Tailspike Protein Research Articles

Combine thermal processing with polyvalent phage LPEK22 to prevent the Escherichia coli and Salmonella enterica contamination in food

Thermal processing is the most frequently used method to destruct bacteria in food processing. However, insufficient thermal processing may lead to the outbreak of foodborne illness. This study combined thermal processing with thermostable phage to prevent food contamination. The thermostable phages were screened which can retain activity at 70 °C for 1 h. Among them, the polyvalent phage LPEK22 was obtained to lyse Escherichia coli and Salmonella enterica, especially several multi-drug resistant bacteria. In milk (liquid food matrix), LPEK22 significantly reduced the E. coli by 5.00 ± 0.18 log10 CFU/mL and S. enterica by 4.20 ± 0.23 log10 CFU/mL after thermal processing at 63 °C for 30 min. For beef sausage (solid food matrix), LPEK22 significantly reduced the E. coli by 2.34 ± 0.17 log10 CFU/cm2 and S. enterica by 1.54 ± 0.13 log10 CFU/cm2 after thermal processing at 66 °C for 90 s. Genome analysis revealed that LPEK22 was a novel phage with a unique tail spike protein belonging to the family of Ackermannviridae. LPEK22 did not contain lysogenic, drug-resistant, and virulent genes that may compromise the safety of food application. These results determined that LPEK22, a novel polyvalent Ackermannviridae phage, could combine with thermal processing to prevent drug-resistant E. coli and S. enterica both in vitro and in foods.

Structural insights into a spindle-shaped archaeal virus with a sevenfold symmetrical tail

Archaeal viruses with a spindle-shaped virion are abundant and widespread in extremely diverse environments. However, efforts to obtain the high-resolution structure of a spindle-shaped virus have been unsuccessful. Here, we present the structure of SSV19, a spindle-shaped virus infecting the hyperthermophilic archaeon Sulfolobus sp. E11-6. Our near-atomic structure reveals an unusual sevenfold symmetrical virus tail consisting of the tailspike, nozzle, and adaptor proteins. The spindle-shaped capsid shell is formed by seven left-handed helical strands, constructed of the hydrophobic major capsid protein, emanating from the highly glycosylated tail assembly. Sliding between adjacent strands is responsible for the variation of a virion in size. Ultrathin sections of the SSV19-infected cells show that SSV19 virions adsorb to the host cell membrane through the tail after penetrating the S-layer. The tailspike harbors a putative endo-mannanase domain, which shares structural similarity to a Bacteroides thetaiotaomicro endo-mannanase. Molecules of glycerol dibiphytanyl glycerol tetraether lipid were observed in hydrophobic clefts between the tail and the capsid shell. The nozzle protein resembles the stem and clip domains of the portals of herpesviruses and bacteriophages, implying an evolutionary relationship among the archaeal, bacterial, and eukaryotic viruses.

Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice.

Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 μg/mouse) of depolymerase intraperitoneal injection protected 90%–100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.

Ultrasensitive and Selective Bacteria Sensors Based on Functionalized Graphene Transistors

We propose here a graphene biosensor based on the field-effect transistor (FET) architecture for continuous and real-time monitoring of bacteria, with beneficial features including facile operation, low-cost, selectivity, and high sensitivity. Our sensing device consists of the chemical-vapor-deposition (CVD) graphene monolayer, functionalized by the phage tail spike proteins (TSPs) that form specific binding sites to capture E. coli bacteria. We have investigated effects of surface functionalization and bacteria binding on the conductance of atomically thin graphene that determines transfer characteristics of a graphene FET (GFET). We have experimentally demonstrated that the concentration of E. coli bacteria can be selectively and accurately detected (at the single bacterium level) by a TSP-functionalized GFET. The proposed graphene biosensor may be of great interest for rapid, efficient detection of bacterial pathogens that could potentially pose a severe threat to human, animal, or plant health.

Bacteriophage Sf6 host range mutant that infects Shigella flexneri serotype 2a2 strains.

