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CRISPR-Cas adaptive immune systems are widespread among bacteria and archaea. Recent studies have shown that these systems have minimal long-term evolutionary effects in limiting horizontal gene transfer. This suggests that the ability to evade CRISPR-Cas immunity must also be widespread in phages and other mobile genetic elements. In this Progress article, we discuss recent discoveries that highlight how phages inactivate CRISPR-Cas systems by using anti-CRISPR proteins, and we outline evolutionary and biotechnological implications of their activity.

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system., (Copyright © 2017 Pawluk et al.)

Bacteria and archaea use CRISPR-Cas adaptive immune systems to defend themselves from infection by bacteriophages (phages). These RNA-guided nucleases are powerful weapons in the fight against foreign DNA, such as phages and plasmids, as well as a revolutionary gene editing tool. Phages are not passive bystanders in their interactions with CRISPR-Cas systems, however; recent discoveries have described phage genes that inhibit CRISPR-Cas function. More than 20 protein families, previously of unknown function, have been ascribed anti-CRISPR function. Here, we discuss how these CRISPR-Cas inhibitors were discovered and their modes of action were elucidated. We also consider the potential impact of anti-CRISPRs on bacterial and phage evolution. Finally, we speculate about the future of this field.

CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Clustered, regularly interspaced, short, palindromic repeats (CRISPR) loci, together with their CRISPR-associated (Cas) proteins, provide bacteria and archaea with adaptive immunity against invasion by bacteriophages, plasmids, and other mobile genetic elements. These host defenses impart selective pressure on phages and mobile elements to evolve countermeasures against CRISPR immunity. As a consequence of this pressure, phages and mobile elements have evolved 'anti-CRISPR' proteins that function as direct inhibitors of diverse CRISPR-Cas effector complexes. Some of these CRISPR-Cas complexes can be deployed as genome engineering platforms, and anti-CRISPRs could therefore be useful in exerting spatial, temporal, or conditional control over genome editing and related applications. Here we describe the discovery of anti-CRISPRs, the range of CRISPR-Cas systems that they inhibit, their mechanisms of action, and their potential utility in biotechnology., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target., (Copyright © 2017 Elsevier Inc. All rights reserved.)

CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The activity of bacteriophages poses a major threat to bacterial survival. Upon infection, a temperate phage can either kill the host cell or be maintained as a prophage. In this state, the bacteria carrying the prophage is at risk of superinfection, where another phage injects its genetic material and competes for host cell resources. To avoid this, many phages have evolved mechanisms that alter the bacteria and make it resistant to phage superinfection. The mechanisms underlying these phentoypic conversions and the fitness consequences for the host are poorly understood, and systematic studies of superinfection exclusion mechanisms are lacking. In this study, we examined a wide range of Pseudomonas aeruginosa phages and found that they mediate superinfection exclusion through a variety of mechanisms, some of which affected the type IV pilus and O-antigen, and others that functioned inside the cell. The strongest resistance mechanism was a surface modification that we showed is cost-free for the bacterial host in a natural soil environment and in a Caenorhabditis. elegans infection model. This study represents the first systematic approach to address how a population of prophages influences phage resistance and bacterial behavior in P. aeruginosa.

Bacterial CRISPR-Cas adaptive immune systems use small guide RNAs to protect against phage infection and invasion by foreign genetic elements. We previously demonstrated that a group of Pseudomonas aeruginosa phages encode anti-CRISPR proteins that inactivate the type I-F and I-E CRISPR-Cas systems using distinct mechanisms. Here, we present the three-dimensional structure of an anti-CRISPR protein and map a functional surface that is critical for its potent inhibitory activity. The interaction of the anti-CRISPR protein with the CRISPR-Cas complex through this functional surface is proposed to prevent the binding of target DNA.

