23540272
Cryo-Electron Microscopy Structure of Lactococcal Siphophage 1358 Virion
ABSTRACT Lactococcus lactis, a Gram+ lactic acid-producing bacterium used for the manufacture of several fermented dairy products, is subject to infection by diverse virulent tailed phages, leading to industrial fermentation failures. This constant viral risk has led to a sustained interest in the study of their biology, diversity, and evolution. Lactococcal phages now constitute a wide ensemble of at least 10 distinct genotypes within the Caudovirales order, many of them belonging to the Siphoviridae family. Lactococcal siphophage 1358, currently the only member of its group, displays a noticeably high genomic similarity to some Listeria phages as well as a host range limited to a few L. lactis strains. These genomic and functional characteristics stimulated our interest in this phage. Here, we report the cryo-electron microscopy structure of the complete 1358 virion. Phage 1358 exhibits noteworthy features, such as a capsid with dextro handedness and protruding decorations on its capsid and tail. Observations of the baseplate of virion particles revealed at least two conformations, a closed and an open, activated form. Functional assays uncovered that the adsorption of phage 1358 to its host is Ca2+ independent, but this cation is necessary to complete its lytic cycle. Taken together, our results provide the complete structural picture of a unique lactococcal phage and expand our knowledge on the complex baseplate of phages of the Siphoviridae family. IMPORTANCE Phages of Lactococcus lactis are investigated mainly because they are sources of milk fermentation failures in the dairy industry. Despite the availability of several antiphage measures, new phages keep emerging in this ecosystem. In this study, we provide the cryo-electron microscopy reconstruction of a unique lactococcal phage that possesses genomic similarity to particular Listeria phages and has a host range restricted to only a minority of L. lactis strains. The capsid of phage 1358 displays the almost unique characteristic of being dextro handed. Its capsid and tail exhibit decorations that we assigned to nonspecific sugar binding modules. We observed the baseplate of 1358 in two conformations, a closed and an open form. We also found that the adsorption to its host, but not infection, is Ca2+ independent. Overall, this study advances our understanding of the adhesion mechanisms of siphophages.
19887164
Bacteriophage electron microscopy.
Since the advent of the electron microscope approximately 70 years ago, bacterial viruses and electron microscopy are inextricably linked. Electron microscopy proved that bacteriophages are particulate and viral in nature, are complex in size and shape, and have intracellular development cycles and assembly pathways. The principal contribution of electron microscopy to bacteriophage research is the technique of negative staining. Over 5500 bacterial viruses have so far been characterized by electron microscopy, making bacteriophages, at least on paper, the largest viral group in existence. Other notable contributions are cryoelectron microcopy and three-dimensional image reconstruction, particle counting, and immunoelectron microscopy. Scanning electron microscopy has had relatively little impact. Transmission electron microscopy has provided the basis for the recognition and establishment of bacteriophage families and is one of the essential criteria to classify novel viruses into families. It allows for instant diagnosis and is thus the fastest diagnostic technique in virology. The most recent major contribution of electron microscopy is the demonstration that the capsid of tailed phages is monophyletic in origin and that structural links exist between some bacteriophages and viruses of vertebrates and archaea. DNA sequencing cannot replace electron microscopy and vice versa.
45967175
Determination of bacteriophage lambda tail length by a protein ruler.
How the size and shape of living structures are determined by genetic information is one of the fundamental problems in biology. Here I describe a study in which the size of a biological supramolecular structure was changed in a predictable way by in vitro genetics, with the size both before and after manipulation being exactly determined. I have studied the tail of bacteriophage lambda, whose length is determined by the length of the 'ruler protein', the product of gene H. The length of the tail can be decreased or increased by deleting the middle part of gene H or by forming a small duplication there, and the length of the tail is proportional to the size of the protein. These results can be regarded as a special case of protein engineering, namely supramolecular protein engineering.
28902573
Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: the Structure of Lactococcal Phage TP901-1
ABSTRACT Tailed phages are genome delivery machines exhibiting unequaled efficiency acquired over more than 3 billion years of evolution. Siphophages from the P335 and 936 families infect the Gram-positive bacterium Lactococcus lactis using receptor-binding proteins anchored to the host adsorption apparatus (baseplate). Crystallographic and electron microscopy (EM) studies have shed light on the distinct adsorption strategies used by phages of these two families, suggesting that they might also rely on different infection mechanisms. Here, we report electron microscopy reconstructions of the whole phage TP901-1 (P335 species) and propose a composite EM model of this gigantic molecular machine. Our results suggest conservation of structural proteins among tailed phages and add to the growing body of evidence pointing to a common evolutionary origin for these virions. Finally, we propose that host adsorption apparatus architectures have evolved in correlation with the nature of the receptors used during infection.