Shigella flexneri serotype 2a2 (II:9;10) is the most prevalent strain in causing bacillary dysentery in developing countries. Chemical modifications such as glucosylation, O-acetylation, and phosphoethanolamine modifications of lipopolysaccharide (LPS) O antigen (Oag) contribute to the emergence of various serotypes. Sf6 is a Shigella-specific bacteriophage that infects only a limited range of S. flexneri serotypes [X, Y]. LPS Oag is the primary receptor for bacteriophage Sf6 where it uses its tailspike protein (TSP) in binding and hydrolysing LPS Oags. Sf6TSP has recently been shown to be capable of hydrolysing the LPS Oag of Type II strains, albeit modestly. Phage therapy has regained attention in recent years as an alternative therapeutic approach. Therefore, this study aimed to expand the host range of Sf6 to the prevalent S. flexneri serotype 2a2 strain. We discovered a new lytic Sf6 host range mutant that is capable of infecting S. flexneri serotype 2a2 and identified residues in Sf6TSP that may potentially be involved in binding and hydrolysing serotype 2a2 LPS Oag. This work increased the limited Shigella-specific bacteriophage collection and may be useful in the future for phage therapy and/or biocontrolling of S. flexneri in contaminated food and water.

Structure of Escherichia coli O157:H7 bacteriophage CBA120 tailspike protein 4 baseplate anchor and tailspike assembly domains (TSP4-N)

Four tailspike proteins (TSP1-4) of Escherichia coli O157:H7 bacteriophage CBA120 enable infection of multiple hosts. They form a branched complex that attaches to the tail baseplate. Each TSP recognizes a different lipopolysaccharide on the membrane of a different bacterial host. The 335 N-terminal residues of TSP4 promote the assembly of the TSP complex and anchor it to the tail baseplate. The crystal structure of TSP4-N335 reveals a trimeric protein comprising four domains. The baseplate anchor domain (AD) contains an intertwined triple-stranded β-helix. The ensuing XD1, XD2 and XD3 β-sheet containing domains mediate the binding of TSP1-3 to TSP4. Each of the XD domains adopts the same fold as the respective XD domains of bacteriophage T4 gp10 baseplate protein, known to engage in protein–protein interactions via its XD2 and XD3 domains. The structural similarity suggests that XD2 and XD3 of TSP4 also function in protein–protein interactions. Analytical ultracentrifugation analyses of TSP4-N335 and of domain deletion proteins showed how TSP4-N335 promotes the formation of the TSP quaternary complex. TSP1 and TSP2 bind directly to TSP4 whereas TSP3 binding requires a pre-formed TSP4-N335:TSP2 complex. A 3-dimensional model of the bacteriophage CBA120 TSP complex has been developed based on the structural and ultracentrifuge information.

A Novel Phage Indirectly Regulates Diatom Growth by Infecting a Diatom-Associated Biofilm-Forming Bacterium.

Algae and heterotrophic bacteria have close and intricate interactions, which are regulated by multiple factors in the natural environment. Phages are the major factor determining bacterial mortality rates. However, their impacts on the alga-associated bacteria and thus on the alga-bacterium interactions are poorly understood. Here, we obtained a diatom-associated bacterium, Stappia indica SNL01, that could form a biofilm and had an inhibitory effect on the growth of the diatom Thalassiosira pseudonana. Meanwhile, phage SI01, with a double-stranded circular DNA genome (44,247 bp), infecting S. indica SNL01 was isolated. Phylogenetic analysis revealed that phage SI01 represents a novel member of the Podoviridae family. The phage contained multiple lysis genes encoding cell wall-lysing muramidase and spore cortex-lysing SleB, as well as depolymerase-like tail spike protein. By lysing the host bacterium and inhibiting the formation of biofilm, this phage could indirectly promote the growth of the diatom. Our results provide new insights into how phages indirectly regulate algal growth by infecting bacteria that are closely associated with algae or in the phycosphere. IMPORTANCE The impact of phage infection on the alga-bacterium relationship in the ocean is poorly understood. Here, a novel phage infecting the diatom-associated bacterium Stappia indica SNL01 was isolated. This bacterium could form a biofilm and had a negative effect on diatom growth. We revealed that this phage contained multiple lysis genes and could inhibit the formation of the bacterial biofilm, thus indirectly promoting diatom growth. This study suggests that phages not only are important regulators of bacteria but also have substantial indirect effects on algae and the alga-bacterium relationship.

Proteolytic Analysis of Epsilon 34 Phage Tailspike Protein Indicates Partial Sensitivity to Proteinase K

Proteolytic Analysis of Epsilon 34 Phage Tailspike Protein Indicates Partial Sensitivity to Proteinase K

Novel Phage-Derived Depolymerase with Activity against Proteus mirabilis Biofilms.

