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.     

Identification of a New P335 Subgroup through Molecular Analysis of Lactococcal Phages Q33 and BM13

Lactococcal dairy starter strains are under constant threat from phages in dairy fermentation facilities, especially by members of the so-called 936, P335, and c2 species. Among these three phage groups, members of the P335 species are the most genetically diverse. Here, we present the complete genome sequences of two P335-type phages, Q33 and BM13, isolated in North America and representing a novel lineage within this phage group. The Q33 and BM13 genomes exhibit homology, not only to P335-type, but also to elements of the 936-type phage sequences. The two phage genomes also have close relatedness to phages infecting Enterococcus and Clostridium, a heretofore unknown feature among lactococcal P335 phages. The Q33 and BM13 genomes are organized in functionally related clusters with genes encoding functions such as DNA replication and packaging, morphogenesis, and host cell lysis. Electron micrographic analysis of the two phages highlights the presence of a baseplate more reminiscent of the baseplate of 936 phages than that of the majority of members of the P335 group, with the exception of r1t and LC3.     

Argentinean Lactococcus lactis bacteriophages: genetic characterization and adsorption studies

Aims: Characterization of four virulent Lactococcus lactis phages (CHD, QF9, QF12 and QP4) isolated from whey samples obtained from Argentinean cheese plants. Methods and Results: Phages were characterized by means of electron microscopy, host range and DNA studies. The influence of Ca²⁺, physiological cell state, pH and temperature on cell adsorption was also investigated. The double‐stranded DNA genomes of these lactococcal phages showed distinctive restriction patterns. Using a multiplex PCR, phage QP4 was classified as a member of the P335 polythetic species while the three others belong to the 936 group. Ca²⁺ was not needed for phage adsorption but indispensable to complete cell lysis by phage QF9. The lactococci phages adsorbed normally between pH 5 and pH 8, and from 0°C to 40°C, with the exception of phage QF12 which had an adsorption rate significantly lower at pH 8 and 0°C. Conclusions: Lactococcal phages from Argentina belong to the same predominant groups of phages found in other countries and they have the same general characteristics. Significance and Impact of the Study: This work is the first study to characterize Argentinean L. lactis bacteriophages.     

Identification of the Receptor-Binding Protein in 936-Species Lactococcal Bacteriophages

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.     

Listeria phage A511, a model for the contractile tail machineries of SPO1‐related bacteriophages

Recognition of the bacterial host and attachment to its surface are two critical steps in phage infection. Here we report the identification of Gp108 as the host receptor‐binding protein of the broad host‐range, virulent Listeria phage A511. The ligands for Gp108 were found to be N‐acetylglucosamine and rhamnose substituents of the wall teichoic acids of the bacterial cell wall. Transmission electron microscopy and immunogold‐labelling allowed us to create a model of the A511 baseplate in which Gp108 forms emanating short tail fibres. Data obtained for related phages, such as Staphylococcus phages ISP and Twort, demonstrate the evolutionary conservation of baseplate components and receptor‐binding proteins within the Spounavirinae subfamily, and contractile tail machineries in general. Our data reveal key elements in the infection process of large phages infecting Gram‐positive bacteria and generate insights into the complex adsorption process of phage A511 to its bacterial host.     

Revisiting the host adhesion determinants of Streptococcus thermophilus siphophages

Available 3D structures of bacteriophage modules combined with predictive bioinformatic algorithms enabled the identification of adhesion modules in 57 siphophages infecting Streptococcus thermophilus (St). We identified several carbohydrate-binding modules (CBMs) in so-called evolved distal tail (Dit) and tail-associated lysozyme (Tal) proteins of St phage baseplates. We examined the open reading frame (ORF) downstream of the Tal-encoding ORF and uncovered the presence of a putative p2-like receptor-binding protein (RBP). A 21 Å resolution electron microscopy structure of the baseplate of cos-phage STP1 revealed the presence of six elongated electron densities, surrounding the core of the baseplate, that harbour the p2-like RBPs at their tip. To verify the functionality of these modules, we expressed GFP- or mCherry-coupled Tal and putative RBP CBMs and observed by fluorescence microscopy that both modules bind to their corresponding St host, the putative RBP CBM with higher affinity than the Tal-associated one. The large number of CBM functional domains in St phages suggests that they play a contributory role in the infection process, a feature that we previously described in lactococcal phages and beyond, possibly representing a universal feature of the siphophage host-recognition apparatus.     

Genomic,Proteomic, Morphological,and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia – Salmonella genera.     

The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.     

Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Since 2008, a global outbreak of P. syringae pv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance in P. syringae pv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active against P. syringae pv. actinidiae. Extensive host range testing on P. syringae pv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to the Caudovirales and were analyzed based on morphology and genome size, which showed them to be representatives of Myoviridae, Podoviridae, and Siphoviridae. Twenty-one Myoviridae members have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of these Myoviridae members were sequenced, and each was unique. The most closely related sequenced phages were a group infecting Pseudomonas aeruginosa and characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization of P. syringae pv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker.     