26138922
The tail structure of bacteriophage T4 and its mechanism of contraction
Bacteriophage T4 and related viruses have a contractile tail that serves as an efficient mechanical device for infecting bacteria. A three-dimensional cryo-EM reconstruction of the mature T4 tail assembly at 15-Å resolution shows the hexagonal dome-shaped baseplate, the extended contractile sheath, the long tail fibers attached to the baseplate and the collar formed by six whiskers that interact with the long tail fibers. Comparison with the structure of the contracted tail shows that tail contraction is associated with a substantial rearrangement of the domains within the sheath protein and results in shortening of the sheath to about one-third of its original length. During contraction, the tail tube extends beneath the baseplate by about one-half of its total length and rotates by 345°, allowing it to cross the host's periplasmic space.
5539832
Genome Organization and Characterization of the Virulent Lactococcal Phage 1358 and Its Similarities to Listeria Phages
ABSTRACT Virulent phage 1358 is the reference member of a rare group of phages infecting Lactococcus lactis. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the Siphoviridae family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two Listeria monocytogenes phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.
665110
Structures and host-adhesion mechanisms of lactococcal siphophages
The Siphoviridae family of bacteriophages is the largest viral family on earth and comprises members infecting both bacteria and archaea. Lactococcal siphophages infect the Gram-positive bacterium Lactococcus lactis, which is widely used for industrial milk fermentation processes (e.g., cheese production). As a result, lactococcal phages have become one of the most thoroughly characterized class of phages from a genomic standpoint. They exhibit amazing and intriguing characteristics. First, each phage has a strict specificity toward a unique or a handful of L. lactis host strains. Second, most lactococcal phages possess a large organelle at their tail tip (termed the baseplate), bearing the receptor binding proteins (RBPs) and mediating host adsorption. The recent accumulation of structural and functional data revealed the modular structure of their building blocks, their different mechanisms of activation and the fine specificity of their RBPs. These results also illustrate similarities and differences between lactococcal Siphoviridae and Gram-negative infecting Myoviridae.
19459349
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 β-sandwich attached to the phage; the neck, an interlaced β-prism; and the receptor-recognition head, a seven-stranded β-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.
14700376
Receptor-Binding Protein of Lactococcus lactis Phages: Identification and Characterization of the Saccharide Receptor-Binding Site
ABSTRACT 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-Å resolution structure of RBP, was found to bind tightly (Kd = 0.26 μM) 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.
5455253
Crystal Structure of ORF12 from Lactococcus lactis Phage p2 Identifies a Tape Measure Protein Chaperone
ABSTRACT 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-Å 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.
26722693
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 ∼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 Ca2+, a cation mandatory for infection, the RBPs rotated 200° 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.
27196531
Modular Structure of the Receptor Binding Proteins of Lactococcus lactis Phages
Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the “receptors.” We report here the 1.65 Å resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced β-prism, and a β-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages.
863683
Structure of the phage TP901-1 1.8 MDa baseplate suggests an alternative host adhesion mechanism
Phages of the Caudovirales order possess a tail that recognizes the host and ensures genome delivery upon infection. The X-ray structure of the approximately 1.8 MDa host adsorption device (baseplate) from the lactococcal phage TP901-1 shows that the receptor-binding proteins are pointing in the direction of the host, suggesting that this organelle is in a conformation ready for host adhesion. This result is in marked contrast with the lactococcal phage p2 situation, whose baseplate is known to undergo huge conformational changes in the presence of Ca2+ to reach its active state. In vivo infection experiments confirmed these structural observations by demonstrating that Ca2+ ions are required for host adhesion among p2-like phages (936-species) but have no influence on TP901-1-like phages (P335-species). These data suggest that these two families rely on diverse adhesion strategies which may lead to different signaling for genome release.
33331946
Viral infection modulation and neutralization by camelid nanobodies
Significance Lactococcal siphophages infect Lactococcus lactis, a Gram-positive bacterium used in commercial dairy fermentations. The phage TP901-1 baseplate (BP) recognizes and binds specifically to polysaccharides covering the host cell. We raised llama nanobodies against the BP as tools to dissect the molecular determinants of phage infection. Using complementary techniques, we identified BP binders and determined their affinity and epitopes for their targets and their impact on phage infectivity. X-ray structures revealed that two nanobodies block the BP saccharide binding site, and viral infection assays showed that they neutralize infection, a possible way to circumvent phages detrimental effect on dairy fermentation. Lactococcal phages belong to a large family of Siphoviridae and infect Lactococcus lactis, a Gram-positive bacterium used in commercial dairy fermentations. These phages are believed to recognize and bind specifically to pellicle polysaccharides covering the entire bacterium. The phage TP901-1 baseplate, located at the tip of the tail, harbors 18 trimeric receptor binding proteins (RBPs) promoting adhesion to a specific lactococcal strain. Phage TP901-1 adhesion does not require major conformational changes or Ca2+, which contrasts other lactococcal phages. Here, we produced and characterized llama nanobodies raised against the purified baseplate and the Tal protein of phage TP901-1 as tools to dissect the molecular determinants of phage TP901-1 infection. Using a set of complementary techniques, surface plasmon resonance, EM, and X-ray crystallography in a hybrid approach, we identified binders to the three components of the baseplate, analyzed their affinity for their targets, and determined their epitopes as well as their functional impact on TP901-1 phage infectivity. We determined the X-ray structures of three nanobodies in complex with the RBP. Two of them bind to the saccharide binding site of the RBP and are able to fully neutralize TP901-1 phage infectivity, even after 15 passages,. These results provide clear evidence for a practical use of nanobodies in circumventing lactococcal phages viral infection in dairy fermentation.
