Mature Phage Research Articles

How Viruses Enter Cells: A Story behind Bacteriophage T4

Viruses are the most abundant biological agents in nature that need to enter living cells to successfully replicate (1). Although cells are protected by membranes, viruses have developed elaborate mechanisms to overcome this defense. One of the most known and fascinating examples is bacteriophage T4, which infects Escherichia coli bacteria (2). The structure of this virus has been well investigated (2–6). It consists of three main parts that are assembled independently and then joined together to produce a mature phage (2).

Tiny Answers to Big Questions

Tiny Answers to Big Questions

Forced Bacteriophage Uncorking: Viral DNA Ejection Triggered by a Sensitive Mechanical Switch

The foremost event of bacteriophage infection is the ejection of genomic DNA into the host bacterium after virus binding to surface receptor sites. The DNA ejection triggering mechanism is yet largely unknown. We investigated the role of mechanical force in triggering DNA ejection of the T7 bacteriophage. Mature phages containing tightly packed 40 kbp dsDNA were studied. We employed atomic force microscopy (AFM) to visualize the topographical structure and mechanically-induced changes of the phage particles covalently attached to mica surface. The sample was gently tapped so as to prevent the dislodging or fracture of the T7 capsids. We found that tapping the capsid wall with an oscillating AFM cantilever triggered a rapid DNA ejection via the tail complex. The forces employed were calculated to cause a very small (0.1-0.2%) change in the internal pressure of the capsid. Triggering rate increased exponentially as a function of force, following transition-state kinetics with an activation barrier of 23 kcal/mol at 1.2 nm along the reaction coordinate. The configuration of the ejected DNA molecule revealed that it had been exposed to a propulsive force. This propulsive force, which is thought to arise from intra-capsid pressure, assists in initiating the ejection process and the transfer of DNA across spatial dimensions beyond that of the virion. Furthermore, the internal pressure likely contributes to mechanically pre-loading the ejection machinery, thereby tilting the energy landscape towards DNA ejection; in this state a small additional, external force is sufficient to trigger the process with an apparently switch-like mechanism.

Crystal Structure of the Maturation Protein from Bacteriophage Qβ

Virions of the single-stranded RNA bacteriophages contain a single copy of the maturation protein, which is bound to the phage genome and is required for the infectivity of the particles. The maturation protein mediates the adsorption of the virion to bacterial pili and the subsequent release and penetration of the genome into the host cell. Here, we report a crystal structure of the maturation protein from bacteriophage Qβ. The protein has a bent, highly asymmetric shape and spans 110Å in length. Apart from small local substructures, the overall fold of the maturation protein does not resemble that of other known proteins. The protein is organized in two distinct regions, an α-helical part with a four-helix core, and a β stranded part that contains a seven-stranded sheet in the central part and a five-stranded sheet at the tip of the protein. The Qβ maturation protein has two distinct, positively charged areas at opposite sides of the α-helical part, which are involved in genomic RNA binding. The maturation protein binds to each of the surrounding coat protein dimers in the capsid differently, and the interaction is considerably weaker compared to coat protein interdimer contacts. The coat protein- or RNA-binding residues are not preserved among different ssRNA phage maturation proteins; instead, the distal end of the α-helical part is the most evolutionarily conserved, suggesting the importance of this region for maintaining the functionality of the protein.

Resolution and Probabilistic Structural Models of Subcomponents Derived from CryoEM Maps of Mature P22 Bacteriophage

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and thus it can be very useful to be able to estimate resolution in specific components. Also, measuring the uncertainty in structural models built based on density maps is becoming increasingly important especially at medium to low resolutions. Here, we present procedures to 1) estimate resolution in subcomponents by gold-standard FSC, 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting, and 3) build probabilistic models that merge high-accuracy priors (such as crystallographic structures) with medium resolution information (the cryoEM densities they are fitted to). The main goal of such procedures is to better understand what information and insights can be obtained from cryoEM density maps, particularly at medium resolution. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22 virion, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-stadard FSC show the highest accuracies are in the coat region (7.6A), while portal, hub, adhesin, and needle components are seen at lower resolutions (9.2A, 8.5A, 10.9A, and 10.5A respectively). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Using loop modeling and flexible fitting to augment and mold crystallographic models based on observed density, we then build probabilistic models of some of the subcomponents including the portal, hub, and adhesin proteins, leading to some new insights into the structure and composition of the P22 virion.

