Evolved distal tail carbohydrate binding modules of Lactobacillus phage J‐1: a novel type of anti‐receptor widespread among lactic acid bacteria phages

Bacteriophage replication requires specific host‐recognition. Some siphophages harbour a large complex, the baseplate, at the tip of their non‐contractile tail. This baseplate holds receptor binding proteins (RBPs) that can recognize the host cell‐wall polysaccharide (CWPS) and specifically attach the phage to its host. While most phages possess a dedicated RBP, the phage J‐1 that infects Lactobacillus casei seemed to lack one. It has been shown that the phage J‐1 distal tail protein (Dit) plays a role in host recognition and that its sequence comprises two inserted modules compared with ‘classical’ Dits. The first insertion is similar to carbohydrate‐binding modules (CBMs), whereas the second insertion remains undocumented. Here, we determined the structure of the second insertion and found it also similar to several CBMs. Expressed insertion CBM2, but not CBM1, binds to L. casei cells and neutralize phage attachment to the bacterial cell wall and the isolated and purified CWPS of L. casei BL23 prevents CBM2 attachment to the host. Electron microscopy single particle reconstruction of the J‐1 virion baseplate revealed that CBM2 is projected at the periphery of Dit to optimally bind the CWPS receptor. Taken together, these results identify J‐1 evolved Dit as the phage RBP.     

Mapping the Tail Fiber as the Receptor Binding Protein Responsible for Differential Host Specificity of Pseudomonas aeruginosa Bacteriophages PaP1 and JG004

The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.     

The targeted recognition of Lactococcus lactis phages to their polysaccharide receptors

Each phage infects a limited number of bacterial strains through highly specific interactions of the receptor‐binding protein (RBP) at the tip of phage tail and the receptor at the bacterial surface. Lactococcus lactis is covered with a thin polysaccharide pellicle (hexasaccharide repeating units), which is used by a subgroup of phages as a receptor. Using L. lactis and phage 1358 as a model, we investigated the interaction between the phage RBP and the pellicle hexasaccharide of the host strain. A core trisaccharide (TriS), derived from the pellicle hexasaccharide repeating unit, was chemically synthesised, and the crystal structure of the RBP/TriS complex was determined. This provided unprecedented structural details of RBP/receptor site‐specific binding. The complete hexasaccharide repeating unit was modelled and found to aptly fit the extended binding site. The specificity observed in in vivo phage adhesion assays could be interpreted in view of the reported structure. Therefore, by combining synthetic carbohydrate chemistry, X‐ray crystallography and phage plaquing assays, we suggest that phage adsorption results from distinct recognition of the RBP towards the core TriS or the remaining residues of the hexasacchride receptor. This study provides a novel insight into the adsorption process of phages targeting saccharides as their receptors.     

A receptor‐binding protein of Campylobacter jejuni bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino‐modified pseudaminic acid

Bacteriophage receptor‐binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far‐western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino‐modified pseudaminic acid. This binding activity is localized to the C‐terminal quarter of the protein and both wild‐type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.     

A mutant bacteriophage evolved to infect resistant bacteria gained a broader host range

Bacteriophages (phages) are the most abundant entities in nature, yet little is known about their capacity to acquire new hosts and invade new niches. By exploiting the Gram‐positive soil bacterium Bacillus subtilis (B. subtilis) and its lytic phage SPO1 as a model, we followed the coevolution of bacteria and phages. After infection, phage‐resistant bacteria were readily isolated. These bacteria were defective in production of glycosylated wall teichoic acid (WTA) polymers that served as SPO1 receptor. Subsequently, a SPO1 mutant phage that could infect the resistant bacteria evolved. The emerging phage contained mutations in two genes, encoding the baseplate and fibers required for host attachment. Remarkably, the mutant phage gained the capacity to infect non‐host Bacillus species that are not infected by the wild‐type phage. We provide evidence that the evolved phage lost its dependency on the species‐specific glycosylation pattern of WTA polymers. Instead, the mutant phage gained the capacity to directly adhere to the WTA backbone, conserved among different species, thereby crossing the species barrier.     

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.     

Structural and Functional Studies of gpX of Escherichia coli Phage P2 Reveal a Widespread Role for LysM Domains in the Baseplates of Contractile-Tailed Phages

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.     

Structural characterization and assembly of the distal tail structure of the temperate lactococcal bacteriophage TP901-1

The tail structures of bacteriophages infecting gram-positive bacteria are largely unexplored, although the phage tail mediates the initial interaction with the host cell. The temperate Lactococcus lactis phage TP901-1 of the Siphoviridae family has a long noncontractile tail with a distal baseplate. In the present study, we investigated the distal tail structures and tail assembly of phage TP901-1 by introducing nonsense mutations into the late transcribed genes dit (orf46), tal(TP901-1) (orf47), bppU (orf48), bppL (orf49), and orf50. Transmission electron microscopy examination of mutant and wild-type TP901-1 phages showed that the baseplate consisted of two different disks and that a central tail fiber is protruding below the baseplate. Evaluation of the mutant tail morphologies with protein profiles and Western blots revealed that the upper and lower baseplate disks consist of the proteins BppU and BppL, respectively. Likewise, Dit and Tal(TP901-1) were shown to be structural tail proteins essential for tail formation, and Tal(TP901-1) was furthermore identified as the tail fiber protein by immunogold labeling experiments. Determination of infection efficiencies of the mutant phages showed that the baseplate is fundamental for host infection and the lower disk protein, BppL, is suggested to interact with the host receptor. In contrast, ORF50 was found to be nonessential for tail assembly and host infection. A model for TP901-1 tail assembly, in which the function of eight specific proteins is considered, is presented.     