9001047
Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2
ABSTRACT 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.
38287751
Cell Surface of Lactococcus lactis Is Covered by a Protective Polysaccharide Pellicle*
In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.
18902675
Differences in Lactococcal Cell Wall Polysaccharide Structure Are Major Determining Factors in Bacteriophage Sensitivity
ABSTRACT Analysis of the genetic locus encompassing a cell wall polysaccharide (CWPS) biosynthesis operon of eight strains of Lactococcus lactis, identified as belonging to the same CWPS type C genotype, revealed the presence of a variable region among the strains examined. The results allowed the identification of five subgroups of the C type named subtypes C1 to C5. This variable region contains genes encoding glycosyltransferases that display low or no sequence homology between the subgroups. In this study, we purified an acidic polysaccharide from the cell wall of L. lactis 3107 (subtype C2) and confirmed that it is structurally different from the previously established CWPS of subtype C1 L. lactis MG1363. The CWPS of L. lactis 3107 is composed of pentasaccharide repeating units linked by phosphodiester bonds with the structure 6-α-Glc-3-β-Galf-3-β-GlcNAc-2-β-Galf-6-α-GlcNAc-1-P. Combinations of genes from the variable region of subtype C2 were introduced into a mutant of subtype C1 L. lactis NZ9000 deficient in CWPS biosynthesis. The resulting recombinant mutant synthesized a polysaccharide with a composition characteristic of that of subtype C2 L. lactis 3107 and not wild-type C1 L. lactis NZ9000. By challenging the recombinant mutant with various lactococcal phages, we demonstrated that CWPS is the host cell surface receptor of tested bacteriophages of both the P335 and 936 groups and that differences between the CWPS structures play a crucial role in determining phage host range. IMPORTANCE Despite the efforts of nearly 80 years of lactococcal phage research, the precise nature of the cell surface receptors of the P335 and 936 phage group receptors has remained elusive. This work demonstrates the molecular nature of a P335 group receptor while bolstering the evidence of its role in host recognition by phages of the 936 group and at least partially explains why such phages have a very narrow host range. The information generated will be instrumental in understanding the molecular mechanisms of how phages recognize specific saccharidic receptors located on the surface of their bacterial host. Despite the efforts of nearly 80 years of lactococcal phage research, the precise nature of the cell surface receptors of the P335 and 936 phage group receptors has remained elusive. This work demonstrates the molecular nature of a P335 group receptor while bolstering the evidence of its role in host recognition by phages of the 936 group and at least partially explains why such phages have a very narrow host range. The information generated will be instrumental in understanding the molecular mechanisms of how phages recognize specific saccharidic receptors located on the surface of their bacterial host.
10824832
Molecular Insights on the Recognition of a Lactococcus lactis Cell Wall Pellicle by the Phage 1358 Receptor Binding Protein
ABSTRACT The Gram-positive bacterium Lactococcus lactis is used for the production of cheeses and other fermented dairy products. Accidental infection of L. lactis cells by virulent lactococcal tailed phages is one of the major risks of fermentation failures in industrial dairy factories. Lactococcal phage 1358 possesses a host range limited to a few L. lactis strains and strong genomic similarities to Listeria phages. We report here the X-ray structures of phage 1358 receptor binding protein (RBP) in complex with monosaccharides. Each monomer of its trimeric RBP is formed of two domains: a “shoulder” domain linking the RBP to the rest of the phage and a jelly roll fold “head/host recognition” domain. This domain harbors a saccharide binding crevice located in the middle of a monomer. Crystal structures identified two sites at the RBP surface, ∼8 Å from each other, one accommodating a GlcNAc monosaccharide and the other accommodating a GlcNAc or a glucose 1-phosphate (Glc1P) monosaccharide. GlcNAc and GlcNAc1P are components of the polysaccharide pellicle that we identified at the cell surface of L. lactis SMQ-388, the host of phage 1358. We therefore modeled a galactofuranose (Galf) sugar bridging the two GlcNAc saccharides, suggesting that the trisaccharidic motif GlcNAc-Galf-GlcNAc (or Glc1P) might be common to receptors of genetically distinct lactococcal phages p2, TP091-1, and 1358. Strain specificity might therefore be elicited by steric clashes induced by the remaining components of the pellicle hexasaccharide. Taken together, these results provide a first insight into the molecular mechanism of host receptor recognition by lactococcal phages. IMPORTANCE Siphophages infecting the Gram-positive bacterium Lactococcus lactis are sources of milk fermentation failures in the dairy industry. We report here the structure of the pellicle polysaccharide from L. lactis SMQ-388, the specific host strain of phage 1358. We determined the X-ray structures of the lytic lactococcal phage 1358 receptor binding protein (RBP) in complex with monosaccharides. The positions and nature of monosaccharides bound to the RBP are in agreement with the pellicle structure and suggest a general binding mode of lactococcal phages to their pellicle saccharidic receptor.
