.e., that the DNA is able to continuously relax to a free-energy minimum state (3–5, 19–21). The DNA arrangement is generally assumed to be an inverse spool with local hexagonal close packing between DNA segments, as suggested by electron microscopy and X-ray scattering studies (22, 23). Such models yield exact analytical predictions that reproduce many of the experimental trends, including the sharp rise in resistance during the latter stages of packaging (3–5, 20).Dynamic simulations, however, predict differing results. Depending on model and simulation protocol, some predict rapid equilibration into ordered spool or folded toroid conformations, whereas others predict nonequilibrium dynamics and disordered conformations (3, 6, 24–31). The packaged DNA conformation also depends on ionic conditions, capsid size and shape, and shape of the internal core structure found in some phages (6, 30). Notably, some electron microscopy studies have also been interpreted as suggesting ordered spooled conformations (22), whereas others have been interpreted as suggesting partly disordered conformations (29). Although some simulations predict nonequilibrium dynamics, several potential caveats are that (i) the DNA has been represented by coarse-grained polymer models with various approximations for physical interactions (6), (ii) the packaging rate used in the simulations is >10⁵ times higher than the measured packaging rate due to computational constraints (3, 26–28), and (iii) it has been pointed out by some authors that simulation timescale cannot be directly related to experimental timescale because of the use of coarse-grained models for DNA (25, 28). As noted in early modeling studies, the calculations based on quasistatic models may represent a lower bound on the required packaging forces due to dissipative dynamic losses (4). Whether nonequilibrium dynamics play a significant role in real systems has thus remained an important open question.     

Type VI secretion system sheaths as nanoparticles for antigen display

The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction. T6SS sheaths are cytoplasmic tubular structures composed of stable VipA-VipB (named for ClpV-interacting protein A and B) heterodimers. Here, the structure of the VipA/B sheath was exploited to generate immunogenic multivalent particles for vaccine delivery. Sheaths composed of VipB and VipA fused to an antigen of interest were purified from Vibrio cholerae or Escherichia coli and used for immunization. Sheaths displaying heterologous antigens generated better immune responses against the antigen and different IgG subclasses compared with soluble antigen alone. Moreover, antigen-specific antibodies raised against sheaths presenting Neisseria meningitidis factor H binding protein (fHbp) antigen were functional in a serum bactericidal assay. Our results demonstrate that multivalent nanoparticles based on the T6SS sheath represent a versatile scaffold for vaccine applications.The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between effector cells and target cells (1–4). It is conserved in 25% of Gram-negative bacteria, including Vibrio cholerae, Pseudomonas aeruginosa and Escherichia coli. The T6SS plays a crucial role in bacterial pathogenicity and symbiosis, targeting either eukaryotic cells or competitor bacterial cells (5). The assembled and functional T6SS apparatus has structural homology to bacteriophage T4 phage tail components and can be divided into two distinct assemblies: a contractile phage tail-like structure and a transmembrane complex (6). V. cholerae VipA and VipB (named for ClpV-interacting protein A and B) and orthologous proteins in other bacteria build within the cytosol of effector cells a tubular sheath structure that is anchored to the various layers of the cell envelope through its association with the T6SS transmembrane complex (7).VipA/B sheaths are composed of six protofilaments arranged as a right-handed six-start helix similar to early T4 tail sheaths (8). Each protofilament is formed by a VipA/B heterodimer, and the atomic-resolution structure of a native contracted V. cholerae sheath has been recently determined by cryo-electron microscopy (9). Stable expression of VipB in V. cholerae requires the presence of VipA, and VipA/B heterodimers can be recruited into assembled tubular sheath structures spontaneously (10, 11). Because both ends of VipA are exposed on the external surface of the sheath tubules, a C-terminal fusion of VipA protein with superfolded green fluorescent protein (sfGFP) is functional in T6SS sheath assembly and activity, as previously demonstrated (3).Because these tubular structures are assembled in cytoplasm and can be purified from bacteria (3), we explored the possibility that T6SS sheaths could be used as a new particle-based delivery system for vaccine antigens. It is thought that particulate structures used for vaccine formulations are efficiently targeted for uptake by antigen-presenting cells (APCs) and interact directly with antigen-specific B cells generating humoral responses (12). Although particulate protein antigens may be more resistant to degradation, they are eventually proteolytically processed, and the resulting peptides are presented by the major histocompatibility complex (MHC) class I and class II molecules in a process that leads to activation of CD4⁺ and CD8⁺ T-cell helper and effector responses. Examples of particulate vaccine delivery systems include lipid-based systems [emulsions, immune-stimulating complexes (ISCOMs), liposomes, virosomes], polymer-based structures (e.g., nano-/microparticles), and virus-like particles (VLPs), with each of these systems presenting their own spectrum of advantages and disadvantages for practical use as human immunogens (13).In this work, VipA/B sheaths displaying heterologous protein antigens on the surface were generated and tested as a particulate vaccine antigen delivery system. Our results show that sheath-like structures displaying different antigens were immunogenic and that antibodies elicited against one of these, the Neisseria meningitidis factor H binding protein (fHbp), were functional in a serum bactericidal assay. The T6SS antigen delivery system demonstrates potential as a multivalent particle to deliver one or more antigens simultaneously into the same antigen-presenting cell. Moreover, the use of heterologous VipA and VipB sheaths displaying a common antigen in sequential vaccine booster regimens minimizes immune responses against the delivery system itself and focuses the immune responses against the common antigen of interest.     