Genome Mining and Comparative Genomic Analysis of Five Coagulase- Negative Staphylococci (CNS) Isolated from Human Colon and Gall Bladder

Coagulase-negative Staphylococci (CNS) are known to cause distinct types of infections in humans like endocarditis and urinary tract infections (UTI). Surprisingly, there is a lack of genome analysis data in literature against CNS particularly of human origin. In light of this, we performed genome mining and comparative genomic analysis of CNS strains Staphylococcus cohnii subsp. cohnii strain GM22B2, Staphylococcus equorum subsp. strain equorum G8HB1, Staphylococcus pasteuri strain BAB3 isolated from gall bladder and Staphylococcus haemolyticus strain 1HT3, Staphylococcus warneri strain 1DB1 isolated from colon. We identified 29% of shared virulence determinants in the CNS strains which involved resistance to antibiotics and toxic compounds, bacteriocins and ribosomally synthesized peptides, adhesion, invasion, intracellular resistance, prophage regions, pathogenicity islands. 10 unique virulence factors involved in adhesion, negative transcriptional regulation, resistance to copper and cadmium, phage maturation were also present in our strains. Apart from comparing the genome homology, size and G + C content, we also showed the presence 10 different CRISPR-cas genes in the CNS strains. Further, KAAS based annotation revealed the presence of CNS genes in different pathways involved in human diseases. In conclusion, this study is a first attempt to unveil the pathogenomics of CNS isolated from two distinct body organs and highlights the importance of CNS as emerging pathogens of health care sector.

Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.

Viruses in bottom sediments in a eutrophic reservoir (Ivankovo Reservoir, Upper Volga)

The total number of viruses, morphological and size composition of viriobenthos, number of bacterial cells infected by viruses, number of mature phages inside bacterial cells, and virus-induced mortality of bacteriobenthos have been estimated in bottom sediments of the Ivankovo Reservoir. The total number of viriobenthos in the reservoir range from 10.3 to 83.2 (31.5 ± 6.2 on average) billion particles/cm3. The total number of virus-to-bacteria ratio in the Ivan’kovo reservoir varies between 0.3 and 1.7 (0.7 ± 0.1 on average). A positive correlation between viral and bacterial abundance is found (R = 0.61, p = 0.05). In different parts of the reservoir, viral infection of bacteria is not observed or is very low. The number of infected bacteria ranges from 1.0 to 3.5% (1.7 ± 0.2% on average) of the total viriobenthos abundance and mortality due to viral lysis accounts for 1.0–3.7% (1.8 ± 0.2% on average) of the total bacterial mortality. The number of phages found inside bacterial cells average 8 ± 1 phages/cell. Thus, a phenomenon known as “infection paradox” is observed in the Ivankovo Reservoir when, at a very high viral abundance, no viral infection of bacteria is recorded or it was very low.

The Protein Interactome of Mycobacteriophage Giles Predicts Functions for Unknown Proteins.