The adherence of Proteus mirabilis to the surface of urinary catheters leads to colonization and eventual blockage of the catheter lumen by unique crystalline biofilms produced by these opportunistic pathogens, making P. mirabilis one of the leading causes of catheter-associated urinary tract infections. The Proteus biofilms reduce efficiency of antibiotic-based treatment, which in turn increases the risk of antibiotic resistance development. Bacteriophages and their enzymes have recently become investigated as alternative treatment options. In this study, a novel Proteus bacteriophage (vB_PmiS_PM-CJR) was isolated from an environmental sample and fully characterized. The phage displayed depolymerase activity and the subsequent genome analysis revealed the presence of a pectate lyase domain in its tail spike protein. The protein was heterologously expressed and purified; the ability of the purified tail spike to degrade Proteus biofilms was tested. We showed that the application of the tail spike protein was able to reduce the adherence of bacterial biofilm to plastic pegs in a MBEC (minimum biofilm eradication concentration) assay and improve the survival of Galleria mellonella larvae infected with Proteus mirabilis. Our study is the first to successfully isolate and characterize a biofilm depolymerase from a Proteus phage, demonstrating the potential of this group of enzymes in treatment of Proteus infections.

Perturbation of ACE2 Structural Ensembles by SARS-CoV-2 Spike Protein Binding.

The human ACE2 enzyme serves as a critical first recognition point of coronaviruses, including SARS-CoV-2. In particular, the extracellular domain of ACE2 interacts directly with the S1 tailspike protein of the SARS-CoV-2 virion through a broad protein–protein interface. Although this interaction has been characterized by X-ray crystallography, these structures do not reveal significant differences in the ACE2 structure upon S1 protein binding. In this work, using several all-atom molecular dynamics simulations, we show persistent differences in the ACE2 structure upon binding. These differences are determined with the linear discriminant analysis (LDA) machine learning method and validated using independent training and testing datasets, including long trajectories generated by D. E. Shaw Research on the Anton 2 supercomputer. In addition, long trajectories for 78 potent ACE2-binding compounds, also generated by D. E. Shaw Research, were projected onto the LDA classification vector in order to determine whether the ligand-bound ACE2 structures were compatible with S1 protein binding. This allows us to predict which compounds are “apo-like” versus “complex-like” and to pinpoint long-range ligand-induced allosteric changes in the ACE2 structure.

Characterization and Food Application of the Novel Lytic Phage BECP10: Specifically Recognizes the O-polysaccharide of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a global concern that causes serious diseases, such as hemolytic uremic syndrome and bloody diarrhea. To control E. coli O157:H7 in food, a novel siphophage, BECP10, that targets the O157 serotype was isolated and characterized. Unlike other E. coli phages, BECP10 can only infect E. coli O157 strains, and thus, did not infect other strains. The 48 kbp genome of BECP10 contained 76 open reading frames (ORFs), including 33 putative functional ORFs. The phage did not contain lysogeny-related modules or toxin-associated genes, suggesting that the phage might be strictly lytic. The tail spike protein (TSP) sequence had very low homology with the reported T1-like phages, indicating that TSP might be related to this unique host spectrum. The specific O-antigen residue of E. coli O157:H7 may be a key factor for phage infection by adsorption and receptor identification. The phage exhibited strong antibacterial activity against E. coli O157:H7 over a broad pH range and showed little development of phage-insensitive mutants. The phage sustained viability on the burger patties and reduced E. coli O157:H7 to a non-detectable level without the emergence of resistant cells at low temperatures for five days. Therefore, phage BECP10 might be a good biocontrol agent for E. coli O157:H7-contaminated food matrices.

Phage φAB6-Borne Depolymerase Combats Acinetobacter baumannii Biofilm Formation and Infection.

Biofilm formation is one of the main causes of increased antibiotic resistance in Acinetobacter baumannii infections. Bacteriophages and their derivatives, such as tail proteins with depolymerase activity, have shown considerable potential as antibacterial or antivirulence agents against bacterial infections. Here, we gained insights into the activity of a capsular polysaccharide (CPS) depolymerase, derived from the tailspike protein (TSP) of φAB6 phage, to degrade A. baumannii biofilm in vitro. Recombinant TSP showed enzymatic activity and was able to significantly inhibit biofilm formation and degrade formed biofilms; as low as 0.78 ng, the inhibition zone can still be formed on the bacterial lawn. Additionally, TSP inhibited the colonization of A. baumannii on the surface of Foley catheter sections, indicating that it can be used to prevent the adhesion of A. baumannii to medical device surfaces. Transmission and scanning electron microscopy demonstrated membrane leakage of bacterial cells treated with TSP, resulting in cell death. The therapeutic effect of TSP in zebrafish was also evaluated and the results showed that the survival rate was significantly improved (80%) compared with that of the untreated control group (10%). Altogether, we show that TSP derived from φAB6 is expected to become a new antibiotic against multi-drug resistant A. baumannii and a biocontrol agent that prevents the formation of biofilms on medical devices.