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

Contrasted coevolutionary dynamics between a bacterial pathogen and its bacteriophages

Many antagonistic interactions between hosts and their parasites result in coevolution. Although coevolution can drive diversity and specificity within species, it is not known whether coevolutionary dynamics differ among functionally similar species. We present evidence of coevolution within simple communities of Pseudomonas aeruginosa PAO1 and a panel of bacteriophages. Pathogen identity affected coevolutionary dynamics. For five of six phages tested, time-shift assays revealed temporal peaks in bacterial resistance and phage infectivity, consistent with frequency-dependent selection (Red Queen dynamics). Two of the six phages also imposed additional directional selection, resulting in strongly increased resistance ranges over the entire length of the experiment (ca. 60 generations). Cross-resistance to these two phages was very high, independent of the coevolutionary history of the bacteria. We suggest that coevolutionary dynamics are associated with the nature of the receptor used by the phage for infection. Our results shed light on the coevolutionary process in simple communities and have practical application in the control of bacterial pathogens through the evolutionary training of phages, increasing their virulence and efficacy as therapeutics or disinfectants.Many host–parasite associations coevolve, and patterns in this antagonistic interaction are influenced by biology and environment (1–3). In single-species host–parasite interactions, parasite genotypes show differences in their host ranges and specificities on host genotypes, providing the basis for such coevolution (4–8). Arms race dynamics (ARD) driven by directional selection favors a broader resistance range in the host against a greater number of parasite genotypes and an increased host range in the parasite allowing more host genotypes to be infected (2, 9). In contrast, fluctuating selection dynamics (FSD), in which there is no directional change in the evolution of the host resistance range, is governed by negative frequency-dependent selection, favoring hosts that resist the most frequently encountered parasite genotypes and parasites that infect the most common host genotypes (9–11). It has been suggested that ARD predominates during the initial stages of coevolution, when adaptations to the coevolving opponent are largely cost-free, whereas FDS is more significant at later stages, when attack/defense alleles accumulate in the genome and impose costs (12). Thus, when systems shift from ARD to FSD, dynamic coevolutionary equilibria may arise, with constant numbers of attack/defense alleles at the individual level (13) and the continuous frequency-dependent (re)cycling of alleles at the population level (14).Coevolutionary dynamics between hosts and parasites is increasingly investigated using experimental evolution (15) and more specifically time-shift assays (16–18), in which hosts or parasites from a given time point are compared with their counterparts from the past or future, enabling the examination of putative reciprocal adaptations (19, 20). Some of the prime experimental models are bacteria and their lytic phages, which exhibit rapid evolution and are amenable to time-shift tests. Recent study indicates that coevolving populations may exhibit either ARD or FSD (12, 21–24), and there is some evidence for the genetic mechanism involved [e.g., mutations at tail fiber genes (12, 25)] and for the expansion of the phage host range as coevolution proceeds (6). Most studies of bacteria–phage coevolution involve the model system Pseudomonas fluorescens SBW25 and its lytic phage ϕ2 (15). Although these studies are important for an in-depth understanding of this process, their restriction to a single host–parasite pair is unfortunate, given the immense diversity of bacteria and phages in both terrestrial and aquatic ecosystems (26–28) and the importance of many of these organisms in human health (29). Thus, the generality of previous results to other bacterial species and to different phages parasitizing a given bacterium remains an open question.Coevolution may be particularly important in an applied context, namely when predators or parasitic organisms are used for biocontrol (30). For instance, Conrad et al. (31) recently argued that a community perspective with its ecological and evolutionary underpinnings is needed to explore the usefulness of phages as antimicrobial agents to treat cystic fibrosis patients infected with several bacteria and notably strains of Pseudomonas aeruginosa, a congeneric to the model organism P. fluorescens. Different phages naturally have different host ranges (8, 32, 33), but whether phage taxonomic origin influences impacts on P. aeruginosa is not known. Despite the study of phage mixtures to control P. aeruginosa infections (34), the nature of bacterial cross-resistance to phages other than that with which the bacterium evolved has not been addressed.Previous studies are inconclusive regarding whether P. aeruginosa coevolves with bacteriophages (35, 36). Given the ubiquity of this model organism in natural habitats (37) and as a widespread pathogen in hospitals (38, 39), it is important to know whether phage parasitism influences P. aeruginosa population biology and adaptation, whether coevolution between these antagonists actually occurs, and, if so, whether there are general patterns shared by different phages. Here, we test coevolutionary dynamics and their consistency in a panel of lytic bacteriophages and their host P. aeruginosa PAO1. We allowed this bacterium to interact and potentially coevolve with each of six different phage isolates separately, four from the Podoviridae and two from the Myoviridae (Table S1), for a total of 10 serial transfers (∼60 generations). Using bacteria and phages isolated from different time points, we conducted time-shift assays of resistance to infer patterns of the coevolutionary process. Finally, to assess specificity, we performed a cross-resistance assay with evolved bacteria and the six ancestral phage isolates to compare resistance of the bacteria to their “own” phage and to “foreign” phages with which they had not coevolved.     