Contractile phage tails are powerful cell puncturing nanomachines that have been co-opted by bacteria for self-defense against both bacteria and eukaryotic cells. The tail of phage T4 has long served as the paradigm for understanding contractile tail-like systems despite its greater complexity compared with other contractile-tailed phages. Here, we present a detailed investigation of the assembly of a "simple" contractile-tailed phage baseplate, that of Escherichia coli phage Mu. By coexpressing various combinations of putative Mu baseplate proteins, we defined the required components of this baseplate and delineated its assembly pathway. We show that the Mu baseplate is constructed through the independent assembly of wedges that are organized around a central hub complex. The Mu wedges are comprised of only three protein subunits rather than the seven found in the equivalent structure in T4. Through extensive bioinformatic analyses, we found that homologs of the essential components of the Mu baseplate can be identified in the majority of contractile-tailed phages and prophages. No T4-like prophages were identified. The conserved simple baseplate components were also found in contractile tail-derived bacterial apparatuses, such as type VI secretion systems, Photorhabdus virulence cassettes, and R-type tailocins. Our work highlights the evolutionary connections and similarities in the biochemical behavior of phage Mu wedge components and the TssF and TssG proteins of the type VI secretion system. In addition, we demonstrate the importance of the Mu baseplate as a model system for understanding bacterial phage tail-derived systems., Competing Interests: The authors declare no conflict of interest.

CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids(1,2). They are present in approximately half of all sequenced prokaryotes(3) and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function(4,5). We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.

Background: Prospective orthopedic residency applicants commonly use one of three databases to identify potential programs: Accreditation Council of Graduate Medical Education (ACGME), American Medical Association (FREIDA), or Orthogate. org. In addition, institutional websites are typically the primary source of information once programs are identified. We sought to evaluate the databases and websites used by prospective orthopedic surgery applicants for content and accessibility. We hypothesized that information would be more available in comparison to previous studies but would still fail to provide complete, up to date program information for the prospective applicant., Methods: Three online databases were queried in December 2014 to compile a list of orthopedic residency programs in the United States. This combined list was used as a basis for evaluating individual institution websites. Previously described criteria were used to evaluate the availability of information contained within orthopedic surgery residency websites., Results: At the time of online review, 157 programs were identified. Depending on the database in question, up to 33% of programs either did not provide a link or listed a non-functioning link. Among the variety of evaluated criteria, inclusion of the information varied between 12% and 97% for the individual program websites., Conclusions: Online databases are useful in listing programs, but individual program details and direct functional links are lacking. Most program websites contain varying degrees of desired information; however, not all programs maintain websites which consistently provide information to satisfy the evaluated criteria in this study. Improved online accessibility and availability of information for residency programs would increase their visibility and utility for prospective applicants.

CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR-Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR-Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR-Cas systems., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

Phages play critical roles in the spread of virulence factors and control of bacterial populations through their predation of bacteria. An essential step in the phage lifecycle is genome entry, where the infecting phage must productively interact with the components of the bacterial cell envelope in order to transmit its genome out of the viral particle and into the host cell cytoplasm. In this study, we characterize this process for the Escherichia coli phage HK97. We have discovered that HK97 genome injection requires the activities of the inner membrane glucose transporter protein, PtsG, and the periplasmic chaperone, FkpA. The requirements for PtsG and FkpA are determined by the sequence of the phage tape measure protein (TMP). We also identify a region of the TMP that mediates inhibition of phage genome injection by the HK97 superinfection exclusion protein, gp15. This region of the TMP also determines the PtsG requirement, and we show that gp15-mediated inhibition requires PtsG. Based on these data, we present a model for the in vivo genome injection process of phage HK97 and postulate a mechanism by which the inhibitory action of gp15 is reliant upon PtsG., (© 2014 John Wiley & Sons Ltd.)

