Decoding the Structure-Function Relationship of the Muramidase Domain in E. coli O157.H7 Bacteriophage Endolysin: A Potential Building Block for Chimeric Enzybiotics.

Bacteriophage endolysins are potential alternatives to conventional antibiotics for treating multidrug-resistant gram-negative bacterial infections. However, their structure-function relationships are poorly understood, hindering their optimization and application. In this study, we focused on the individual functionality of the C-terminal muramidase domain of Gp127, a modular endolysin from E. coli O157:H7 bacteriophage PhaxI. This domain is responsible for the enzymatic activity, whereas the N-terminal domain binds to the bacterial cell wall. Through protein modeling, docking experiments, and molecular dynamics simulations, we investigated the activity, stability, and interactions of the isolated C-terminal domain with its ligand. We also assessed its expression, solubility, toxicity, and lytic activity using the experimental data. Our results revealed that the C-terminal domain exhibits high activity and toxicity when tested individually, and its expression is regulated in different hosts to prevent self-destruction. Furthermore, we validated the muralytic activity of the purified refolded protein by zymography and standardized assays. These findings challenge the need for the N-terminal binding domain to arrange the active site and adjust the gap between crucial residues for peptidoglycan cleavage. Our study shed light on the three-dimensional structure and functionality of muramidase endolysins, thereby enriching the existing knowledge pool and laying a foundation for accurate in silico modeling and the informed design of next-generation enzybiotic treatments.

Characterization of a novel phage depolymerase specific to Escherichia coli O157:H7 and biofilm control on abiotic surfaces.

The increasing prevalence of foodborne diseases caused by Escherichia coli O157:H7 as well as its ability to form biofilms poses major threats to public health worldwide. With increasing concerns about the limitations of current disinfectant treatments, phage-derived depolymerases may be used as promising biocontrol agents. Therefore, in this study, the characterization, purification, and application of a novel phage depolymerase, Dpo10, specifically targeting the lipopolysaccharides of E. coli O157, was performed. Dpo10, with a molecular mass of 98 kDa, was predicted to possess pectate lyase activity via genome analysis and considered to act as a receptor-binding protein of the phage. We confirmed that the purified Dpo10 showed O-polysaccharide degrading activity only for the E. coli O157 strains by observing its opaque halo. Dpo10 maintained stable enzymatic activities across a wide range of temperature conditions under 55°C and mild basic pH. Notably, Dpo10 did not inhibit bacterial growth but significantly increased the complement-mediated serum lysis of E. coli O157 by degrading its O-polysaccharides. Moreover, Dpo10 inhibited the biofilm formation against E. coli O157 on abiotic polystyrene by 8-fold and stainless steel by 2.56 log CFU/coupon. This inhibition was visually confirmed via fieldemission scanning electron microscopy. Therefore, the novel depolymerase from E. coli siphophage exhibits specific binding and lytic activities on the lipopolysaccharide of E. coli O157 and may be used as a promising anti-biofilm agent against the E. coli O157:H7 strain.

Circulation of Fluorescently Labelled Phage in a Murine Model.

Interactions between bacteriophages and mammals strongly affect possible applications of bacteriophages. This has created a need for tools that facilitate studies of phage circulation and deposition in tissues. Here, we propose red fluorescent protein (RFP)-labelled E. coli lytic phages as a new tool for the investigation of phage interactions with cells and tissues. The interaction of RFP-labelled phages with living eukaryotic cells (macrophages) was visualized after 20 min of co-incubation. RFP-labeled phages were applied in a murine model of phage circulation in vivo. Phages administered by three different routes (intravenously, orally, rectally) were detected through the course of time. The intravenous route of administration was the most efficient for phage delivery to multiple body compartments: 20 min after administration, virions were detected in lymph nodes, lungs, and liver; 30 min after administration, they were detectable in muscles; and 1 h after administration, phages were detected in spleen and lymph nodes. Oral and rectal administration of RFP-labelled phages allowed for their detection in the gastrointestinal (GI) tract only.

HK97 gp74 Possesses an α-Helical Insertion in the ßßα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities.

The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ßßα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two ß-strands of the ßßα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ßßα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle.IMPORTANCE This study demonstrates that residues outside the canonical ßßα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.

Targeted Elimination of Tumorigenic Human Pluripotent Stem Cells Using Suicide-Inducing Virus-like Particles.