Identification of divergent type VI secretion effectors using a conserved chaperone domain

The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.Protein secretion systems play a pivotal role in bacterial interspecies interaction and virulence (1, 2). Of the known secretion systems in Gram-negative bacteria, the type VI secretion system (T6SS) enables bacteria to compete with both eukaryotic and prokaryotic species through delivery of toxic effectors (2–4). The T6SS is a multicomponent nanomachine analogous to the contractile bacteriophage tail (5). First characterized in Vibrio cholerae (6) and Pseudomonas aeruginosa (7), the T6SS has now been identified in ∼25% of Gram-negative bacteria, including many important pathogens (2, 8), and has been implicated as a critical factor in niche competition (9–11).The T6SS structure is composed of an Hcp inner tube, a VipAB outer sheath that wraps around the Hcp tube, a tip complex consisting of VgrG and PAAR proteins, and a membrane-bound baseplate (2, 4, 12). Sheath contraction drives the inner Hcp tube and the tip proteins, VgrG and PAAR, outward into the environment and neighboring cells (13, 14). The contracted sheath is then dissembled by an ATPase ClpV and recycled for another T6SS assembly and contraction event (12, 15, 16). Two essential T6SS baseplate components, VasF and VasK, are homologous to the DotU and IcmF proteins of the type IV secretion system (T4SS) in Legionella pneumophila (17).Bacteria often possess multiple copies of VgrG and PAAR genes that form the tip of T6SS, and deletion of VgrG and PAAR genes abolishes T6SS secretion (14). Some VgrG and PAAR proteins carry functional extension domains and thus act as secreted T6SS effectors, as exemplified by the VgrG1 actin cross-linking domain (6), VgrG3 lysozyme domain in V. cholerae (18, 19), and the nuclease domain of the PAAR protein RhsA in Dickeya dadantii (20). Known T6SS effectors can target a number of essential cellular components, including the actin and membrane of eukaryotic cells (18, 21, 22) and the cell wall, membrane, and DNA of bacterial cells (3, 18–20, 23, 24). Each antibacterial effector coexists with an antagonistic immunity protein that confers protection during T6SS-mediated attacks between sister cells (3, 18, 24). Interestingly, T6SS-mediated lethal attacks induce the generation of reactive oxygen species in the prey cells (25), similar to cells treated with antibiotics (26, 27).For non-VgrG/PAAR–related effectors, their translocation requires either binding to the inner tube Hcp proteins as chaperones or binding to the tip VgrG proteins (2, 14, 28). T6SS-dependent effectors can be experimentally identified by comparing the secretomes of WT and T6SS mutants (3, 29–31) and by screening for T6SS-encoded immunity proteins (18). Because known effectors lack a common secretion signal, bioinformatic identification of T6SS effectors is challenging. A heuristic approach based on the physical properties of effectors has been used to identify a superfamily of peptidoglycan-degrading effectors in bacteria (32). A recent study identified a common N-terminal motif in a number of T6SS effectors (31). However, this motif does not exist in the T6SS effector TseL in V. cholerae (18).In this study, we report that VC1417, the gene upstream of tseL, encodes a protein with a highly conserved domain, DUF4123. We show that VC1417 is required for TseL delivery and interacts with VgrG1 (VC1416) and TseL. Because of the genetic linkage of VC1417 and TseL and its importance for TseL secretion, we postulated that genes encoding the conserved DUF4123 domain proteins are generally located upstream of genes encoding putative T6SS effectors. Using the conserved domain sequence, we bioinformatically predicted a large family of effector proteins with diverse functions in Gram-negative bacteria. We validated our prediction by the identification and characterization of a new secreted effector TseC and its antagonistic immunity protein TsiC in Aeromonas hydrophila SSU. Our results demonstrate a new effective approach to identify T6SS effectors with highly divergent sequences.     

