In silico study of cox protein from P2 type enteric bacteriophages based on sequence, structure and dynamics to understand its functional integrity.

Cox protein plays a critical role in deciding the lytic-lysogenic switch of P2 enteric phages. This phenomenon makes Cox protein one of the most important candidates in developing novel phage-based therapeutics against antibacterial resistant pathogens. The principle focus concerning protein and its decision making is a DNA binding event, which helps to regulate differential promoter expression. In the current study, we have attempted to understand the sequence, structural and dynamic features associated with Cox protein and its DNA binding. Unavailability of information was a big burden in further proceedings. We have done an extensive literature search to develop a database of Cox with relevant information. That information coupled with the methods of Sequence-based phylogenetic and conservation studies, Homology Modelling, Atomic-level Docking and Molecular Dynamics (MD) Simulation (50 ns each for 10 systems, i.e. total of 500 ns) were performed in the current study. Analysis of those extensive studies has provided us the required sequence to structure to dynamics to functional understanding. Our present study would indeed be very helpful in understanding the biochemical mechanism of Cox activation as well as designing potential phage therapeutics.

Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems.

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.

Structure, adsorption to host, and infection mechanism of virulent lactococcal phage p2.

Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower k(off) values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.

Sequence variability of P2-like prophage genomes carrying the cytolethal distending toxin V operon in Escherichia coli O157.

Cytolethal distending toxins (CDT) are potent cytotoxins of several Gram-negative pathogenic bacteria, including Escherichia coli, in which five types (CDT-I to CDT-V) have been identified so far. CDT-V is frequently associated with Shiga-toxigenic E. coli (STEC), enterohemorrhagic E. coli (EHEC) O157 strains, and strains not fitting any established pathotypes. In this study, we were the first to sequence and annotate a 31.2-kb-long, noninducible P2-like prophage carrying the cdt-V operon from an stx- and eae-negative E. coli O157:H43 strain of bovine origin. The cdt-V operon is integrated in the place of the tin and old phage immunity genes (termed the TO region) of the prophage, and the prophage itself is integrated into the bacterial chromosome between the housekeeping genes cpxP and fieF. The presence of P2-like genes (n = 20) was investigated in a further five CDT-V-positive bovine E. coli O157 strains of various serotypes, three EHEC O157:NM strains, four strains expressing other variants of CDT, and eight CDT-negative strains. All but one CDT-V-positive atypical O157 strain uniformly carried all the investigated genomic regions of P2-like phages, while the EHEC O157 strains missed three regions and the CDT-V-negative strains carried only a few P2-like sequences. Our results suggest that P2-like phages play a role in the dissemination of cdt-V between E. coli O157 strains and that after integration into the bacterial chromosome, they adapted to the respective hosts and became temperate.

The DNA binding mechanism of a SSB protein from Lactococcus lactis siphophage p2.

Virulent lactococcal phages of the Siphoviridae family are responsible for the industrial milk fermentation failures worldwide. Lactococcus lactis, a Gram-positive bacterium widely used for the manufacture of fermented dairy products, is subjected to infections by virulent phages, predominantly those of the 936 group, including phage p2. Among the proteins coded by lactococcal phage genomes, of special interest are those expressed early, which are crucial to efficiently carry out the phage lytic cycle. We previously identified and solved the 3D structure of lactococcal phage p2 ORF34, a single stranded DNA binding protein (SSBp2). Here we investigated the molecular basis of ORF34 binding mechanism to DNA. DNA docking on SSBp2 and Molecular Dynamics simulations of the resulting complex identified R15 as a crucial residue for ssDNA binding. Electrophoretic Mobility Shift Assays (EMSA) and Atomic Force Microscopy (AFM) imaging revealed the inability of the Arg15Ala mutant to bind ssDNA, as compared to the native protein. Since R15 is highly conserved among lactococcal SSBs, we propose that its role in the SSBp2/DNA complex stabilization might be extended to all the members of this protein family.