Background: Limited data exist on pediatric subaxial cervical spine injuries. The goal of this study was to characterize the injuries and initial treatment of a large consecutive series of patients with injuries from C3 to C7., Methods: Medical records and radiographs of consecutive patients admitted with cervical spine fractures and/or dislocations at a single level 1 pediatric trauma center from 2003 to 2013 were reviewed. Data abstracted included age, injury type and level, mechanism of injury, associated nonspine injuries, neurological status, length of hospitalization, and initial treatment., Results: Fifty-one patients were grouped into 3 age ranges: infant, 0 to 3 years (2); youth, 4 to 12 years (13); and adolescent, 13 to 16 years (36). Isolated fractures were identified in both infants and accounted for most of injuries in youths (85%) and adolescents (86%). Single vertebra or single vertebral level injuries were present in 65% of patients, most commonly at C7 (36%) or C6 (29%). No correlation existed between cervical level injured and patient age. Multiple cervical spine injuries occurred in 1 infant, 3 youths, and 14 adolescents. Other concomitant thoracic and/or lumbar spine injuries were found in 1 infant and 3 adolescents. The most common mechanisms of injury were motor vehicle accidents (53%) and sports (14%). High-energy trauma was associated with higher rates of noncontiguous spinal injuries and associated nonspinal injuries, with a longer length of hospitalization. Neurological deficits were observed in 8 patients: 1 infant, 2 youths, and 5 adolescents, of which 5 resulted from high-energy trauma. One infant and all youth patients were treated nonoperatively; 26 adolescents (73%) were treated in a cervical collar or with observation, 1 was treated with halo-vest immobilization, and 9 had surgical treatment., Conclusions: Most subaxial cervical spine injuries in pediatric and adolescent patients are isolated fractures at C6 and C7. High-energy mechanisms are associated with noncontiguous spinal injuries and other nonspine injuries. Most patients can be treated in a cervical collar, but adolescent patients are more likely to require halo placement or surgical intervention., Level of Evidence: Level IV-retrospective, diagnostic.

Background: Pediatric orthopaedic fellowship applicants commonly use online-based resources for information on potential programs. Two primary sources are the San Francisco Match (SF Match) database and the Pediatric Orthopaedic Society of North America (POSNA) database. We sought to determine the accessibility and quality of information that could be obtained by using these 2 sources., Methods: The online databases of the SF Match and POSNA were reviewed to determine the availability of embedded program links or external links for the included programs. If not available in the SF Match or POSNA data, Web sites for listed programs were located with a Google search. All identified Web sites were analyzed for accessibility, content volume, and content quality., Results: At the time of online review, 50 programs, offering 68 positions, were listed in the SF Match database. Although 46 programs had links included with their information, 36 (72%) of them simply listed http://www.sfmatch.org as their unique Web site. Ten programs (20%) had external links listed, but only 2 (4%) linked directly to the fellowship web page. The POSNA database does not list any links to the 47 programs it lists, which offer 70 positions. On the basis of a Google search of the 50 programs listed in the SF Match database, web pages were found for 35. Of programs with independent web pages, all had a description of the program and 26 (74%) described their application process. Twenty-nine (83%) listed research requirements, 22 (63%) described the rotation schedule, and 12 (34%) discussed the on-call expectations. A contact telephone number and/or email address was provided by 97% of programs. Twenty (57%) listed both the coordinator and fellowship director, 9 (26%) listed the coordinator only, 5 (14%) listed the fellowship director only, and 1 (3%) had no contact information given., Conclusions: The SF Match and POSNA databases provide few direct links to fellowship Web sites, and individual program Web sites either do not exist or do not effectively convey information about the programs., Clinical Relevance: Improved accessibility and accurate information online would allow potential applicants to obtain information about pediatric fellowships in a more efficient manner.

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.

CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. IMPORTANCE The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts.

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR associated) systems provide a sophisticated adaptive immune system that offers protection against foreign DNA. These systems are widely distributed in prokaryotes and exert an important influence on bacterial behavior and evolution. However, interpreting the biological effects of a CRISPR-Cas system within a given species can be complicated because the outcome of rejecting foreign DNA does not always provide a fitness advantage, as foreign DNA uptake is sometimes beneficial. To address these issues, here we review data pertaining to the potential in vivo costs and benefits of CRISPR-Cas systems, novel functions for these systems, and how they may be inactivated., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Most organisms on the planet have viruses that infect them. Viral infection may lead to cell death, or to a symbiotic relationship where the genomes of both virus and host replicate together. In the symbiotic state, both virus and cell potentially experience increased fitness as a result of the other. The viruses that infect bacteria, called bacteriophages (or phages), well exemplify the symbiotic relationships that can develop between viruses and their host. In this review, we will discuss the many ways that prophages, which are phage genomes integrated into the genomes of their hosts, influence bacterial behavior and virulence.

Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.

A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the "minimal" myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis.