Specific Interaction of Novel Friunavirus Phages Encoding Tailspike Depolymerases with Corresponding Acinetobacter baumannii Capsular Types.

Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organisation refers it to its «critical priority» category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-depolymerasing enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89 and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae The linear double-stranded DNA phage genomes of 41,105-42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K-units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that cleave specifically the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K-units.IMPORTANCE Acinetobacter baumannii, a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K-types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidate to the cocktail designed to control A. baumannii with different K-types.

Subtypes of tail spike proteins predicts the host range of Ackermannviridae phages

Phages belonging to the Ackermannviridae family encode up to four tail spike proteins (TSPs), each recognizing a specific receptor of their bacterial hosts. Here, we determined the TSPs diversity of 99 Ackermannviridae phages by performing a comprehensive in silico analysis. Based on sequence diversity, we assigned all TSPs into distinctive subtypes of TSP1, TSP2, TSP3 and TSP4, and found each TSP subtype to be specifically associated with the genera (Kuttervirus, Agtrevirus, Limestonevirus, Taipeivirus) of the Ackermannviridae family. Further analysis showed that the N-terminal XD1 and XD2 domains in TSP2 and TSP4, hinging the four TSPs together, are preserved. In contrast, the C-terminal receptor binding modules were only conserved within TSP subtypes, except for some Kuttervirus TSP1s and TSP3s that were similar to specific TSP4s. A conserved motif in TSP1, TSP3 and TSP4 of Kuttervirus phages may allow recombination between receptor binding modules, thus altering host recognition. The receptors for numerous uncharacterized phages expressing TSPs in the same subtypes were predicted using previous host range data. To validate our predictions, we experimentally determined the host recognition of three of the four TSPs expressed by kuttervirus S117. We confirmed that S117 TSP1 and TSP2 bind to their predicted host receptors, and identified the receptor for TSP3, which is shared by 51 other Kuttervirus phages. Kuttervirus phages were thus shown encode a vast genetic diversity of potentially exchangeable TSPs influencing host recognition. Overall, our study demonstrates that comprehensive in silico and host range analysis of TSPs can predict host recognition of Ackermannviridae phages.

Pantoea stewartii WceF is a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like fold

Pathogenic microorganisms often reside in glycan-based biofilms. Concentration and chain length distribution of these mostly anionic exopolysaccharides (EPS) determine the overall biophysical properties of a biofilm and result in a highly viscous environment. Bacterial communities regulate this biofilm state via intracellular small-molecule signaling to initiate EPS synthesis. Reorganization or degradation of this glycan matrix, however, requires the action of extracellular glycosidases. So far, these were mainly described for bacteriophages that must degrade biofilms for gaining access to host bacteria. The plant pathogen Pantoea stewartii (P. stewartii) encodes the protein WceF within its EPS synthesis cluster. WceF has homologs in various biofilm forming plant pathogens of the Erwinia family. In this work, we show that WceF is a glycosidase active on stewartan, the main P. stewartii EPS biofilm component. WceF has remarkable structural similarity with bacteriophage tailspike proteins (TSPs). Crystal structure analysis showed a native trimer of right-handed parallel β-helices. Despite its similar fold, WceF lacks the high stability found in bacteriophage TSPs. WceF is a stewartan hydrolase and produces oligosaccharides, corresponding to single stewartan repeat units. However, compared with a stewartan-specific glycan hydrolase of bacteriophage origin, WceF showed lectin-like autoagglutination with stewartan, resulting in notably slower EPS cleavage velocities. This emphasizes that the bacterial enzyme WceF has a role in P. stewartii biofilm glycan matrix reorganization clearly different from that of a bacteriophage exopolysaccharide depolymerase.

Crystal structure of the MyRF ICA domain with its upstream β-helical stalk reveals the molecular mechanisms underlying its trimerization and self-cleavage.