Identification of the lower baseplate protein as the antireceptor of the temperate lactococcal bacteriophages TP901-1 and Tuc2009

The first step in the infection process of tailed phages is recognition and binding to the host receptor. This interaction is mediated by the phage antireceptor located in the distal tail structure. The temperate Lactococcus lactis phage TP901-1 belongs to the P335 species of the Siphoviridae family, which also includes the related phage Tuc2009. The distal tail structure of TP901-1 is well characterized and contains a double-disk baseplate and a central tail fiber. The structural tail proteins of TP901-1 and Tuc2009 are highly similar, but the phages have different host ranges and must therefore encode different antireceptors. In order to identify the antireceptors of TP901-1 and Tuc2009, a chimeric phage was generated in which the gene encoding the TP901-1 lower baseplate protein (bppL(TP901-1)) was exchanged with the analogous gene (orf53(2009)) of phage Tuc2009. The chimeric phage (TP901-1C) infected the Tuc2009 host strain efficiently and thus displayed an altered host range compared to TP901-1. Genomic analysis and sequencing verified that TP901-1C is a TP901-1 derivative containing the orf53(2009) gene in exchange for bppL(TP901-1); however, a new sequence in the late promoter region was also discovered. Protein analysis confirmed that TP901-1C contains ORF53(2009) and not the lower baseplate protein BppL(TP901-1), and it was concluded that BppL(TP901-1) and ORF53(2009) constitute antireceptor proteins of TP901-1 and Tuc2009, respectively. Electron micrographs revealed altered baseplate morphology of TP901-1C compared to that of the parental phage.     

Structure and Functional Analysis of the Host Recognition Device of Lactococcal Phage Tuc2009

Many phages employ a large heteropolymeric organelle located at the tip of the tail, termed the baseplate, for host recognition. Contrast electron microscopy (EM) of the lactococcal phage Tuc2009 baseplate and its host-binding subunits, the so-called tripods, allowed us to obtain a low-resolution structural image of this organelle. Structural comparisons between the baseplate of the related phage TP901-1 and that of Tuc2009 demonstrated that they are highly similar, except for the presence of an additional protein in the Tuc2009 baseplate (BppA_Tuc2009), which is attached to the top of the Tuc2009 tripod structure. Recombinantly produced Tuc2009 or TP901-1 tripods were shown to bind specifically to their particular host cell surfaces and are capable of almost fully and specifically eliminating Tuc2009 or TP901-1 phage adsorption, respectively. In the case of Tuc2009, such adsorption-blocking ability was reduced in tripods that lacked BppA_Tuc2009, indicating that this protein increases the binding specificity and/or affinity of the Tuc2009 tripod to its host receptor.     

Not a barrier but a key: How bacteriophages exploit host's O‐antigen as an essential receptor to initiate infection

Tailed bacteriophages specific for Gram‐negative bacteria encounter lipopolysaccharide (LPS) during the first infection steps. Yet, it is not well understood how biochemistry of these initial interactions relates to subsequent events that orchestrate phage adsorption and tail rearrangements to initiate cell entry. For many phages, long O‐antigen chains found on the LPS of smooth bacterial strains serve as essential receptor recognized by their tailspike proteins (TSP). Many TSP are depolymerases and O‐antigen cleavage was described as necessary step for subsequent orientation towards a secondary receptor. However, O‐antigen specific host attachment must not always come along with O‐antigen degradation. In this issue of Molecular Microbiology Prokhorov et al. report that coliphage G7C carries a TSP that deacetylates O‐antigen but does not degrade it, whereas rough strains or strains lacking O‐antigen acetylation remain unaffected. Bacteriophage G7C specifically functionalizes its tail by attaching the deacetylase TSP directly to a second TSP that is nonfunctional on the host's O‐antigen. This challenges the view that bacteriophages use their TSP only to clear their way to a secondary receptor. Rather, O‐antigen specific phages may employ enzymatically active TSP as a tool for irreversible LPS membrane binding to initiate subsequent infection steps.     

Structure of the central hub of bacteriophage Mu baseplate determined by X-ray crystallography of gp44

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.     

The flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi‐like protein tail fibre

The discovery of a Salmonella‐targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi‐like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi‐like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.     