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., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

The assembly of long non-contractile phage tails begins with the formation of the tail tip complex (TTC). TTCs are multi-functional protein structures that mediate host cell adsorption and genome injection. The TTC of phage λ is assembled from multiple copies of eight different proteins, including gpL. Purified preparations of gpL and several homologues all displayed a distinct reddish color, suggesting the binding of iron by these proteins. Further characterization of the gpL homologue from phage N15, which was most amenable to in vitro analyses, showed that it contains two domains. The C-terminal domain was demonstrated to coordinate an iron-sulfur cluster, providing the first example of a viral structural protein binding to this type of metal group. We characterized the iron-sulfur cluster using inductively coupled plasma-atomic emission spectroscopy, absorbance spectroscopy, and electron paramagnetic resonance spectroscopy and found that it is an oxygen-sensitive [4Fe-4S](2+) cluster. Four highly conserved cysteine residues were shown to be required for coordinating the iron-sulfur cluster, and substitution of any of these Cys residues with Ser or Ala within the context of λ gpL abolished biological activity. These data imply that the intact iron-sulfur cluster is required for function. The presence of four conserved Cys residues in the C-terminal regions of very diverse gpL homologues suggest that utilization of an iron-sulfur cluster is a widespread feature of non-contractile tailed phages that infect Gram-negative bacteria. In addition, this is the first example of a viral structural protein that binds an iron-sulfur cluster., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes. Similar to RNA interference in eukaryotes, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes. Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct 'anti-CRISPR' genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.

Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function.

Prophage-encoded genes can provide a variety of benefits for their bacterial hosts. These beneficial genes are often contained within "moron" elements. Morons, thus termed as the insertion of the DNA encoding them adds "more on" the genome in which they are found, are independent transcriptional units disseminated among phage genomes through horizontal gene transfer. Morons have been identified in the majority of phage genomes and they have been found to play diverse roles in bacterial physiology. At present, we are only beginning to ascribe functions to the many proteins encoded within these ubiquitous genetic elements. Recently, we discovered that the first described moron-encoded protein, gp15 of phage HK97, is expressed from the HK97 prophage and functions as a superinfection exclusion protein, protecting its host from genome injection by other phages. This work and the growing body of data pertaining to other morons challenges the traditional view of phages as purely parasites of bacteria and emphasizes the symbiotic relationship between bacteria and prophages.

One of the final steps in the morphogenetic pathway of phage λ is the packaging of a single genome into a preformed empty head structure. In addition to the terminase enzyme, the packaging chaperone, FI protein (gpFI), is required for efficient DNA packaging. In this study, we demonstrate an interaction between gpFI and the major head protein, gpE. Amino acid substitutions in gpFI that reduced the strength of this interaction also decreased the biological activity of gpFI, implying that this head binding activity is essential for the function of gpFI. We also show that gpFI is a two-domain protein, and the C-terminal domain is responsible for the head binding activity. Using nuclear magnetic resonance spectroscopy, we determined the three-dimensional structure of the C-terminal domain and characterized the helical nature of the N-terminal domain. Through structural comparisons, we were able to identify two previously unannotated prophage-encoded proteins with tertiary structures similar to gpFI, although they lack significant pairwise sequence identity. Sequence analysis of these diverse homologues led us to identify related proteins in a variety of myo- and siphophages, revealing that gpFI function has a more highly conserved role in phage morphogenesis than was previously appreciated. Finally, we present a novel model for the mechanism of gpFI chaperone activity in the DNA packaging reaction of phage λ.