Mycobacteriophages are viruses that infect mycobacterial hosts and are prevalent in the environment. Nearly 700 mycobacteriophage genomes have been completely sequenced, revealing considerable diversity and genetic novelty. Here, we have determined the protein complement of mycobacteriophage Giles by mass spectrometry and mapped its genome-wide protein interactome to help elucidate the roles of its 77 predicted proteins, 50% of which have no known function. About 22,000 individual yeast two-hybrid (Y2H) tests with four different Y2H vectors, followed by filtering and retest screens, resulted in 324 reproducible protein-protein interactions, including 171 (136 nonredundant) high-confidence interactions. The complete set of high-confidence interactions among Giles proteins reveals new mechanistic details and predicts functions for unknown proteins. The Giles interactome is the first for any mycobacteriophage and one of just five known phage interactomes so far. Our results will help in understanding mycobacteriophage biology and aid in development of new genetic and therapeutic tools to understand Mycobacterium tuberculosis. Mycobacterium tuberculosis causes over 9 million new cases of tuberculosis each year. Mycobacteriophages, viruses of mycobacterial hosts, hold considerable potential to understand phage diversity, evolution, and mycobacterial biology, aiding in the development of therapeutic tools to control mycobacterial infections. The mycobacteriophage Giles protein-protein interaction network allows us to predict functions for unknown proteins and shed light on major biological processes in phage biology. For example, Giles gp76, a protein of unknown function, is found to associate with phage packaging and maturation. The functions of mycobacteriophage-derived proteins may suggest novel therapeutic approaches for tuberculosis. Our ORFeome clone set of Giles proteins and the interactome data will be useful resources for phage interactomics.

Impact of viruses on mortality of heterotrophic bacteria in rivers flowing through a large city (Cherepovets, Upper Volga)

The abundance and production of planktonic viruses, frequency of infected bacterial cells and number of mature phages in infected bacterial cells, and virus-induced mortality of bacterioplankton were studied in two small rivers and the large Sheksna River flowing through a large city in April–October, 2011. High positive correlations have been determined between the abundance of planktonic viruses and bacteria and the number of viruses attached to bacterial cell walls and the frequency of infected bacteria. The number of planktonic viruses and the mortality of bacteria caused by virus-induced lysis in hypertrophic and eutrophic small rivers are higher than in the mesotrophic Sheksna River.

Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation.

We report a novel, modular approach to immuno-detection based on antibody recognition and PCR read-out that employs antibody-conjugated bacteriophage and easily-manipulated non-pathogenic viruses as affinity agents. Our platform employs phage genetically tagged for in vivo biotinylation during phage maturation that can easily be linked, through avidin, to any biotinylated affinity agent, including full-length antibodies, peptides, lectins or aptamers. The presence of analyte is reported with high sensitivity through real-time PCR. This approach avoids the need to clone antibody-encoding DNA fragments, allows the use of full-length, high affinity antibodies and, by having DNA reporters naturally encapsulated inside the bacteriophage, greatly reduces nonspecific binding of DNA. We validate the efficacy of this new approach through the detection of Vascular Endothelial Growth Factor, a known angiogenic cancer biomarker protein, at attomolar concentrations in bronchoalveolar lavage fluid.

Transient Contacts on the Exterior of the HK97 Procapsid That Are Essential for Capsid Assembly

The G-loop is a 10-residue glycine-rich loop that protrudes from the surface of the mature bacteriophage HK97 capsid at the C-terminal end of the long backbone helix of major capsid protein subunits. The G-loop is essential for assembly, is conserved in related capsid and encapsulin proteins, and plays its role during HK97 capsid assembly by making crucial contacts between the hill-like hexamers and pentamers in precursor proheads. These contacts are not preserved in the flattened capsomers of the mature capsid. Aspartate 231 in each of the ~400 G-loops interacts with lysine 178 of the E-loop (extended loop) of a subunit on an adjacent capsomer. Mutations disrupting this interaction prevented correct assembly and, in some cases, induced abnormal assembly into tubes, or small, incomplete capsids. Assembly remained defective when D231 and K178 were replaced with larger charged residues or when their positions were exchanged. Second-site suppressors of lethal mutants containing substitution D231L replaced the ionic interaction with new interactions between neutral and hydrophobic residues of about the same size: D231L/K178V, D231L/K178I, and D231L/K178N. We conclude that it is not the charge but the size and shape of the side chains of residues 178 and 231 that are important. These two residues control the geometry of contacts between the E-loop and the G-loop, which apparently must be precisely spaced and oriented for correct assembly to occur. We present a model for how the G-loop could control HK97 assembly and identify G-loop-like protrusions in other capsid proteins that may play analogous roles.