A topological model of the baseplate of lactococcal phage Tuc2009

Phages infecting Lactococcus lactis, a Gram-positive bacterium, are a recurrent problem in the dairy industry. Despite their economical importance, the knowledge on these phages, belonging mostly to Siphoviridae, lags behind that accumulated for members of Myoviridae. The three-dimensional structures of the receptor-binding proteins (RBP) of three lactococcal phages have been determined recently, illustrating their modular assembly and assigning the nature of their bacterial receptor. These RBPs are attached to the baseplate, a large phage organelle, located at the tip of the tail. Tuc2009 baseplate is formed by the products of 6 open read frames, including the RBP. Because phage binding to its receptor induces DNA release, it has been postulated that the baseplate might be the trigger for DNA injection. We embarked on a structural study of the lactococcal phages baseplate, ultimately to gain insight into the triggering mechanism following receptor binding. Structural features of the Tuc2009 baseplate were established using size exclusion chromatography coupled to on-line UV-visible absorbance, light scattering, and refractive index detection (MALS/UV/RI). Combining the results of this approach with literature data led us to propose a "low resolution" model of Tuc2009 baseplate. This model will serve as a knowledge base to submit relevant complexes to crystallization trials.     

Bacteriophages drive strain diversification in a marine Flavobacterium: implications for phage resistance and physiological properties

Genetic, structural and physiological differences between strains of the marine bacterium Cellulophaga baltica MM#3 (Flavobacteriaceae) developing in response to the activity of two virulent bacteriophages, ΦS_M and ΦS_T, was investigated during 3 weeks incubation in chemostat cultures. A distinct strain succession towards increased phage resistance and a diversification of the metabolic properties was observed. During the incubation the bacterial population diversified from a single strain, which was sensitive to 24 tested Cellulophaga phages, into a multistrain and multiresistant population, where the dominant strains had lost susceptibility to up to 22 of the tested phages. By the end of the experiment the cultures reached a quasi steady state dominated by ΦS_T‐resistant and ΦS_M + ΦS_T‐resistant strains coexisting with small populations of phage‐sensitive strains sustaining both phages at densities of > 10⁶ plaque forming units (pfu) ml^?1. Loss of susceptibility to phage infection was associated with a reduction in the strains' ability to metabolize various carbon sources as demonstrated by BIOLOG assays. This suggested a cost of resistance in terms of reduced physiological capacity. However, there was no direct correlation between the degree of resistance and the loss of metabolic properties, suggesting either the occurrence of compensatory mutations in successful strains or that the cost of resistance in some strains was associated with properties not resolved by the BIOLOG assay. The study represents the first direct demonstration of phage‐driven generation of functional diversity within a marine bacterial host population with significant implications for both phage susceptibility and physiological properties. We propose, therefore, that phage‐mediated selection for resistant strains contributes significantly to the extensive microdiversity observed within specific bacterial species in marine environments.     

Lactococcal 949 Group Phages Recognize a Carbohydrate Receptor on the Host Cell Surface

Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages.     

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.     

Site-specific recombination of T2 phage using IP008 long tail fiber genes provides a targeted method for expanding host range while retaining lytic activity

The application of bacteriophages (phages) in therapy urgently requires the production of wide-host-range recombinant phages that possess strong lytic activity. The wide-host-range IP008 phage was classified by transmission electron microscopy analysis as an A2 morphotype member of the Myoviridae family of the order Caudovirales . IP008 showed a high homology (99.4% similarity in the amino acid alignment of the major capsid protein Gp 23) with KEP10, another wide-host-range phage. The long tail fiber genes (genes 37 and 38 ) from the genome of T2 were replaced with those of the IP008 phage by homologous recombination. The host range of the recombinant phages was identical to that of IP008. Furthermore, the recombinant phage bacterial lytic activity was restored. Future analyses of host-range mutants of the closely related phages T2 and IP008 could lead to a more precise localization of the genetic factors responsible for receptor specificity.     

Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

Bacteriophages engage in complex dynamic interactions with their bacterial hosts and with each other. Bacteria have numerous mechanisms to resist phage infection, and phages must co‐evolve by overcoming bacterial resistance or by choosing an alternative host. Phages also compete with each other, both during lysogeny by prophage‐mediated defense against viral attack and by superinfection exclusion during lytic replication. Phages are enormously diverse genetically and are replete with small genes of unknown function, many of which are not required for lytic growth, but which may modulate these bacteria–phage and phage–phage dynamics. Using cellular toxicity of phage gene overexpression as an assay, we identified the 93‐residue protein gp52 encoded by Cluster F mycobacteriophage Fruitloop. The toxicity of Fruitloop gp52 overexpression results from interaction with and inactivation of Wag31 (DivIVA), an essential Mycobacterium smegmatis protein organizing cell wall biosynthesis at the growing cellular poles. Fruitloop gene 52 is expressed early in lytic growth and is not required for normal Fruitloop lytic replication but interferes with Subcluster B2 phages such as Hedgerow and Rosebush. We conclude that Hedgerow and Rosebush are Wag31‐dependent phages and that Fruitloop gp52 confers heterotypic superinfection exclusion by inactivating Wag31.