Biodiversity and classification of lactococcal phages

For this study, an in-depth review of the classification of Lactococcus lactis phages was performed. Reference phages as well as unclassified phages from international collections were analyzed by stringent DNA-DNA hybridization studies, electron microscopy observations, and sequence analyses. A new classification scheme for lactococcal phages is proposed that reduces the current 12 groups to 8. However, two new phages (Q54 and 1706), which are unrelated to known lactococcal phages, may belong to new emerging groups. The multiplex PCR method currently used for the rapid identification of phages from the three main lactococcal groups (936, c2, and P335) was improved and tested against the other groups, none of which gave a PCR product, confirming the specificity of this detection tool. However, this method does not detect all members of the highly diverse P335 group. The lactococcal phages characterized here were deposited in the Félix d'Hérelle Reference Center for Bacterial Viruses and represent a highly diverse viral community from the dairy environment.     

The terminally redundant, nonpermuted genome of Listeria bacteriophage A511: a model for the SPO1-like myoviruses of gram-positive bacteria

Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting low-G+C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (approximately 6 kb) and K (approximately 20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G+C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.     

Immune electron microscopy in the study of biological matter

A brief review of literature data and our investigations on the antibodies used for specific labeling in electron microscopy is presented. Considered are the problems connected with structure and function of separate components of bacterial viruses revealed by means of specific antibodies. The results of fine differentiation of antigenic components in the case of phages of the colidysentery group allowed to elucidate the functional role of the adsorption apparatus in the course of phage interaction with the bacterial cell. The topology of structural proteins (gene-products 35, 36, 37) of the tail's long strands for phages T4, DDVI+h and DDVIh is determined. Antigenic properties of proteins that are found in the composition of two forms of Bacillus mycoides are demonstrated immune-electronmicroscopically. On the basis of this finding, a conclusion was made that one of these phages acted as precursor, the other--as satellite during the simultaneous development of these phages in the bacterial cell. It was also established that temperate and virulent phages are related antigenically, which proves that lysogenic bacteria can be one of the phage sources on the environmental conditions. Visualization of non-ribosomal genes of procaryots that code for structural proteins of a defective phage proves the efficiency of the immune-electronmicroscopic method for investigating of biological objects.     

A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages.

Direct visualization of filamentous phage infection in Escherichia coli (E. coli) was attempted using biotinylated phages (BIO-phages). The biotinylation of the phages did not influence their infectivity into E. coli. E. coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E. coli could be directly observed by using the avidin-biotin-peroxidase complex method with electron microscopy. This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin-avidin interaction. This simple and powerful method is applicable to the study of infection by various viruses.     

Early events in polyoma virus infection: attachment, penetration, and nuclear entry.

The plaque-assay technique was used as a tool to determine the optimal conditions for adsorption of polyoma virions to host cells. Using these optimal conditions of adsorption, an electron microscopy study of the early events of infection was performed. By electron microscopy and autoradiography, it was demonstrated that both the viral coat proteins and DNA arrive simultaneously in the nucleus as early as 15 min postinfection. When horseradish peroxidase-labeled virions, pseudovirions, and capsids were used to infect cells, only the particles with nucleic acid or a factor(s) associated with the nucleic acid, i.e., histones, appeared to enter the nucleus. Moreover, when virions were used to infect either permissive or nonpermissive cells, identical early events of viral infection, i.e., adsorption, penetration, and nuclear transport, were observed, suggesting that these early events of infection are a property of the virion and not the host cell.     

Reorganization of the Endosomal System in Salmonella-Infected Cells: The Ultrastructure of Salmonella-Induced Tubular Compartments

During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella.     

Crystal Structure of Bacteriophage SPP1 Distal Tail Protein (gp19.1): A BASEPLATE HUB PARADIGM IN GRAM-POSITIVE INFECTING PHAGES*

Siphophage SPP1 infects the Gram-positive bacterium Bacillus subtilis using its long non-contractile tail and tail-tip. Electron microscopy (EM) previously allowed a low resolution assignment of most orf products belonging to these regions. We report here the structure of the SPP1 distal tail protein (Dit, gp19.1). The combination of x-ray crystallography, EM, and light scattering established that Dit is a back-to-back dimer of hexamers. However, Dit fitting in the virion EM maps was only possible with a hexamer located between the tail-tube and the tail-tip. Structure comparison revealed high similarity between Dit and a central component of lactophage baseplates. Sequence similarity search expanded its relatedness to several phage proteins, suggesting that Dit is a docking platform for the tail adsorption apparatus in Siphoviridae infecting Gram-positive bacteria and that its architecture is a paradigm for these hub proteins. Dit structural similarity extends also to non-contractile and contractile phage tail proteins (gpV_N and XkdM) as well as to components of the bacterial type 6 secretion system, supporting an evolutionary connection between all these devices.     

Host genetic requirements for DNA release of lactococcal phage TP901-1

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.     

Narrow-Host-Range Bacteriophages That Infect Rhizobium etli Associate with Distinct Genomic Types

In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ∼45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species.     

Identification of the receptor-binding protein in 936-species lactococcal bacteriophages

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and phi 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.