Sensitization to prodrugs via transgenic expression of suicide genes is a leading strategy for the selective elimination of potentially tumorigenic human pluripotent stem cells (hPSCs) in regenerative medicine, but transgenic modification poses safety risks such as deleterious mutagenesis. We describe here an alternative method of delivering suicide-inducing molecules explicitly to hPSCs using virus-like particles (VLPs) and demonstrate its use in eliminating undifferentiated hPSCs in vitro. VLPs were engineered from Qß bacteriophage capsids to contain enhanced green fluorescent protein (EGFP) or cytosine deaminase (CD) and to simultaneously display multiple IgG-binding ZZ domains. After labeling with antibodies against the hPSC-specific surface glycan SSEA-5, EGFP-containing particles were shown to specifically bind undifferentiated cells in culture, and CD-containing particles were able to eliminate undifferentiated hPSCs with virtually no cytotoxicity to differentiated cells upon treatment with the prodrug 5-fluorocytosine.

Solvent Networks Tune Thermodynamics of Oligosaccharide Complex Formation in an Extended Protein Binding Site.

The principles of protein-glycan binding are still not well understood on a molecular level. Attempts to link affinity and specificity of glycan recognition to structure suffer from the general lack of model systems for experimental studies and the difficulty to describe the influence of solvent. We have experimentally and computationally addressed energetic contributions of solvent in protein-glycan complex formation in the tailspike protein (TSP) of E. coli bacteriophage HK620. HK620TSP is a 230 kDa native trimer of right-handed, parallel beta-helices that provide extended, rigid binding sites for bacterial cell surface O-antigen polysaccharides. A set of high-affinity mutants bound hexa- or pentasaccharide O-antigen fragments with very similar affinities even though hexasaccharides introduce an additional glucose branch into an occluded protein surface cavity. Remarkably different thermodynamic binding signatures were found for different mutants; however, crystal structure analyses indicated that no major oligosaccharide or protein topology changes had occurred upon complex formation. This pointed to a solvent effect. Molecular dynamics simulations using a mobility-based approach revealed an extended network of solvent positions distributed over the entire oligosaccharide binding site. However, free energy calculations showed that a small water network inside the glucose-binding cavity had the most notable influence on the thermodynamic signature. The energy needed to displace water from the glucose binding pocket depended on the amino acid at the entrance, in agreement with the different amounts of enthalpy-entropy compensation found for introducing glucose into the pocket in the different mutants. Studies with small molecule drugs have shown before that a few active water molecules can control protein complex formation. HK620TSP oligosaccharide binding shows that similar fundamental principles also apply for glycans, where a small number of water molecules can dominate the thermodynamic signature in an extended binding site.

The Odd "RB" Phage-Identification of Arabinosylation as a New Epigenetic Modification of DNA in T4-Like Phage RB69.

In bacteriophages related to T4, hydroxymethylcytosine (hmC) is incorporated into the genomic DNA during DNA replication and is then further modified to glucosyl-hmC by phage-encoded glucosyltransferases. Previous studies have shown that RB69 shares a core set of genes with T4 and relatives. However, unlike the other “RB” phages, RB69 is unable to recombine its DNA with T4 or with the other “RB” isolates. In addition, despite having homologs to the T4 enzymes used to synthesize hmC, RB69 has no identified homolog to known glucosyltransferase genes. In this study we sought to understand the basis for RB69’s behavior using high-pH anion exchange chromatography (HPAEC) and mass spectrometry. Our analyses identified a novel phage epigenetic DNA sugar modification in RB69 DNA, which we have designated arabinosyl-hmC (ara-hmC). We sought a putative glucosyltranserase responsible for this novel modification and determined that RB69 also has a novel transferase gene, ORF003c, that is likely responsible for the arabinosyl-specific modification. We propose that ara-hmC was responsible for RB69 being unable to participate in genetic exchange with other hmC-containing T-even phages, and for its described incipient speciation. The RB69 ara-hmC also likely protects its DNA from some anti-phage type-IV restriction endonucleases. Several T4-related phages, such as E. coli phage JS09 and Shigella phage Shf125875 have homologs to RB69 ORF003c, suggesting the ara-hmC modification may be relatively common in T4-related phages, highlighting the importance of further work to understand the role of this modification and the biochemical pathway responsible for its production.

The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region.

