Structure of the phage TP901-1 1.8 MDa baseplate suggests an alternative host adhesion mechanism.
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ABSTRACT:
SUBMITTER: Veesler D
PROVIDER: S-EPMC3384155 | biostudies-literature | 2012 Jun
REPOSITORIES: biostudies-literature
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Project description:P335 lactococcal phages infect the gram(+) bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized on the host cell surface. We report here the electron microscopic structure of the phage TP901-1 wild-type BP as well as those of two mutants bppL (-) and bppU(-), lacking BppL (the RBPs) or both peripheral BP components (BppL and BppU), respectively. We also achieved an electron microscopic reconstruction of a partial BP complex, formed by BppU and BppL. This complex exhibits a tripod shape and is composed of nine BppLs and three BppUs. These structures, combined with light-scattering measurements, led us to propose that the TP901-1 BP harbors six tripods at its periphery, located around the central tube formed by ORF46 (Dit) hexamers, at its proximal end, and a ORF47 (Tal) trimer at its distal extremity. A total of 54 BppLs (18 RBPs) are thus available to mediate host anchoring with a large apparent avidity. TP901-1 BP exhibits an infection-ready conformation and differs strikingly from the lactococcal phage p2 BP, bearing only 6 RBPs, and which needs a conformational change to reach its activated state. The comparison of several Siphoviridae structures uncovers a close organization of their central BP core whereas striking differences occur at the periphery, leading to diverse mechanisms of host recognition.
| S-EPMC2998104 | biostudies-literature
Project description:UNLABELLED:The Gram-positive bacterium Lactococcus lactis, used for the production of cheeses and other fermented dairy products, falls victim frequently to fortuitous infection by tailed phages. The accompanying risk of dairy fermentation failures in industrial facilities has prompted in-depth investigations of these phages. Lactococcal phage Tuc2009 possesses extensive genomic homology to phage TP901-1. However, striking differences in the baseplate-encoding genes stimulated our interest in solving the structure of this host's adhesion device. We report here the X-ray structures of phage Tuc2009 receptor binding protein (RBP) and of a "tripod" assembly of three baseplate components, BppU, BppA, and BppL (the RBP). These structures made it possible to generate a realistic atomic model of the complete Tuc2009 baseplate that consists of an 84-protein complex: 18 BppU, 12 BppA, and 54 BppL proteins. The RBP head domain possesses a different fold than those of phages p2, TP901-1, and 1358, while the so-called "stem" and "neck" domains share structural features with their equivalents in phage TP901-1. The BppA module interacts strongly with the BppU N-terminal domain. Unlike other characterized lactococcal phages, Tuc2009 baseplate harbors two different carbohydrate recognition sites: one in the bona fide RBP head domain and the other in BppA. These findings represent a major step forward in deciphering the molecular mechanism by which Tuc2009 recognizes its saccharidic receptor(s) on its host. IMPORTANCE:Understanding how siphophages infect Lactococcus lactis is of commercial importance as they cause milk fermentation failures in the dairy industry. In addition, such knowledge is crucial in a general sense in order to understand how viruses recognize their host through protein-glycan interactions. We report here the lactococcal phage Tuc2009 receptor binding protein (RBP) structure as well as that of its baseplate. The RBP head domain has a different fold than those of phages p2, TP901-1, and 1358, while the so-called "stem" and "neck" share the fold characteristics also found in the equivalent baseplate proteins of phage TP901-1. The baseplate structure contains, in contrast to other characterized lactococcal phages, two different carbohydrate binding modules that may bind different motifs of the host's surface polysaccharide.
| S-EPMC4742702 | biostudies-literature
Project description: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.
| S-EPMC9733650 | biostudies-literature
Project description:Siphoviridae is the most abundant viral family on earth which infects bacteria as well as archaea. All known siphophages infecting gram+ Lactococcus lactis possess a baseplate at the tip of their tail involved in host recognition and attachment. Here, we report analysis of the p2 phage baseplate structure by X-ray crystallography and electron microscopy and propose a mechanism for the baseplate activation during attachment to the host cell. This approximately 1 MDa, Escherichia coli-expressed baseplate is composed of three protein species, including six trimers of the receptor-binding protein (RBP). RBPs host-recognition domains point upwards, towards the capsid, in agreement with the electron-microscopy map of the free virion. In the presence of Ca(2+), a cation mandatory for infection, the RBPs rotated 200 degrees downwards, presenting their binding sites to the host, and a channel opens at the bottom of the baseplate for DNA passage. These conformational changes reveal a novel siphophage activation and host-recognition mechanism leading ultimately to DNA ejection.