Generation of reactive oxygen species by lethal attacks from competing microbes

Whether antibiotics induce the production of reactive oxygen species (ROS) that contribute to cell death is an important yet controversial topic. Here, we report that lethal attacks from bacterial and viral species also result in ROS production in target cells. Using soxS as an ROS reporter, we found soxS was highly induced in Escherichia coli exposed to various forms of attacks mediated by the type VI secretion system (T6SS), P1vir phage, and polymyxin B. Using a fluorescence ROS probe, we found enhanced ROS levels correlate with induced soxS in E. coli expressing a toxic T6SS antibacterial effector and in E. coli treated with P1vir phage or polymyxin B. We conclude that both contact-dependent and contact-independent interactions with aggressive competing bacterial species and viruses can induce production of ROS in E. coli target cells.Microbial species exist predominantly in complex communities in the natural environment and animal hosts. To survive in a multispecies environment, bacteria have developed various strategies to compete with other species. For example, some bacteria can exert long-range inhibitory effects by secreting diffusible molecules, such as antibiotics, bacteriocins, and H₂O₂ (1), whereas others require direct cell-to-cell contact to kill nearby organisms (2, 3). One such contact-dependent inhibitory system is the type VI secretion system (T6SS), a protein translocating nanomachine expressed by many Gram-negative bacterial pathogens that can kill both bacterial and eukaryotic cells (3–5). Structurally analogous to an inverted bacteriophage tail, the T6SS delivers effectors into target cells by using a contractile sheath to propel an inner tube out of the producer cell and into nearby target cells. The inner tube (composed of Hcp protein) is thought to carry toxic effector proteins within its lumen or on its tip, which is decorated with VgrG and PAAR proteins (4, 6, 7). Given that some cells can detect T6SS attack but not suffer any measurable loss in viability (8, 9), it would seem that cell killing is likely due to the toxicity of effectors rather than membrane disruptions caused by insertion of the spear-like VgrG/PAAR/Hcp tube complex. T6SS-dependent effectors can attack a number of essential cellular targets, including the cell wall (10, 11), membranes (11, 12), and nucleic acids (13), and thus can mimic the actions of antibiotics and bacteriocins. As a model prey or target organism, Escherichia coli can be killed by the T6SS activities of a number of bacteria including Vibrio cholerae (14), Pseudomonas aeruginosa (10, 15), and Acinetobacter baylyi ADP1 (7).Collins and coworkers (16–18) have reported that antibiotic treatment of E. coli elicits the production of reactive oxygen species (ROS) resulting from a series of events involving perturbation of the central metabolic pathway, NADPH depletion, and the Fenton reaction. ROS can cause lethal damage to DNA, lipid, and proteins (19, 20) and thus can contribute to cell death in combination with the deleterious effects of antibiotics on their primary targets. The idea that antibiotics kill bacterial cells, in part, through the action of ROS has been supported by a number of follow-up studies (18, 21–23) but has also been challenged by others as a result of observations contradictory to a model where ROS is the sole mediator of antibiotic lethality (24–26). These observations include the fact that antibiotics kill under anaerobic conditions, oxidation of the hydroxyphenyl fluorescein fluorescence dye used to measure ROS levels is nonspecific, and the extracellular level of H₂O₂ is not elevated by antibiotic treatment (24, 26). To address these concerns, Dwyer et al. (27) used a panel of ROS-detection fluorescence dyes, a defined growth medium under stringent anaerobic conditions, and an in vivo H₂O₂ enzymatic assay to study the effects of antibiotics on cells. The results further support that antibiotics induce ROS generation, which contributes to the efficacy of antibiotics in addition to their primary lethal actions (18, 27, 28).     