43513971
Comparative Genome Analysis of Listeria Bacteriophages Reveals Extensive Mosaicism, Programmed Translational Frameshifting, and a Novel Prophage Insertion Site
ABSTRACT The genomes of six Listeria bacteriophages were sequenced and analyzed. Phages A006, A500, B025, P35, and P40 are members of the Siphoviridae and contain double-stranded DNA genomes of between 35.6 kb and 42.7 kb. Phage B054 is a unique myovirus and features a 48.2-kb genome. Phage B025 features 3′ overlapping single-stranded genome ends, whereas the other viruses contain collections of terminally redundant, circularly permuted DNA molecules. Phages P35 and P40 have a broad host range and lack lysogeny functions, correlating with their virulent lifestyle. Phages A500, A006, and B025 integrate into bacterial tRNA genes, whereas B054 targets the 3′ end of translation elongation factor gene tsf. This is the first reported case of phage integration into such an evolutionarily conserved genetic element. Peptide fingerprinting of viral proteins revealed that both A118 and A500 utilize +1 and −1 programmed translational frameshifting for generating major capsid and tail shaft proteins with C termini of different lengths. In both cases, the unusual +1 frameshift at the 3′ ends of the tsh coding sequences is induced by overlapping proline codons and cis-acting shifty stops. Although Listeria phage genomes feature a conserved organization, they also show extensive mosaicism within the genome building blocks. Of particular interest is B025, which harbors a collection of modules and sequences with relatedness not only to other Listeria phages but also to viruses infecting other members of the Firmicutes. In conclusion, our results yield insights into the composition and diversity of Listeria phages and provide new information on their function, genome adaptation, and evolution.
1117447
EMAN2: an extensible image processing suite for electron microscopy.
EMAN is a scientific image processing package with a particular focus on single particle reconstruction from transmission electron microscopy (TEM) images. It was first released in 1999, and new versions have been released typically 2-3 times each year since that time. EMAN2 has been under development for the last two years, with a completely refactored image processing library, and a wide range of features to make it much more flexible and extensible than EMAN1. The user-level programs are better documented, more straightforward to use, and written in the Python scripting language, so advanced users can modify the programs' behavior without any recompilation. A completely rewritten 3D transformation class simplifies translation between Euler angle standards and symmetry conventions. The core C++ library has over 500 functions for image processing and associated tasks, and it is modular with introspection capabilities, so programmers can add new algorithms with minimal effort and programs can incorporate new capabilities automatically. Finally, a flexible new parallelism system has been designed to address the shortcomings in the rigid system in EMAN1.
10592588
SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs
This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.
10097682
Image processing for electron microscopy single-particle analysis using XMIPP
We describe a collection of standardized image processing protocols for electron microscopy single-particle analysis using the XMIPP software package. These protocols allow performing the entire processing workflow starting from digitized micrographs up to the final refinement and evaluation of 3D models. A particular emphasis has been placed on the treatment of structurally heterogeneous data through maximum-likelihood refinements and self-organizing maps as well as the generation of initial 3D models for such data sets through random conical tilt reconstruction methods. All protocols presented have been implemented as stand-alone, executable python scripts, for which a dedicated graphical user interface has been developed. Thereby, they may provide novice users with a convenient tool to quickly obtain useful results with minimum efforts in learning about the details of this comprehensive package. Examples of applications are presented for a negative stain random conical tilt data set on the hexameric helicase G40P and for a structurally heterogeneous data set on 70S Escherichia coli ribosomes embedded in vitrified ice.
2350192
Fourier shell correlation threshold criteria.