Unravelling bacteriophage ϕ11 requirements for packaging and transfer of mobile genetic elements in Staphylococcus aureus

Bacteriophages play a major role in spreading mobile genetic elements (MGEs)-encoded genes among bacterial populations. In spite of this, the molecular requirements for building phage transducing particles have not been completely deciphered. Here, we systematically inactivated each ORF from the packaging and lysis modules of the staphylococcal phage ϕ11, used as a model for the Siphoviridae phages infecting Gram-positive bacteria, and determined their functional role in transferring different MGEs including plasmids, staphylococcal pathogenicity islands (SaPIs) and the phage itself. In a previous report, we identified seven of these ORFs as being required for the production of functional phage or SaPI particles. In this report, we have completed the mutational analysis and have identified and characterized 15 additional phage-encoded proteins required for the production of mature phage, SaPI, or transducing particles. Apart from these, we have not yet ascertained any specific function for the six remaining ϕ11 genes, though they are highly conserved among the staphylococcal bacteriophages. To the best of our knowledge, this study represents the first systematic deletion analysis of all the ORFs comprising the morphogenetic and lysis modules of a phage, clearly defining the molecular requirements involved in phage-mediated MGEs transfer.

Plate Tectonics of Virus Shell Assembly and Reorganization in Phage Φ8, a Distant Relative of Mammalian Reoviruses

SummaryThe hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity.

New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium

Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene from Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.

Popping the cork: mechanisms of phage genome ejection

Sixty years after Hershey and Chase showed that nucleic acid is the major component of phage particles that is ejected into cells, we still do not fully understand how the process occurs. Advances in electron microscopy have revealed the structure of the condensed DNA confined in a phage capsid, and the mechanisms and energetics of packaging a phage genome are beginning to be better understood. Condensing DNA subjects it to high osmotic pressure, which has been suggested to provide the driving force for its ejection during infection. However, forces internal to a phage capsid cannot, alone, cause complete genome ejection into cells. Here, we describe the structure of the DNA inside mature phages and summarize the current models of genome ejection, both in vitro and in vivo.

Identification and Characterization of Spontaneous Deletions within the Sp11-Sp12 Prophage Region of Escherichia coli O157:H7 Sakai

Prophages make up 12% of the enterohemorrhagic Escherichia coli genome and play prominent roles in the evolution and virulence of this food-borne pathogen. Acquisition and loss of and rearrangements within prophage regions are the primary causes of differences in pulsed-field gel electrophoresis (PFGE) patterns among strains of E. coli O157:H7. Sp11 and Sp12 are two tandemly integrated and putatively defective prophages carried by E. coli O157:H7 strain Sakai. In this study, we identified 3 classes of deletions that occur within the Sp11-Sp12 region, at a frequency of ca. 7.74 × 10(-4). One deletion resulted in a precise excision of Sp11, and the other two spanned the junction of Sp11 and Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE. We sequenced the inducible prophage pool of Sakai but did not identify any mature phage particles corresponding to either Sp11 or Sp12. Deletions containing pchB and psrC, which are Sp11-carried genes encoding proteins known or suspected to regulate type III secretion, did not affect the secretion levels of the EspA or EspB effector. Alignment of the Sp11-Sp12 DNA sequence with its corresponding regions in other E. coli O157:H7 and O55:H7 strains suggested that homologous recombination rather than integrase-mediated excision is the mechanism behind these deletions. Therefore, this study provides a mechanism behind the previously observed genetic instability of this genomic region of E. coli O157:H7.