Bacteriophage endolysin is one of the most promising antibiotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidoglycans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in catalytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall binding domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to obtain an engineered lysin that can lyse bacteria from the outside. Our results showed that lysep3-D8 could lyse both Gramnegative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 µg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bactericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 µg/ml. The cell wall binding domain of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic domain to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.

Inactivation of Viruses and Bacteriophages as Models for Swine Hepatitis E Virus in Food Matrices.

Hepatitis E virus has been recognised as a food-borne virus hazard in pork products, due to its zoonotic properties. This risk can be reduced by adequate treatment of the food to inactivate food-borne viruses. We used a spectrum of viruses and bacteriophages to evaluate the effect of three food treatments: high pressure processing (HPP), lactic acid (LA) and intense light pulse (ILP) treatments. On swine liver at 400 MPa for 10 min, HPP gave log10 reductions of ≥4.2, ≥5.0 and 3.4 for feline calicivirus (FCV) 2280, FCV wildtype (wt) and murine norovirus 1 (MNV 1), respectively. Escherichia coli coliphage ϕX174 displayed a lower reduction of 1.1, while Escherichia coli coliphage MS2 was unaffected. For ham at 600 MPa, the corresponding reductions were 4.1, 4.4, 2.9, 1.7 and 1.3 log10. LA treatment at 2.2 M gave log10 reductions in the viral spectrum of 0.29-2.1 for swine liver and 0.87-3.1 for ham, with ϕX174 and MNV 1, respectively, as the most stable microorganisms. The ILP treatment gave log10 reductions of 1.6-2.8 for swine liver, 0.97-2.2 for ham and 1.3-2.3 for sausage, at 15-60 J cm-2, with MS2 as the most stable microorganism. The HPP treatment gave significantly (p < 0.05) greater virus reduction on swine liver than ham for the viruses at equivalent pressure/time combinations. For ILP treatment, reductions on swine liver were significantly (p < 0.05) greater than on ham for all microorganisms. The results presented here could be used in assessments of different strategies to protect consumers against virus contamination and in advice to food producers. Conservative model indicators for the pathogenic viruses could be suggested.

Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Therefore, sensitive, reliable and easy to use methods for the concentration, detection and quantification of microorganisms associated with the safety and quality of water are needed. In this study, we performed a field evaluation of an anion exchange resin-based method to concentrate male-specific (F+) RNA coliphages (FRNA), fecal indicator organisms, from diverse environmental waters that were suspected to be contaminated with feces. In this system, FRNA coliphages are adsorbed to anion exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase (RT)-PCR detection. Matrix-dependent inhibition of this method was evaluated using known quantities of spiked FRNA coliphages belonging to four genogroups (GI, GII, GII and GIV). RT-PCR-based detection was successful in 97%, 72%, 85% and 98% of the samples spiked (106 pfu/l) with GI, GII, GIII and GIV, respectively. Differential FRNA coliphage genogroup detection was linked to inhibitors that altered RT-PCR assay efficiency. No association between inhibition and the physicochemical properties of the water samples was apparent. Additionally, the anion exchange resin method facilitated detection of naturally present FRNA coliphages in 40 of 65 environmental water samples (61.5%), demonstrating the viability of this system to concentrate FRNA coliphages from water.

Incomplete LPS Core-Specific Felix01-Like Virus vB_EcoM_VpaE1.

Bacteriophages represent a valuable source for studying the mechanisms underlying virus-host interactions. A better understanding of the host-specificity of viruses at the molecular level can promote various phage applications, including bacterial diagnostics, antimicrobial therapeutics, and improve methods in molecular biology. In this study, we describe the isolation and characterization of a novel coliphage, vB_EcoM_VpaE1, which has different host specificity than its relatives. Morphology studies, coupled with the results of genomic and proteomic analyses, indicate that vB_EcoM_VpaE1 belongs to the newly proposed genus Felix01likevirus in the family Myoviridae. The genus Felix01likevirus comprises a group of highly similar phages that infect O-antigen-expressing Salmonella and Escherichia coli (E. coli) strains. Phage vB_EcoM_VpaE1 differs from the rest of Felix01-like viruses, since it infects O-antigen-deficient E. coli strains with an incomplete core lipopolysaccharide (LPS). We show that vB_EcoM_VpaE1 can infect mutants of E. coli that contain various truncations in their LPS, and can even recognize LPS that is truncated down to the inner-core oligosaccharide, showing potential for the control of rough E. coli strains, which usually emerge as resistant mutants upon infection by O-Ag-specific phages. Furthermore, VpaE1 can replicate in a wide temperature range from 9 to 49 °C, suggesting that this virus is well adapted to harsh environmental conditions. Since the structural proteins of such phages tend to be rather robust, the receptor-recognizing proteins of VpaE1 are an attractive tool for application in glycan analysis, bacterial diagnostics and antimicrobial therapeutics.