The resolution value claimed for an electron microscopical three-dimensional reconstruction indicates the overall quality of the experiment. The Fourier shell correlation (FSC) criterion has now become the standard quality measure. However, what has continued to be controversial is the issue of the FSC threshold level at which one defines the reproducible resolution. Here, we discuss the theoretical behaviour of the FSC in conjunction with the various factors which influence it: the number of "voxels" in a given Fourier shell, the symmetry of the structure, and the size of the structure within the reconstruction volume. Both the theoretical considerations and our model experiments show that fixed-valued FSC threshold (like "0.5") may never be used in a reproducible criterion. Fixed threshold values are-as we show here-simply the result of incorrect assumptions in the basic statistics. Two families of FSC threshold curves are discussed: the sigma-factor curves and the new family of bit-based information threshold curves. Whereas sigma-factor curves indicate the resolution level at which one has collected information significantly above the noise level, the information curves indicate the resolution level at which enough information has been collected for interpretation.
2059275
The iterative helical real space reconstruction method: surmounting the problems posed by real polymers.
Many important biological macromolecules exist as helical polymers. Examples are actin, tubulin, myosin, RecA, Rad51, flagellin, pili, and filamentous bacteriophage. The first application of three-dimensional reconstruction from electron microscopic images was to a helical polymer, and a number of laboratories today are using helical tubes of integral membrane proteins for solving the structure of these proteins in the electron microscope at near atomic resolution. We have developed a method to analyze and reconstruct electron microscopic images of macromolecular helical polymers, the iterative helical real space reconstruction (IHRSR) algorithm. We can show that when there is disorder or heterogeneity, when the specimens diffract weakly, or when Bessel functions overlap, we can do far better with our method than can be done using traditional Fourier-Bessel approaches. In many cases, structures that were not even amenable to analysis can be solved at fairly high resolution using our method. The problems inherent in the traditional approach are discussed, and examples are presented illustrating how the IHRSR approach surmounts these problems.
32039818
Image analysis of helical objects: the Brandeis Helical Package.
Although the fundamental steps in Fourier-based image analysis of electron micrographs of helical structures have not changed significantly since the techniques were developed 30 years ago, there have been developments which aid both the analysis itself and the interpretation of results. Increases in computational resources have allowed the automation of many of the repetitive steps in image processing. We describe here a set of computer programs which have been developed at Brandeis University over the past 10 or more years. These programs, referred to as the Brandeis Helical Package, are designed to operate independently of each other with a simple and more uniform protocol for the flow of information between them. The programs are now easier to understand and to use and therefore represent a good tool for the analysis of helical structures.
34934821
Improved medium for lactic streptococci and their bacteriophages.
[This corrects the article on p. 807 in vol. 29.].
45738118
Restriction and Modification in Group N Streptococci: Effect of Heat on Development of Modified Lytic Bacteriophage
The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 × 10−7 for tr plaqued on 799 and 2.1 × 10−7 for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50°C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.
30212788
Evolution of Lactococcus lactis Phages within a Cheese Factory
ABSTRACT We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3′ overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.
18322514
Structural Aspects of the Interaction of Dairy Phages with Their Host Bacteria
Knowledge of phage-host interactions at a fundamental level is central to the design of rational strategies for the development of phage-resistant strains that may be applied in industrial settings. Phages infecting lactic acid bacteria, in particular Lactococcus lactis and Streptococcus thermophilus, negatively impact on dairy fermentation processes with serious economic implications. In recent years a wealth of information on structural protein assembly and topology has become available relating to phages infecting Escherichia coli, Bacillus subtilis and Lactococcus lactis, which act as models for structural analyses of dairy phages. In this review, we explore the role of model tailed phages, such as T4 and SPP1, in advancing our knowledge regarding interactions between dairy phages and their hosts. Furthermore, the potential of currently investigated dairy phages to in turn serve as model systems for this particular group of phages is discussed.
2241888
Solution and electron microscopy characterization of lactococcal phage baseplates expressed in Escherichia coli.
We report here the characterization of several large structural protein complexes forming the baseplates (or part of them) of Siphoviridae phages infecting Lactococcus lactis: TP901-1, Tuc2009 and p2. We revisited a "block cloning" expression strategy and extended this approach to genomic fragments encoding proteins whose interacting partners have not yet been clearly identified. Biophysical characterization of some of these complexes using circular dichroism and size exclusion chromatography, coupled with on-line light scattering and refractometry, demonstrated that the over-produced recombinant proteins interact with each other to form large (up to 1.9MDa) and stable baseplate assemblies. Some of these complexes were characterized by electron microscopy confirming their structural homogeneity as well as providing a picture of their overall molecular shapes and symmetry. Finally, using these results, we were able to highlight similarities and differences with the well characterized much larger baseplate of the myophage T4.