Functional analysis of a class I holin, P2 Y.

Y is the putative holin gene of the paradigm coliphage P2 and encodes a 93-amino-acid protein. Y is predicted to be an integral membrane protein that adopts an N-out C-in membrane topology with 3 transmembrane domains (TMDs) and a highly charged C-terminal cytoplasmic tail. The same features are observed in the canonical class I lambda holin, the S105 protein of phage lambda, which controls lysis by forming holes in the plasma membrane at a programmed time. S105 has been the subject of intensive genetic, cellular, and biochemical analyses. Although Y is not related to S105 in its primary structure, its characterization might prove useful in discerning the essential traits for holin function. Here, we used physiological and genetic approaches to show that Y exhibits the essential holin functional criteria, namely, allele-specific delayed-onset lethality and sensitivity to the energization of the membrane. Taken together, these results suggest that class I holins share a set of unusual features that are needed for their remarkable ability to program the end of the phage infection cycle with precise timing. However, Y holin function requires the integrity of its short cytoplasmic C-terminal domain, unlike for S105. Finally, instead of encoding a second translational product of Y as an antiholin, as shown for lambda S107, the P2 lysis cassette encodes another predicted membrane protein, LysA, which is shown here to have a Y-specific antiholin character.

Comparative analysis of P2-type remnant prophage loci in Xenorhabdus bovienii and Xenorhabdus nematophila required for xenorhabdicin production.

The xnp1 remnant P2-type prophage of Xenorhabdus nematophila produces xenorhabdicin that is active against closely related species. Xenorhabdicin had not been characterized previously in other Xenorhabdus species. Here, we show xenorhabdicin production in six different strains of Xenorhabdus bovienii. The sequenced genome of X. bovienii SS-2004 was found to possess a highly conserved remnant P2-type cluster (xbp1). Inactivation of the xbpS1 sheath gene resulted in loss of bacteriocin activity, indicating that the xbp1 locus was required for xenorhabdicin production. xbp1 and xnp1 contain a CI-type repressor, a dinI gene involved in stabilization of ssDNA-RecA complexes and are inducible with mitomycin C, suggesting that both loci are regulated by cleavage of the CI repressor. Both xnp1 and xbp1 lack typical P2-type lysis genes but contain a predicted endolysin gene (enp) that may be involved in cell lysis. The main tail fibers of xnp1 and xbp1 are mosaic structures with divergent C-terminal regions suggesting they differ in host specificity. Several genes encoding C-terminal tail fiber fragments are present in the same position in xnp1 and xbp1. Recombination between the main fiber genes and the C-terminal fragments could potentially expand the host range specificity of xenorhabdicin in the respective strains.

The host-binding domain of the P2 phage tail spike reveals a trimeric iron-binding structure.

The adsorption and infection of bacteriophage P2 is mediated by tail fibres and tail spikes. The tail spikes on the tail baseplate are used to irreversibly adsorb to the host cells. Recently, a P2 phage tail-spike protein, gpV, was purified and it was shown that a C-terminal domain, Ser87-Leu211, is sufficient for the binding of gpV to host Escherichia coli membranes [Kageyama et al. (2009), Biochemistry, 48, 10129-10135]. In this paper, the crystal structure of the C-terminal domain of P2 gpV is reported. The structure is a triangular pyramid and looks like a spearhead composed of an intertwined ß-sheet, a triple ß-helix and a metal-binding region containing iron, calcium and chloride ions.

Crystal structure of Bacillus subtilis SPP1 phage gp22 shares fold similarity with a domain of lactococcal phage p2 RBP.

SPP1 is a siphophage infecting the gram-positive bacterium Bacillus subtilis. It is constituted by an icosahedric head and a long non-contractile tail formed by gene products (gp) 17-21. A group of 5 small genes (gp 22-24.1) follows in the genome those coding for the main tail components. However, the belonging of the corresponding gp to the tail or to other parts of the phage is not documented. Among these, gp22 lacks sequence identity to any known protein. We report here the gp22 structure solved by X-ray crystallography at 2.35 A resolution. We found that gp22 is a monomer in solution and possesses a significant structural similarity with lactococcal phage p2 ORF 18 N-terminal "shoulder" domain.