| S-EPMC2872406 | biostudies-literature
Project description:BackgroundNarrow host range is a major limitation for phage applications, but phages can evolve expanded host range through adaptations in the receptor-binding proteins.ResultsHere, we report that Pseudomonas phage K8 can evolve broader host range and higher killing efficiency at the cost of virion stability. Phage K8 host range mutant K8-T239A carries a mutant version of the putative baseplate wedge protein GP075, termed GP075m. While phage K8 adsorbs to hosts via the O-specific antigen of bacterial LPS, phage K8-T239A uses GP075m to also bind the bacterial core oligosaccharide, enabling infection of bacterial strains resistant to K8 infection due to modified O-specific antigens. This mutation in GP075 also alters inter-protein interactions among phage proteins, and reduces the stability of phage particles to environmental stressors like heat, acidity, and alkalinity. We find that a variety of mutations in gp075 are widespread in K8 populations, and that the gp075-like genes are widely distributed among the domains of life.ConclusionOur data show that a typical life history tradeoff occurs between the stability and the host range in the evolution of phage K8. Reservoirs of viral gene variants may be widely present in phage communities, allowing phages to rapidly adapt to any emerging environmental stressors. Video Abstract.
| S-EPMC9887876 | biostudies-literature
Project description:We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1.
| S-EPMC4892519 | biostudies-literature
Project description:Bacteriophages belonging to the Caudovirales order possess a tail acting as a molecular machine used during infection to recognize the host and ensure high-efficiency genome delivery to the cell cytoplasm. They bear a large and sophisticated multiprotein organelle at their distal tail end, either a baseplate or a tail-tip, which is the control center for infectivity. We report here insights into the baseplate assembly pathways of two lactoccocal phages (p2 and TP901-1) using electrospray ionization-mass spectrometry. Based on our "block cloning" strategy we have expressed large complexes of their baseplates as well as several significant structural subcomplexes. Previous biophysical characterization using size-exclusion chromatography coupled with on-line light scattering and refractometry demonstrated that the overproduced recombinant proteins interact with each other to form large (up to 1.9 MDa) and stable assemblies. The structures of several of these complexes have been determined by x-ray diffraction or by electron microscopy. In this contribution, we demonstrate that electrospray ionization-mass spectrometry yields accurate mass measurements for the different baseplate complexes studied from which their stoichiometries can be discerned, and that the subspecies observed in the spectra provide valuable information on the assembly mechanisms of these large organelles.
| S-EPMC3186816 | biostudies-literature
Project description: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.
| S-EPMC1317572 | biostudies-literature
Project description:A promoter active in the late phase of the lytic cycle of lactococcal bacteriophage TP901-1 has been identified. The promoter is tightly regulated and requires the product of the phage TP901-1 orf29 for activity. A deletion analysis of the late promoter region showed that a fragment as small as 99 bp contains both the promoter and the region necessary for activation by ORF29. The transcriptional start site of the promoter was identified by primer extension to position 13073 on the TP901-1 genome, thus located 87 bp downstream of orf29 in a 580-bp intergenic region between orf29 and orf30. Furthermore, the region located -85 to -61 bp upstream of the start site was shown to be necessary for promoter activity. During infection, the transcript arising from the late promoter is fully induced at 40 min postinfection, and our results suggest that a certain level of ORF29 must be reached in order to activate transcription of the promoter. Several lactococcal bacteriophages encode ORF29 homologous proteins, indicating that late transcription may be controlled by a similar mechanism in these phages. With the identification of this novel regulator, our results suggest that within the P335 group of lactococcal phages at least two regulatory systems controlling transcription in the late stage of infection exist.
| S-EPMC93353 | biostudies-literature
Project description:Virulent phages infecting L. lactis, an industry-relevant bacterium, pose a significant risk to the quality of the fermented milk products. Phages of the Skunavirus genus are by far the most isolated lactococcal phages in the cheese environments and phage p2 is the model siphophage for this viral genus. The baseplate of phage p2, which is used to recognize its host, was previously shown to display two conformations by X-ray crystallography, a rested state and an activated state ready to bind to the host. The baseplate became only activated and opened in the presence of Ca2+. However, such an activated state was not previously observed in the virion. Here, using nanobodies binding to the baseplate, we report on the negative staining electron microscopy structure of the activated form of the baseplate directly observed in the p2 virion, that is compatible with the activated baseplate crystal structure. Analyses of this new structure also established the presence of a second distal tail (Dit) hexamer as a component of the baseplate, the topology of which differs largely from the first one. We also observed an uncoupling between the baseplate activation and the tail tip protein (Tal) opening, suggesting an infection mechanism more complex than previously expected.
| S-EPMC7472080 | biostudies-literature