19873185
Bacteriophage Tuc2009 Encodes a Tail-Associated Cell Wall-Degrading Activity
ABSTRACT Tuc2009 is a P335-type member of the tailed-phage supergroup Siphoviridae and was originally identified as a resident prophage of the gram-positive bacterium Lactococcus lactis UC509. A Tuc2009 gene designated tal2009 which is located within the morphogenic module was shown to specify a lytic activity within the 3′ portion of its coding region. Comparative sequence analysis indicated that the cell wall-degrading part of Tal2009 is a member of the M37 protein family and that Tal2009 lacks a cell-binding domain, a finding supported by binding studies. Tal2009 appears to undergo self-mediated posttranslational processing in both L. lactis and Escherichia coli. Antibodies directed against a purified C-terminal portion of Tal2009 were used for immunoelectron microscopy, which showed that Tal2009 is located at the tail tip of Tuc2009. Antibody neutralization studies demonstrated that Tal2009-directed antibodies inhibited the ability of phage to mediate host lysis by more than 100-fold. These data indicate that tal2009 encodes a tail-associated lysin involved in localized cell wall degradation, thus allowing the Tuc2009 DNA injection machinery access to the membrane of its bacterial host.
28851499
The Lactococcal Phages Tuc2009 and TP901-1 Incorporate Two Alternate Forms of Their Tail Fiber into Their Virions for Infection Specialization*
Background: Siphoviridae virions often possess lytic domains facilitating host-penetration. Results: Tuc2009 and TP901-1 virions may contain full-length or truncated tail fibers, possessing or lacking a lytic domain, respectively. Conclusion: Phages with a lytic domain infect stationary phase cells better, whereas truncated derivatives have higher adsorption efficiencies. Significance: The heterogeneous phage population serves to most effectively infect bacteria where levels of cell wall cross-linkage differ. Lactococcal phages Tuc2009 and TP901-1 possess a conserved tail fiber called a tail-associated lysin (referred to as Tal2009 for Tuc2009, and Tal901-1 for TP901-1), suspended from their tail tips that projects a peptidoglycan hydrolase domain toward a potential host bacterium. Tal2009 and Tal901-1 can undergo proteolytic processing mid-protein at the glycine-rich sequence GG(S/N)SGGG, removing their C-terminal structural lysin. In this study, we show that the peptidoglycan hydrolase of these Tal proteins is an M23 peptidase that exhibits d-Ala-d-Asp endopeptidase activity and that this activity is required for efficient infection of stationary phase cells. Interestingly, the observed proteolytic processing of Tal2009 and Tal901-1 facilitates increased host adsorption efficiencies of the resulting phages. This represents, to the best of our knowledge, the first example of tail fiber proteolytic processing that results in a heterogeneous population of two phage types. Phages that possess a full-length tail fiber, or a truncated derivative, are better adapted to efficiently infect cells with an extensively cross-linked cell wall or infect with increased host-adsorption efficiencies, respectively.
27103632
Time-resolved molecular dynamics of bacteriophage HK97 capsid maturation interpreted by electron cryo-microscopy and X-ray crystallography.
The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.
28826291
The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 A resolution.
The HK97 bacteriophage capsid is a unique example of macromolecular catenanes: interlocked rings of covalently attached protein subunits. The chain mail organization of the subunits stabilizes a particle in which the maximum thickness of the protein shell is 18A and the maximum diameter is 550A. The electron density has the appearance of a balloon illustrating the extraordinary strength conferred by the unique subunit organization. The refined structure shows novel qualities of the HK97 shell protein, gp5 that, together with the protease gp4, guides the assembly and maturation of the virion. Although gp5 forms hexamers and pentamers and the subunits exist in different structural environments, the tertiary structures of the seven protein molecules in the viral asymmetric unit are closely similar. The interactions of the subunits in the shell are exceptionally complex with each subunit interacting with nine other subunits. The interactions of the N-terminus released after gp5 cleavage appear important for organization of the loops that become crosslinked to the core of a neighboring subunit at the maturation. A comparison with a model of the Prohead II structure revealed that the surfaces of non-covalent contact between the monomers that build up hexamers/pentamers are completely redefined during maturation.