A phage moron is a DNA element inserted between a pair of genes in one phage genome that are adjacent in other related phage genomes. Phage morons are commonly found within phage genomes, and in a number of cases, they have been shown to mediate phenotypic changes in the bacterial host. The temperate phage HK97 encodes a moron element, gp15, within its tail morphogenesis region that is absent in most closely related phages. We show that gp15 is actively expressed from the HK97 prophage and is responsible for providing the host cell with resistance to infection by phages HK97 and HK75, independent of repressor immunity. To identify the target(s) of this gp15-mediated resistance, we created a hybrid of HK97 and the related phage HK022. This hybrid phage revealed that the tail tube or tape measure proteins likely mediate the susceptibility of HK97 to inhibition by gp15. The N terminus of gp15 is predicted with high probability to contain a single membrane-spanning helix by several transmembrane prediction programs. Consistent with this putative membrane localization, gp15 acts to prevent the entry of phage DNA into the cytoplasm, acting in a manner reminiscent of those of several previously characterized superinfection exclusion proteins. The N terminus of gp15 and its phage homologues bear sequence similarity to YebO proteins, a family of proteins of unknown function found ubiquitously in enterobacteria. The divergence of their C termini suggests that phages have co-opted this bacterial protein and subverted its activity to their advantage.

Saccharomyces cerevisiae Actin-Binding Protein 1 (Abp1p) is a member of the Abp1 family of proteins, which are in diverse organisms including fungi, nematodes, flies, and mammals. All proteins in this family possess an N-terminal Actin Depolymerizing Factor Homology (ADF-H) domain, a central Proline-Rich Region (PRR), and a C-terminal SH3 domain. In this study, we employed sequence analysis to identify additional conserved features of the family, including sequences rich in proline, glutamic acid, serine, and threonine amino acids (PEST), which are found in all family members examined, and two motifs, Conserved Fungal Motifs 1 and 2 (CFM1 and CFM2), that are conserved in fungi. We also discovered that, similar to its mammalian homologs, Abp1p is phosphorylated in its PRR. This phosphorylation is mediated by the Cdc28p and Pho85p kinases, and it protects Abp1p from proteolysis mediated by the conserved PEST sequences. We provide evidence for an intramolecular interaction between the PRR region and SH3 domain that may be affected by phosphorylation. Although deletion of CFM1 alone caused no detectable phenotype in any genetic backgrounds or conditions tested, deletion of this motif resulted in a significant reduction of growth when it was combined with a deletion of the ADF-H domain. Importantly, this result demonstrates that deletion of highly conserved domains on its own may produce no phenotype unless the domains are assayed in conjunction with deletions of other functionally important elements within the same protein. Detection of this type of intragenic synthetic lethality provides an important approach for understanding the function of individual protein domains or motifs.

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved β-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state., (Copyright © 2012 Wiley Periodicals, Inc.)

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

At least 30% of human proteins are thought to contain intrinsically disordered regions, which lack stable structural conformation. Despite lacking enzymatic functions and having few protein domains, disordered regions are functionally important for protein regulation and contain short linear motifs (short peptide sequences involved in protein-protein interactions), but in most disordered regions, the functional amino acid residues remain unknown. We searched for evolutionarily conserved sequences within disordered regions according to the hypothesis that conservation would indicate functional residues. Using a phylogenetic hidden Markov model (phylo-HMM), we made accurate, specific predictions of functional elements in disordered regions even when these elements are only two or three amino acids long. Among the conserved sequences that we identified were previously known and newly identified short linear motifs, and we experimentally verified key examples, including a motif that may mediate interaction between protein kinase Cbk1 and its substrates. We also observed that hub proteins, which interact with many partners in a protein interaction network, are highly enriched in these conserved sequences. Our analysis enabled the systematic identification of the functional residues in disordered regions and suggested that at least 5% of amino acids in disordered regions are important for function.

Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture., (Copyright © 2011 Wiley Periodicals, Inc.)

For more than half a century, researchers have studied the basic biology of Adenovirus (Ad), unraveling the subtle, yet profound, interactions between the virus and the host. These studies have uncovered previously unknown proteins and pathways crucial for normal cell function that the virus manipulates to achieve optimal virus replication and gene expression. In the infecting virion, the viral DNA is tightly condensed in a virally encoded protamine-like protein which must be remodeled within the first few hours of infection to allow for efficient expression of virus-encoded genes and subsequent viral DNA replication. This review discusses our current knowledge of Ad DNA-protein complex within the infected cell nucleus, the cellular proteins the virus utilizes to achieve chromatinization, and how this event contributes to efficient gene expression and progression of the virus life cycle.