Escherichia coli nfuA is essential for maintenance of Shiga toxin phage Min27 lysogeny under iron-depleted condition.

It has been earlier hypothesized that lysogenic infection with Stx-encoding phages influences protein expression in the bacterial host, and therefore, some differentially expressed proteins could affect survival characteristics and pathogenicity. We compared the protein expression profiles of the host MG1655 and lysogens by 2D electrophoresis. Four different genes identified were all related to Fe/S subunit production, namely, nfuA, fdoH, sdhB and ftnA. To explore the role of nfuA in the biology of Stx prophage lysogeny, gene knockout experiments and phage lysogenic conversion were performed. The inactivation of nfuA caused the prophage to enter its lytic life cycle, especially under an iron-depleted condition. A similar activity was also detected in the Escherichia coli O157:H7 strain from which the Stx phage Min 27 was originally isolated. NfuA might be the positive regulator of genes controlling lysogenic cycle such as cI, cII and cIII since their transcriptional level was significantly reduced in nfuA deletion mutant as shown by qRT-PCR. We conclude that NfuA is essential for maintenance of Stx phage lysogeny in host's genetic reservoir under iron-deficient condition.

Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex.

Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.

Interaction of human adenoviruses and coliphages with kaolinite and bentonite.

Human adenoviruses (hAdVs) are pathogenic viruses responsible for public health problems worldwide. They have also been used as viral indicators in environmental systems. Coliphages (e.g., MS2, ΦX174) have also been studied as indicators of viral pollution in fecally contaminated water. Our objective was to evaluate the distribution of three viral fecal indicators (hAdVs, MS2, and ΦΧ174), between two different phyllosilicate clays (kaolinite and bentonite) and the aqueous phase. A series of static and dynamic experiments were conducted under two different temperatures (4, 25°C) for a time period of seven days. HAdV adsorption was examined in DNase I reaction buffer (pH=7.6, and ionic strength (IS)=1.4mM), whereas coliphage adsorption in phosphate buffered saline solution (pH=7, IS=2mM). Moreover, the effect of IS on hAdV adsorption under static conditions was evaluated. The adsorption of hAdV was assessed by real-time PCR and its infectivity was tested by cultivation methods. The coliphages MS2 and ΦΧ174 were assayed by the double-layer overlay method. The experimental results have shown that coliphage adsorption onto both kaolinite and bentonite was higher for the dynamic than the static experiments; whereas hAdV adsorption was lower under dynamic conditions. The adsorption of hAdV increased with decreasing temperature, contrary to the results obtained for the coliphages. This study examines the combined effect of temperature, agitation, clay type, and IS on hAdV adsorption onto clays. The results provide useful new information on the effective removal of viral fecal indicators (MS2, ΦX174 and hAdV) from dilute aqueous solutions by adsorption onto kaolinite and bentonite. Factors enabling enteric viruses to penetrate soils, groundwater and travel long distances within aquifers are important public health issues. Because the observed adsorption behavior of surrogate coliphages MS2 and ΦΧ174 is substantially different to that of hAdV, neither MS2 nor ΦΧ174 is recommended as a suitable model for adenovirus.

SDS-PAGE and IR spectroscopy to evaluate modifications in the viral protein profile induced by a cationic porphyrinic photosensitizer.

Reactive oxygen species can be responsible for microbial photodynamic inactivation due to its toxic effects, which include severe damage to proteins, lipids and nucleic acids. In this study, the photo-oxidative modifications of the proteins of a non-enveloped T4-like bacteriophage, induced by the cationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide were evaluated. Two methods were used: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared spectroscopy. SDS-PAGE analysis showed that the phage protein profile was considerably altered after photodynamic treatment. Seven protein bands putatively corresponding to capsid and tail tube proteins were attenuated and two other were enhanced. Infrared spectroscopy confirmed the time-dependent alteration on the phage protein profile detected by SDS-PAGE, indicative of a response to oxidative damage. Infrared analysis showed to be a promising and rapid screening approach for the analysis of the modifications induced on viral proteins by photosensitization. In fact, one single infrared spectrum can highlight the changes induced to all viral molecular structures, overcoming the delays and complex protocols of the conventional methods, in a much simple and cost effective way.