13227878
The First Structure of a Mycobacteriophage, the Mycobacterium abscessus subsp. bolletii Phage Araucaria
ABSTRACT The unique characteristics of the waxy mycobacterial cell wall raise questions about specific structural features of their bacteriophages. No structure of any mycobacteriophage is available, although ∼3,500 have been described to date. To fill this gap, we embarked in a genomic and structural study of a bacteriophage from Mycobacterium abscessus subsp. bolletii, a member of the Mycobacterium abscessus group. This opportunistic pathogen is responsible for respiratory tract infections in patients with lung disorders, particularly cystic fibrosis. M. abscessus subsp. bolletii was isolated from respiratory tract specimens, and bacteriophages were observed in the cultures. We report here the genome annotation and characterization of the M. abscessus subsp. bolletii prophage Araucaria, as well as the first single-particle electron microscopy reconstruction of the whole virion. Araucaria belongs to Siphoviridae and possesses a 64-kb genome containing 89 open reading frames (ORFs), among which 27 could be annotated with certainty. Although its capsid and connector share close similarity with those of several phages from Gram-negative (Gram−) or Gram+ bacteria, its most distinctive characteristic is the helical tail decorated by radial spikes, possibly host adhesion devices, according to which the phage name was chosen. Its host adsorption device, at the tail tip, assembles features observed in phages binding to protein receptors, such as phage SPP1. All together, these results suggest that Araucaria may infect its mycobacterial host using a mechanism involving adhesion to cell wall saccharides and protein, a feature that remains to be further explored.
205310771
The Opening of the SPP1 Bacteriophage Tail, a Prevalent Mechanism in Gram-positive-infecting Siphophages*
The SPP1 siphophage uses its long non-contractile tail and tail tip to recognize and infect the Gram-positive bacterium Bacillus subtilis. The tail-end cap and its attached tip are the critical components for host recognition and opening of the tail tube for genome exit. In the present work, we determined the cryo-electron microscopic (cryo-EM) structure of a complex formed by the cap protein gp19.1 (Dit) and the N terminus of the downstream protein of gp19.1 in the SPP1 genome, gp211–552 (Tal). This complex assembles two back-to-back stacked gp19.1 ring hexamers, interacting loosely, and two gp211–552 trimers interacting with gp19.1 at both ends of the stack. Remarkably, one gp211–552 trimer displays a “closed” conformation, whereas the second is “open” delineating a central channel. The two conformational states dock nicely into the EM map of the SPP1 cap domain, respectively, before and after DNA release. Moreover, the open/closed conformations of gp19.1-gp211–552 are consistent with the structures of the corresponding proteins in the siphophage p2 baseplate, where the Tal protein (ORF16) attached to the ring of Dit (ORF15) was also found to adopt these two conformations. Therefore, the present contribution allowed us to revisit the SPP1 tail distal-end architectural organization. Considering the sequence conservation among Dit and the N-terminal region of Tal-like proteins in Gram-positive-infecting Siphoviridae, it also reveals the Tal opening mechanism as a hallmark of siphophages probably involved in the generation of the firing signal initiating the cascade of events that lead to phage DNA release in vivo.
21515226
Crystal Structure of Bacteriophage SPP1 Distal Tail Protein (gp19.1)
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 (gpVN and XkdM) as well as to components of the bacterial type 6 secretion system, supporting an evolutionary connection between all these devices.
8552669
Structural framework for DNA translocation via the viral portal protein
Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X‐ray structure of the bacteriophage SPP1 portal protein in its isolated 13‐subunit form and the pseudoatomic structure of a 12‐subunit assembly. The first defines the DNA‐interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal–DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a ‘mexican wave’ of positional and conformational changes propagating sequentially along this belt.
29638230
Structure of bacteriophage SPP1 head-to-tail connection reveals mechanism for viral DNA gating
In many bacterial viruses and in certain animal viruses, the double-stranded DNA genome enters and exits the capsid through a portal gatekeeper. We report a pseudoatomic structure of a complete portal system. The bacteriophage SPP1 gatekeeper is composed of dodecamers of the portal protein gp6, the adaptor gp15, and the stopper gp16. The solution structures of gp15 and gp16 were determined by NMR. They were then docked together with the X-ray structure of gp6 into the electron density of the ≈1-MDa SPP1 portal complex purified from DNA-filled capsids. The resulting structure reveals that gatekeeper assembly is accompanied by a large rearrangement of the gp15 structure and by folding of a flexible loop of gp16 to form an intersubunit parallel β-sheet that closes the portal channel. This stopper system prevents release of packaged DNA. Disulfide cross-linking between β-strands of the stopper blocks the key conformational changes that control genome ejection from the virus at the beginning of host infection.
34241848
Structure of bacteriophage SPP1 tail reveals trigger for DNA ejection
The majority of known bacteriophages have long noncontractile tails (Siphoviridae) that serve as a pipeline for genome delivery into the host cytoplasm. The tail extremity distal from the phage head is an adsorption device that recognises the bacterial receptor at the host cell surface. This interaction generates a signal transmitted to the head that leads to DNA release. We have determined structures of the bacteriophage SPP1 tail before and after DNA ejection. The results reveal extensive structural rearrangements in the internal wall of the tail tube. We propose that the adsorption device–receptor interaction triggers a conformational switch that is propagated as a domino‐like cascade along the 1600 Å‐long helical tail structure to reach the head‐to‐tail connector. This leads to opening of the connector culminating in DNA exit from the head into the host cell through the tail tube.