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.As part of a virus life cycle, genetic information needs to be incorporated into the newly produced virus particles. Tailed bacteriophages, which probably form the largest biomass of the planet (1), and many eukaryotic viruses such as herpes viruses use powerful ATPase motors to achieve this (2). These motors generate forces as high as 80–100 pN and translocate DNA into a preformed prohead until a DNA condensate of near crystalline density fills the interior (3).The viral packaging motors share a common architecture with the ASCE (additional strand, conserved E) superfamily of multimeric ring ATPases that perform diverse functions such as chromosome segregation (helicases), protein remodeling (chaperones and proteasomes), and cargo transport (dyneins) (4). Although much has been learned about the mechanochemistry of these motors, little is known about how a functional motor is assembled and its activity is initiated. The packaging motors have the difficult task of precisely inserting the end of a viral genome into the capsid at the time of initiation.In a general virus assembly pathway shared by dsDNA viruses, assembly starts at a unique fivefold vertex of the prohead called the portal vertex, which is formed from 12 molecules of the portal protein (5). A protein shell assembles around a protein scaffold and later becomes an empty prohead after the scaffold leaves, or is degraded (6). In most dsDNA bacteriophages as well as herpes viruses a complex of two proteins, known as small and large “terminase” proteins, recognize a specific sequence of DNA in the concatemeric viral genome (e.g., cos site in phage λ and pac site in phage P22) and make a cut to create a dsDNA end (7, 8). The small terminase is responsible for binding to the cos or pac site, whereas the large terminase makes the cut. However, phage phi29 and adenoviruses do not require DNA cutting because the genome is a unit-length molecule with a covalently attached “terminal protein” at the ends (9). The large terminase, which is also an ATPase, then attaches to the protruding end of the portal and assembles into an oligomeric motor that translocates the DNA genome into the empty prohead through the ∼3.5-nm-diameter portal channel using energy from ATP hydrolysis (7, 8). After packaging one unit-length viral genome (headful packaging), the motor dissociates from the full head and the neck and tail proteins assemble on the portal to make an infectious virus.Bacteriophage T4 has been an important model for tailed bacteriophages as well as herpes viruses (10, 11). The T4 packaging motor, a pentamer of gp17 (70 kDa) (large terminase protein) assembled on the gp20 portal dodecamer (12) is the fastest (packaging rate up to ∼2,000 bp/s) of the viral packaging motors studied (13). Gp17 possesses all of the basic enzymatic activities necessary for generating a DNA-full head: ATPase, nuclease, and translocase (14, 15). An oligomeric small terminase protein, gp16, that forms 11-mer and 12-mer rings recognizes the viral genome in vivo, although it lacks strict sequence specificity and is dispensable for packaging in vitro (16). Cryo-EM reconstruction of the packaging motor in complex with the capsid portal, which we will call the “packaging machine,” shows a ring of five gp17 molecules assembled on the prohead portal into a pentameric configuration with the translocation groove facing the channel (12). An electrostatic force-driven translocation mechanism was proposed in which gp17 subunits alternate between the “tensed” (compact) and “relaxed” (extended) conformational states that is coupled to translocation of DNA in a piston-like fashion (12).Genetic and biochemical studies of several packaging systems have delineated the mechanisms of genome recognition and DNA cutting (17, 18). Structural studies (12, 16, 19) and single-molecule optical tweezers (3, 13) and fluorescence spectroscopy (20, 21) approaches have been used to dissect the mechanochemical steps of DNA translocation. However, the transient nature of DNA and protein interactions at the initiation stage, which involve insertion of the dsDNA end into the prohead and triggering of translocation, has been a major challenge (22). The dynamics of motor assembly, timescales of motor–DNA–portal interactions, and mechanism of initiation are poorly understood in any system.Here, we report a single-molecule fluorescence assay that allowed us to dissect packaging initiation starting from a dsDNA end, in real time, by the phage T4 DNA packaging machine. We reconstituted a fully functional minimal T4 packaging complex and imaged individual packaging machines in real time by total internal reflection fluorescence microscopy. Each machine carried out successive DNA translocations and the times for motor assembly and packaging initiation were quantified. Using this assay we found that packaging initiations occurred in bursts with long pauses in between. We discovered that packaging initiation shows unusual plasticity. It can occur through multiple pathways: motor assembly on the portal followed by interaction with DNA and, unexpectedly, direct interaction of DNA with the portal followed by recruitment of the motor subunits. Finally, subtle changes in the ATP binding Walker A P-loop residues that lower the rate of ATP hydrolysis lead to severe defects in packaging initiation. These results provided insights into the dynamics of interactions that lead to single-molecule encapsidation of DNA by a viral packaging machine.     