There is increasing evidence for the functional importance of multiple dynamically populated states within single proteins. However, peptide binding by protein-protein interaction domains, such as the SH3 domain, has generally been considered to involve the full engagement of peptide to the binding surface with minimal dynamics and simple methods to determine dynamics at the binding surface for multiple related complexes have not been described. We have used NMR spectroscopy combined with isothermal titration calorimetry to comprehensively examine the extent of engagement to the yeast Abp1p SH3 domain for 24 different peptides. Over one quarter of the domain residues display co-linear chemical shift perturbation (CCSP) behavior, in which the position of a given chemical shift in a complex is co-linear with the same chemical shift in the other complexes, providing evidence that each complex exists as a unique dynamic rapidly inter-converting ensemble. The extent the specificity determining sub-surface of AbpSH3 is engaged as judged by CCSP analysis correlates with structural and thermodynamic measurements as well as with functional data, revealing the basis for significant structural and functional diversity amongst the related complexes. Thus, CCSP analysis can distinguish peptide complexes that may appear identical in terms of general structure and percent peptide occupancy but have significant local binding differences across the interface, affecting their ability to transmit conformational change across the domain and resulting in functional differences.

In this chapter, we describe the structure, assembly, function, and evolution of the long, noncontractile tail of the siphophages, which comprise ∼60% of the phages on earth. We place -particular emphasis on features that are conserved among all siphophages, and trace evolutionary connections between these phages and myophages, which possess long contractile tails. The large number of high-resolution structures of tail proteins solved recently coupled to studies of tail-related complexes by electron microscopy have provided many new insights in this area. In addition, the availability of thousands of phage and prophage genome sequences has allowed the delineation of several large families of tail proteins that were previously unrecognized. We also summarize current knowledge pertaining to the mechanisms by which siphophage tails recognize the bacterial cell surface and mediate DNA injection through the cell envelope. We show that phages infecting Gram-positive and Gram-negative bacteria possess distinct families of proteins at their tail tips that are involved in this process. Finally, we speculate on the evolutionary advantages provided by long phage tails.

Altering the expression level of proteins that are subunits of complexes has been proposed to be particularly detrimental because the resulting stoichiometric imbalance among components would lead to misassembly of the complex. Here we show that assembly of the phage HK97 connector complex is severely inhibited by the overexpression of one of its component proteins, gp6. However, this effect is a result of the unusual mechanism by which the oligomerization and assembly of gp6 are controlled. Alteration of this mechanism by single amino acid substitutions leads to a reversal of the response to gp6 overexpression. Surprisingly, the binding partner of gp6 within the phage particle is expressed at a 500-fold higher concentration despite their identical stoichiometry within the complex. Our data emphasize that a generalized prediction of the effects of changes in the expression level of protein complex subunits is very difficult because these effects are dependent upon assembly mechanism.

The yeast Bem1p SH3b and Nbp2p SH3 domains are unusual because they bind to peptides containing the same consensus sequence, yet they perform different functions and display low sequence similarity. In this work, by analyzing the interactions of these domains with six biologically relevant peptides containing the consensus sequence, they are shown to possess finely tuned and distinct binding specificities. We also identify a residue in the Bem1p SH3b domain that inhibits binding, yet is highly conserved for the purpose of preventing nonspecific interactions. Substitution of this residue results in a marked reduction of in vivo function that is caused by titration of the domain away from its proper targets through nonspecific interactions with other proteins. This work provides a clear illustration of the importance of intrinsic binding specificity for the function of protein-protein interaction modules, and the key role of "negative" interactions in determining the specificity of a domain.