16682264
Molecular architecture of the prolate head of bacteriophage T4.
The head of bacteriophage T4 is a prolate icosahedron with one unique portal vertex to which the phage tail is attached. The three-dimensional structure of mature bacteriophage T4 head has been determined to 22-A resolution by using cryo-electron microscopy. The T4 capsid has a hexagonal surface lattice characterized by the triangulation numbers T(end) = 13 laevo for the icosahedral caps and T(mid) = 20 for the midsection. Hexamers of the major capsid protein gene product (gp)23* and pentamers of the vertex protein gp24*, as well as the outer surface proteins highly antigenic outer capsid protein (hoc) and small outer capsid protein (soc), are clearly evident in the reconstruction. The size and shape of the gp23* hexamers are similar to the major capsid protein organization of bacteriophage HK97. The binding sites and shape of the hoc and soc proteins have been established by analysis of the soc(-) and hoc(-)soc(-) T4 structures.
7648146
Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships.
Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses.
27068226
Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29.
Bacteriophage phi29 is one of the smallest and simplest known dsDNA phages, making it amenable to structural investigations. The three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 A resolution by cryo-electron microscopy (cryo-EM), allowing the identification of alpha helices and beta sheets. Their arrangement indicates that the folds of the phi29 and bacteriophage HK97 capsid proteins are similar except for an additional immunoglobulin-like domain of the phi29 protein. An atomic model that incorporates these two domains fits well into the cryo-EM density of the T = 3, fiberless isometric phi29 particle, and cryo-EM structures of fibered isometric and fiberless prolate prohead phi29 particles at resolutions of 8.7 A and 12.7 A, respectively. Thus, phi29 joins the growing number of phages that utilize the HK97 capsid structure, suggesting that this protein fold may be as prevalent in capsids of dsDNA phages as the jelly roll fold is in eukaryotic viruses.
4832773
Ig-like domains on bacteriophages: a tale of promiscuity and deceit.
The immunoglobulin (Ig) fold is one of the most important structures in biology, playing essential roles in the vertebrate immune response, cell adhesion, and many other processes. Through bioinformatic analysis, we have discovered that Ig-like domains are often found in the constituent proteins of tailed double-stranded (ds) DNA bacteriophage particles, and are likely displayed on the surface of these viruses. These phage Ig-like domains fall into three distinct sequence families, which are similar to the classic immunoglobulin domain (I-Set), the fibronectin type 3 repeat (FN3), and the bacterial Ig-like domain (Big2). The phage Ig-like domains are very promiscuous. They are attached to more than ten different functional classes of proteins, and found in all three morphogenetic classes of tailed dsDNA phages. In addition, they reside in phages that infect a diverse set of gram negative and gram positive bacteria. These domains are deceptive because many are added to larger proteins through programmed ribosomal frameshifting, so that they are not always detected by standard protein sequence searching procedures. In addition, the presence of unrecognized Ig-like domains in a variety of phage proteins with different functions has led to gene misannotation. Our results demonstrate that horizontal gene transfer involving Ig-like domain encoding DNA has occurred commonly between diverse classes of both lytic and temperate phages, which otherwise display very limited sequence similarities to one another. We suggest that phage may have been an important vector in the spread of Ig-like domains through diverse species of bacteria. While the function of the phage Ig-like domains is unknown, several lines of evidence suggest that they may play an accessory role in phage infection by weakly interacting with carbohydrates on the bacterial cell surface.
45685977
The solution structure of the C-terminal Ig-like domain of the bacteriophage λ tail tube protein.
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.
35803622
Long noncontractile tail machines of bacteriophages.
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.
34601670
Capsid Structure and Its Stability at the Late Stages of Bacteriophage SPP1 Assembly
ABSTRACT The structure of the bacteriophage SPP1 capsid was determined at subnanometer resolution by cryo-electron microscopy and single-particle analysis. The icosahedral capsid is composed of the major capsid protein gp13 and the auxiliary protein gp12, which are organized in a T=7 lattice. DNA is arranged in layers with a distance of ∼24.5 Å. gp12 forms spikes that are anchored at the center of gp13 hexamers. In a gp12-deficient mutant, the centers of hexamers are closed by loops of gp13 coming together to protect the SPP1 genome from the outside environment. The HK97-like fold was used to build a pseudoatomic model of gp13. Its structural organization remains unchanged upon tail binding and following DNA release. gp13 exhibits enhanced thermostability in the DNA-filled capsid. A remarkable convergence between the thermostability of the capsid and those of the other virion components was found, revealing that the overall architecture of the SPP1 infectious particle coevolved toward high robustness.