Structure of lactococcal phage p2 baseplate and its mechanism of activation.

Siphoviridae is the most abundant viral family on earth which infects bacteria as well as archaea. All known siphophages infecting gram+ Lactococcus lactis possess a baseplate at the tip of their tail involved in host recognition and attachment. Here, we report analysis of the p2 phage baseplate structure by X-ray crystallography and electron microscopy and propose a mechanism for the baseplate activation during attachment to the host cell. This approximately 1 MDa, Escherichia coli-expressed baseplate is composed of three protein species, including six trimers of the receptor-binding protein (RBP). RBPs host-recognition domains point upwards, towards the capsid, in agreement with the electron-microscopy map of the free virion. In the presence of Ca(2+), a cation mandatory for infection, the RBPs rotated 200 degrees downwards, presenting their binding sites to the host, and a channel opens at the bottom of the baseplate for DNA passage. These conformational changes reveal a novel siphophage activation and host-recognition mechanism leading ultimately to DNA ejection.

Observation of the membrane binding activity and domain structure of gpV, which comprises the tail spike of bacteriophage P2.

The P2 phage virion has tail spike proteins beneath the baseplate and uses them to adsorb to the outer membrane of Escherichia coli during the infection process. Previous immunoelectron microscopic studies suggested that the tail spikes are composed of the gene V product (gpV); however, experimental evidence of its membrane binding activity has yet to be reported. In this study, we purified and characterized recombinant full-length gpV and its C-terminal domain. Limited chymotrypsin proteolysis of gpV produced a C-terminal domain composed of Ser86-Leu211. Our experiments demonstrated that the N- and C-terminal domains have very different melting temperatures: 50 and 74 degrees C, respectively. We also found that gpV binds the E. coli membrane via its C-terminal domain. We conclude that the C-terminal domain of gpV is a stable trimer and serves as the receptor-binding domain for the second step in the phage adsorption process.

A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites.

The Cox protein of the coliphage P2 is multifunctional; it acts as a transcriptional repressor of the Pc promoter, as a transcriptional activator of the P(LL) promoter of satellite phage P4, and as a directionality factor for site-specific recombination. The Cox proteins constitute a unique group of directionality factors since they couple the developmental switch with the integration or excision of the phage genome. In this work, the DNA binding characteristics of the Cox protein of WPhi, a P2-related phage, are compared with those of P2 Cox. P2 Cox has been shown to recognize a 9 bp sequence, repeated at least 6 times in different targets. In contrast to P2 Cox, WPhi Cox binds with a strong affinity to the early control region that contains an imperfect direct repeat of 12 nucleotides. The removal of one of the repeats has drastic effects on the capacity of WPhi to bind to the Pe-Pc region. Again in contrast to P2 Cox, WPhi Cox has a lower affinity to attP compared to the Pe-Pc region, and a repeat of 9 bp can be found that has 5 bp in common with the repeat in the Pe-Pc region. WPhi Cox, however, is essential for excisive recombination in vitro. WPhi Cox, like P2 Cox, binds cooperatively with integrase to attP. Both Cox proteins induce a strong bend in their DNA targets upon binding.

Crystal structure of ORF12 from Lactococcus lactis phage p2 identifies a tape measure protein chaperone.

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is detected in the second half of the lytic cycle, indicating that it is a late-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-A resolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface. Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice was identified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeated motif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex between ORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fitted exquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act as a chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of the lactococcal siphophage p2.

Functional domains of the bacteriophage P2 scaffolding protein: identification of residues involved in assembly and protease activity.