Immunoglobulin (Ig)-like domains are found frequently on the surface of tailed double-stranded DNA bacteriophages, yet their functional role remains obscure. Here, we have investigated the structure and function of the C-terminal Ig-like domain of gpV (gpV(C)), the tail tube protein of phage λ. This domain has been predicted through sequence similarity to be a member of the bacterial Ig-like domain 2 (Big_2) family, which is composed of more than 1300 phage and bacterial sequences. Using trypsin proteolysis, we have delineated the boundaries of gpV(C) and have shown that its removal reduces the biological activity of gpV by 100-fold; thus providing a definitive demonstration of a functional role for this domain. Determination of the solution structure of gpV(C) by NMR spectroscopy showed that it adopts a canonical Ig-like fold of the I-set class. This represents the first structure of a phage-encoded Ig-like domain and only the second structure of a Big_2 domain. Structural and sequence comparisons indicate that the gpV(C) structure is more representative of both the phage-encoded Big_2 domains and Big_2 domains in general than the other available Big_2 structure. Bioinformatics analyses have identified two conserved clusters of residues on the surface of gpV(C) that may be important in mediating the function of this domain., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Background: Increasing our understanding of antibiotic resistance mechanisms is critical. To enable progress in this area, methods to rapidly identify and characterize antibiotic resistance conferring enzymes are required., Results: We have constructed a sensitive reporter system in Escherichia coli that can be used to detect and characterize the activity of enzymes that act upon the antibiotic, tetracycline and its derivatives. In this system, expression of the lux operon is regulated by the tetracycline repressor, TetR, which is expressed from the same plasmid under the control of an arabinose-inducible promoter. Addition of very low concentrations of tetracycline derivatives, well below growth inhibitory concentrations, resulted in luminescence production as a result of expression of the lux genes carried by the reporter plasmid. Introduction of another plasmid into this system expressing TetX, a tetracycline-inactivating enzyme, caused a marked loss in luminescence due to enzyme-mediated reduction in the intracellular Tc concentration. Data generated for the TetX enzyme using the reporter system could be effectively fit with the known Km and kcat values, demonstrating the usefulness of this system for quantitative analyses., Conclusion: Since members of the TetR family of repressors regulate enzymes and pumps acting upon almost every known antibiotic and a wide range of other small molecules, reporter systems with the same design as presented here, but employing heterologous TetR-related proteins, could be developed to measure enzymatic activities against a wide range of antibiotics and other compounds. Thus, the assay described here has far-reaching applicability and could be adapted for high-throughput applications.

Evolutionary relationships may exist among very diverse groups of proteins even though they perform different functions and display little sequence similarity. The tailed bacteriophages present a uniquely amenable system for identifying such groups because of their huge diversity yet conserved genome structures. In this work, we used structural, functional, and genomic context comparisons to conclude that the head-tail connector protein and tail tube protein of bacteriophage lambda diverged from a common ancestral protein. Further comparisons of tertiary and quaternary structures indicate that the baseplate hub and tail terminator proteins of bacteriophage may also be part of this same family. We propose that all of these proteins evolved from a single ancestral tail tube protein fold, and that gene duplication followed by differentiation led to the specialized roles of these proteins seen in bacteriophages today. Although this type of evolutionary mechanism has been proposed for other systems, our work provides an evolutionary mechanism for a group of proteins with different functions that bear no sequence similarity. Our data also indicate that the addition of a structural element at the N terminus of the lambda head-tail connector protein endows it with a distinctive protein interaction capability compared with many of its putative homologues.

The tetracycline repressor family transcriptional regulators (TFRs) are homodimeric DNA-binding proteins that generally act as transcriptional repressors. Their DNA-binding activity is allosterically inactivated by the binding of small-molecule ligands. TFRs constitute the third most frequently occurring transcriptional regulator family found in bacteria with more than 10,000 representatives in the nonredundant protein database. In addition, more than 100 unique TFR structures have been solved by X-ray crystallography. In this study, we have used computational and experimental approaches to reveal the variations and conservation present within TFRs. Although TFR structures are very diverse, we were able to identify a conserved central triangle in their ligand-binding domains that forms the foundation of the structure and the framework for the ligand-binding cavity. While the sequences of DNA-binding domains of TFRs are highly conserved across the whole family, the sequences of their ligand-binding domains are so diverse that pairwise sequence similarity is often undetectable. Nevertheless, by analyzing subfamilies of TFRs, we were able to identify distinct regions of conservation in ligand-binding domains that may be important for allostery. To aid in large-scale analyses of TFR function, we have developed a simple and reliable computational approach to predict TFR operator sequences, a temperature melt-based assay to measure DNA binding, and a generic ligand-binding assay that will likely be applicable to most TFRs. Finally, our analysis of TFR structures highlights their flexibility and provides insight into a conserved allosteric mechanism for this family., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 A crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.