The genome and proteome of a Campylobacter coli bacteriophage vB_CcoM-IBB_35 reveal unusual features

BackgroundCampylobacter is the leading cause of foodborne diseases worldwide. Bacteriophages (phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and thus considered an appealing option to control bacterial pathogens. Nevertheless for an effective use of phages as antimicrobial agents, it is important to understand phage biology which renders crucial the analysis of phage genomes and proteomes. The lack of sequence data from Campylobacter phages adds further importance to these studies.MethodsvB_CcoM-IBB_35 is a broad lytic spectrum Myoviridae Campylobacter phage with high potential for therapeutic use. The genome of this phage was obtained by pyrosequencing and the sequence data was further analyzed. The proteomic analysis was performed by SDS-PAGE and Mass spectrometry.Results and conclusionsThe DNA sequence data of vB_CcoM-IBB_35 consists of five contigs for a total of 172,065 bp with an average GC content of 27%. Attempts to close the gaps between contigs were unsuccessful since the DNA preparations appear to contain substances that inhibited Taq and ϕ29 polymerases. From the 210 identified ORFs, around 60% represent proteins that were not functionally assigned. Homology exists with members of the Teequatrovirinae namely for T4 proteins involved in morphogenesis, nucleotide metabolism, transcription, DNA replication and recombination. Tandem mass spectrometric analysis revealed 38 structural proteins as part of the mature phage particle.ConclusionsGenes encoding proteins involved in the carbohydrate metabolism along with several incidences of gene duplications, split genes with inteins and introns have been rarely found in other phage genomes yet are found in this phage. We identified the genes encoding for tail fibres and for the lytic cassette, this later, expressing enzymes for bacterial capsular polysaccharides (CPS) degradation, which has not been reported before for Campylobacter phages.

The Structure and Function of Highly Bent Toroidal DNA in Bacteriophage φ29

Tailed bacteriophages are highly efficient machines for infecting a host cell. A single virion must attach to the host surface, penetrate the cell wall, then release its double-stranded DNA genome. In these remarkable DNA delivery machines, controlling how and when the genome is released is a task of paramount importance. Recently, a three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of mature bacteriophage φ29 revealed an intriguing toroidal DNA structure contained in a small cavity below the viral capsid. This highly bent toroidal DNA supercoil is thought to be 30-40 basepairs of dsDNA and its function remains unknown.In this study, we employ an elastic rod model to simulate highly strained DNA as it is compressed within the protein cavity. The model provides estimates of force and energy required to form the toroid as well as its equilibrium conformation. Results reveal that a toroid can indeed form under the biologically relevant forces for the viral packing motor (up to 100 pN). To understand the effects of the highly stressed toroid on DNA structure, we then employ molecular dynamics (MD) simulations starting with the rod model-computed equilibrium as the initial condition. Following equilibration in MD, we construct an approximate density map using the final predicted toroid to compare with the recently published cryo-EM data. The computed density map correctly predicts the major dimensions of the toroid as well as regions of high and low density. Finally, we simulate the start of the ejection process by integrating the rod model forward in time upon sudden removal of the protein “tail knob.” The resulting fast dynamic collapse of the toroid suggests that its function could be to prime the ejection of the remaining viral genome.

Genomic and Proteomic Characterization of the Broad-Host-Range Salmonella Phage PVP-SE1: Creation of a New Phage Genus

(Bacterio)phage PVP-SE1, isolated from a German wastewater plant, presents a high potential value as a biocontrol agent and as a diagnostic tool, even compared to the well-studied typing phage Felix 01, due to its broad lytic spectrum against different Salmonella strains. Sequence analysis of its genome (145,964 bp) shows it to be terminally redundant and circularly permuted. Its G+C content, 45.6 mol%, is lower than that of its hosts (50 to 54 mol%). We found a total of 244 open reading frames (ORFs), representing 91.6% of the coding capacity of the genome. Approximately 46% of encoded proteins are unique to this phage, and 22.1% of the proteins could be functionally assigned. This myovirus encodes a large number of tRNAs (n=24), reflecting its lytic capacity and evolution through different hosts. Tandem mass spectrometric analysis using electron spray ionization revealed 25 structural proteins as part of the mature phage particle. The genome sequence was found to share homology with 140 proteins of the Escherichia coli bacteriophage rV5. Both phages are unrelated to any other known virus, which suggests that an "rV5-like virus" genus should be created within the Myoviridae to contain these two phages.