The NMR–Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope

Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ∼1 μm and a diameter of ∼7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6–16.7 Å and a tilt of 36.1–36.6° between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.Filamentous bacteriophage are long, thin, and semiflexible rod viruses that infect bacteria (1, 2). These large assemblies (∼15–35 MDa) contain a circular single-stranded (ss) DNA genome encapsulated in a protein shell. All filamentous phage have a similar life cycle and virion structure despite the relatively high number of strains, with DNA sequence homology varying from almost complete to very little. The unique phage properties make them ideal for a large range of applications such as phage display (3), DNA cloning and sequencing (4, 5), nanomaterial fabrication (6–8), and as drug-carrying nanomachines (9). In addition, filamentous viruses form a variety of liquid crystals driving the development of both theory and practice of soft-matter physics (10, 11). Filamentous viruses are also associated with various diseases, e.g., CTXϕ phage in cholera toxin (12) and Pf4 phage in cystic fibrosis (13).Phage belonging to the Ff family (M13, fd, f1) are F-pilus–specific viruses that share almost identical genomes and very similar structures. M13 is a 16-MDa virus having a diameter of ∼7 nm and a length of ∼1 μm. The capsid is composed of several thousand identical copies of a major coat protein subunit arranged in a helical array surrounding a core of a circular ssDNA. The major coat proteins constitute ∼85% of the total virion mass, the ssDNA ∼12%, and all other minor proteins (gp3, gp6, gp7, gp9) that are specific for infection and assembly constitute about 3% of the total virion mass (1, 14).Previous structural models for a small number of phages have been obtained by means of X-ray fiber diffraction (15–19), static solid-state NMR (20, 21), and cryo-EM (22). Structural models for the Ff family have been proposed based on the three methods; however, satisfactory resolution was only obtained for the Y21M mutant of the fd phage (17, 18, 21) (wt fd is related to M13 by one additional mutation, N12D). The only reported model for M13 (23) (no coordinates available) and models of fd-Y21M from different methods differ in detail (24) and lack accuracy in some structural details such as the N-terminus orientation, the nature of DNA–protein interactions, and sidechain interactions, which are the dominant packing elements of the capsid. The most recent model was built using a combination of static NMR and fiber diffraction (17).According to fiber diffraction, the symmetry of the Ff capsid is C₅S₂, also referred to as class I symmetry. That is, a fivefold rotation of the major coat protein subunit around the virion axis (pentamers) and an approximate 36° rotation relating two successive pentamers [in fd-Y21M a precise 36° rotation was reported; for fd, values of −33.23° (18) and −34.62° (22) were reported]. All studies report that the coat protein is mostly right-handed, curved, α-helical, with a flexible or disordered N terminus.Magic-angle spinning (MAS) solid-state NMR has become a popular tool for studying the structure and dynamics of biological molecules (25–27). The method can be implemented on a variety of systems from small peptides to macromolecular biological assemblies. Integrated approaches can be used to resolve structures of large assemblies (28, 29) and recently, the combination of MAS NMR data, cryo-EM, and Rosetta modeling resulted in a detailed atomic structure of the recombinant type III secretion system needle (30, 31). We have previously performed MAS NMR studies on both wt fd and M13 in a precipitated form (32–34). Their chemical shifts pointed to a single homogeneous capsid subunit that is mostly helical and curved with a mobile N terminus. NMR studies of the interactions between the capsid and the DNA reported on the subunit orientation with respect to the viral axis, and indicated that the C terminus undergoes electrostatic interactions with the DNA.In this study, we use homonuclear 2D ¹³C–¹³C correlation experiments on sparsely labeled M13 samples together with our prior backbone and sidechain resonance assignments of the M13 phage to acquire MAS NMR structure restraints. Using the CS-Rosetta fold-and-dock protocol (35) we derive an atomic detailed well-converged quaternary structural model of the intact M13 phage viral capsid. The specific bacteriophage symmetry produces four identical, repeating hydrophobic pockets for each subunit, resulting in tight subunit packing that stabilizes the phage assembly.