Myelin gene regulatory factor (MyRF), a novel membrane transcription factor expressed on the endoplasmic reticulum membrane, functions as a trimer. The trimerization of MyRF is associated with a fragment between the DNA binding domain and transmembrane domain that shares homology with the triple-β-helix and intramolecular chaperone autocleavage (ICA) domain of phage tailspike proteins. The molecular details of these domains in eukaryotes have not been elucidated. Here, we present the crystal structure of the MyRF ICA domain with its upstream β-helical stalk, determined at 2.4Å resolution. The structure showed that its upstream β-helical stalk is different from the triple β-helix reported before. This is the first structure of the mammalian protein with a triple β-helix. Structure analysis demonstrated that the triple α-helical coiled-coil formed at the MyRF ICA domain C-terminal was the main driving force for the trimerization. Additionally, our findings showed that MyRF was cleaved via a highly conserved serine-lysine catalytic dyad mechanism and that cleavage would be activated only if the ICA domains were organized as trimers. In contrast to the viral ICA domain, almost no interaction was found between the MyRF ICA domain and its upstream neighboring β-helix of the stalk; thus, activation of self-cleavage may not be triggered by the upstream region of the ICA domain, contrary to the observations made in phages. These findings provided an important insight into the molecular mechanisms of MyRF trimerization and self-cleavage.

Structural basis of superinfection exclusion by bacteriophage T4 Spackle

A bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages – a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.

Bacteriophage Tail-Spike Proteins Enable Detection of Pseudaminic-Acid-Coated Pathogenic Bacteria and Guide the Development of Antiglycan Antibodies with Cross-Species Antibacterial Activity.

Pseudaminic acid (Pse), a unique carbohydrate in surface-associated glycans of pathogenic bacteria, has pivotal roles in virulence. Owing to its significant antigenicity and absence in mammals, Pse is considered an attractive target for vaccination or antibody-based therapies against bacterial infections. However, a specific and universal probe for Pse, which could also be used in immunotherapy, has not been reported. In a prior study, we used a tail spike protein from a bacteriophage (ΦAB6TSP) that digests Pse-containing exopolysaccharide (EPS) from Acinetobacter baumannii strain 54149 (Ab-54149) to form a glycoconjugate for preparing anti-Ab-54149 EPS serum. We report here that a catalytically inactive ΦAB6TSP (I-ΦAB6TSP) retains binding ability toward Pse. In addition, an I-ΦAB6TSP-DyLight-650 conjugate (Dy-I-ΦAB6TSP) was more sensitive in detecting Ab-54149 than an antibody purified from anti- Ab-54149 EPS serum. Dy-I-ΦAB6TSP also cross-reacted with other pathogenic bacteria containing Pse on their surface polysaccharides (e.g., Helicobacter pylori and Enterobacter cloacae), revealing it to be a promising probe for detecting Pse across bacterial species. We also developed a detection method that employs I-ΦAB6TSP immobilized on microtiter plate. These results suggested that the anti-Ab-54149 EPS serum would exhibit cross-reactivity to Pse on other organisms. When this was tested, this serum facilitated complement-mediated killing of H. pylori and E. cloacae, indicating its potential as a cross-species antibacterial agent. This work opens new avenues for diagnosis and treatment of multidrug resistant (MDR) bacterial infections.

Origin and Evolution of Studiervirinae Bacteriophages Infecting Pectobacterium: Horizontal Transfer Assists Adaptation to New Niches.

Black leg and soft rot are devastating diseases causing up to 50% loss of potential potato yield. The search for, and characterization of, bacterial viruses (bacteriophages) suitable for the control of these diseases is currently a sought-after task for agricultural microbiology. Isolated lytic Pectobacterium bacteriophages Q19, PP47 and PP81 possess a similar broad host range but differ in their genomic properties. The genomic features of characterized phages have been described and compared to other Studiervirinae bacteriophages. Thorough phylogenetic analysis has clarified the taxonomy of the phages and their positioning relative to other genera of the Autographiviridae family. Pectobacterium phage Q19 seems to represent a new genus not described previously. The genomes of the phages are generally similar to the genome of phage T7 of the Teseptimavirus genus but possess a number of specific features. Examination of the structure of the genes and proteins of the phages, including the tail spike protein, underlines the important role of horizontal gene exchange in the evolution of these phages, assisting their adaptation to Pectobacterium hosts. The results provide the basis for the development of bacteriophage-based biocontrol of potato soft rot as an alternative to the use of antibiotics.

Structure and function of bacteriophage CBA120 ORF211 (TSP2), the determinant of phage specificity towards E. coli O157:H7

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.