Intravitreal injection of the chimeric phage endolysin Ply187 protects mice from Staphylococcus aureus endophthalmitis.

The treatment of endophthalmitis is becoming very challenging due to the emergence of multidrug-resistant bacteria. Hence, the development of novel therapeutic alternatives for ocular use is essential. Here, we evaluated the therapeutic potential of Ply187AN-KSH3b, a chimeric phage endolysin derived from the Ply187 prophage, in a mouse model of Staphylococcus aureus endophthalmitis. Our data showed that the chimeric Ply187 endolysin exhibited strong antimicrobial activity against both methicillin-sensitive S. aureus and methicillin-resistant S. aureus (MRSA) strains, as evidenced by MIC determinations, reductions in turbidity, and disruption of biofilms. Moreover, exposure of S. aureus to Ply187 for up to 10 generations did not lead to resistance development. The intravitreal injection of chimeric Ply187 (at 6 or 12 h postinfection) significantly improved the outcome of endophthalmitis, preserved retinal structural integrity, and maintained visual function as assessed by electroretinogram analysis. Furthermore, phage lysin treatment significantly reduced the bacterial burden and the levels of inflammatory cytokines and neutrophil infiltration in the eyes. These results indicate that the intravitreal administration of a phage lytic enzyme attenuates the development of bacterial endophthalmitis in mice. To the best of our knowledge, this is the first study demonstrating the therapeutic use of phage-based antimicrobials in ocular infections.

The solution structures of two prophage homologues of the bacteriophage λ Ea8.5 protein reveal a newly discovered hybrid homeodomain/zinc-finger fold.

A cluster of genes in the exoxis region of bacteriophage λ are capable of inhibiting the initiation of DNA synthesis in Escherichia coli. The most indispensible gene in this region is ea8.5. Here, we report the nuclear magnetic resonance structures of two ea8.5 orthologs from enteropathogenic E. coli and Pseudomonas putida prophages. Both proteins are characterized by a fused homeodomain/zinc-finger fold that escaped detection by primary sequence search methods. While these folds are both associated with a nucleic acid binding function, the amino acid composition suggests otherwise, leading to the possibility that Ea8.5 associates with other viral and host proteins.

Isolation, characterization and complete genome sequence of PhaxI: a phage of Escherichia coli O157 : H7.

Bacteriophages are considered as promising biological agents for the control of infectious diseases. Sequencing of their genomes can ascertain the absence of antibiotic resistance, toxin or virulence genes. The anti-O157 : H7 coliphage, PhaxI, was isolated from a sewage sample in Iran. Morphological studies by transmission electron microscopy showed that it has an icosahedral capsid of 85-86 nm and a contractile tail of 115×15 nm. PhaxI contains dsDNA composed of 156 628 nt with a G+C content of 44.5 mol% that encodes 209 putative proteins. In MS analysis of phage particles, 92 structural proteins were identified. PhaxI lyses Escherichia coli O157 : H7 in Luria-Bertani medium and milk, has an eclipse period of 20 min and a latent period of 40 min, and has a burst size of about 420 particles per cell. PhaxI is a member of the genus 'Viunalikevirus' of the family Myoviridae and is specific for E. coli O157 : H7.

A conserved spiral structure for highly diverged phage tail assembly chaperones.

Tail assembly chaperones (TACs) are a family of proteins likely required for the morphogenesis of all long-tailed phages. In this study, we determined the crystal structure of gp13, the TAC of phage HK97. This structure is similar to that of the TAC from the Lactococcus phage p2 and two unannotated structures of likely TACs encoded in prophage-derived regions of Bacillus subtilis and Bacillus stearothermophilus. Despite the high sequence divergence of these proteins, gp13 forms a ring structure with similar dimensions to the spirals observed in the crystal lattices of these other proteins. Remarkably, these similar quaternary structures are formed through very different interprotomer interactions. We present functional data supporting the biological relevance of these spiral structures and propose that spiral formation has been the primary requirement for these proteins during evolution. This study presents an unusual example of diverged protein sequences and oligomerization mechanisms in the presence of conserved quaternary structure.