Bacteriophage P2 encodes a scaffolding protein, gpO, which is required for correct assembly of P2 procapsids from the gpN major capsid protein. The 284 residue gpO protein also acts as a protease, cleaving itself into an N-terminal fragment, O, that remains in the capsid following maturation. In addition, gpO is presumed to act as the maturation protease for gpN, which is N-terminally processed to N, accompanied by DNA packaging and capsid expansion. The protease activity of gpO resides in the N-terminal half of the protein. We show that gpO is a classical serine protease, with a catalytic triad comprised of Asp 19, His 48 and Ser 107. The C-terminal 90 amino acids of gpO are required and sufficient for capsid assembly. This fragment contains a predicted alpha-helical segment between residues 197 and 257 and exists as a multimer in solution, suggesting that oligomerization is required for scaffolding activity. Correct assembly requires the C-terminal cysteine residue, which is most likely involved in transient gpN interactions. Our results suggest a model for gpO scaffolding action in which the N-terminal half of gpO binds strongly to gpN, while oligomerization of the C-terminal alpha-helical domain of gpO and transient interactions between Cys 284 and gpN lead to capsid assembly.

Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein.

This note presents the (1)H, (13)C, and (15)N resonances assignment of the 22 kDa, dimeric, C-repressor protein from the P2 bacteriophage. The C-repressor controls the genetic switch that determines if the temperate P2 phage should exist in the lytic or lysogenic lifemode.

Receptor-binding protein of Lactococcus lactis phages: identification and characterization of the saccharide receptor-binding site.

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-A resolution structure of RBP, was found to bind tightly (Kd= 0.26 microM) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.

Lactococcal bacteriophage p2 receptor-binding protein structure suggests a common ancestor gene with bacterial and mammalian viruses.

Lactococcus lactis is a Gram-positive bacterium used extensively by the dairy industry for the manufacture of fermented milk products. The double-stranded DNA bacteriophage p2 infects specific L. lactis strains using a receptor-binding protein (RBP) located at the tip of its noncontractile tail. We have solved the crystal structure of phage p2 RBP, a homotrimeric protein composed of three domains: the shoulders, a beta-sandwich attached to the phage; the neck, an interlaced beta-prism; and the receptor-recognition head, a seven-stranded beta-barrel. We used the complex of RBP with a neutralizing llama VHH domain to identify the receptor-binding site. Structural similarity between the recognition-head domain of phage p2 and those of adenoviruses and reoviruses, which invade mammalian cells, suggests that these viruses, despite evolutionary distant targets, lack of sequence similarity and the different chemical nature of their genomes (DNA versus RNA), might have a common ancestral gene.

Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox.

Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites (attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to lambda, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.

Cleavage leads to expansion of bacteriophage P4 procapsids in vitro.

Proteolytic cleavage of the structural proteins is an important part of the maturation process for most bacteriophages and other viruses. In the double-stranded DNA bacteriophages this cleavage is associated with DNA packaging, capsid expansion, and scaffold removal. To understand the role of protein cleavage in the expansion of bacteriophages P2 and P4, we have experimentally cleaved P4 procapsids produced by overexpression of the capsid and scaffolding proteins. The cleavage leads to particle expansion and scaffold removal in vitro. The resulting expanded capsid has a thin-shelled structure similar, but not identical, to that of mature virions.

[Preparation and identification of phage-displayed ICAM-1-mimic peptide].

AIM: To obtain bioactive ICAM-1 mimetic peptide. METHODS: Phages displaying P1 and P2 were prepared by phage amplication and PEG precipitation. The binding between phage-displayed peptides and anti-ICAM-1 mAb 15.2 was evaluated by sandwich ELISA and competitive ELISA. Bioactivities of P1 and P2 was detected by immunohistochemical staining. RESULTS: Phage-displayed peptides P1 and P2 could specifically bind to mAb 15.2, and the binding could be competitively inhibited by ICAM-1. Immunohistochemical staining showed that P1 and P2 could mimic the binding of ICAM-1 to its receptor LFA-1. CONCLUSION: Phage-displayed peptides P1 and P2 are bioactive just as native ICAM-1.