T6SS-associated Rhs toxin-encapsulating shells: Structural and bioinformatical insights into bacterial weaponry and self-protection.

Bacteria use the type VI secretion system (T6SS) to secrete toxins into pro- and eukaryotic cells via machinery consisting of a contractile sheath and a rigid tube. Rearrangement hotspot (Rhs) proteins represent one of the most common T6SS effectors. The Rhs C-terminal toxin domain displays great functional diversity, while the Rhs core is characterized by YD repeats.

An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano.

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness.

Neck and capsid architecture of the robust Agrobacterium phage Milano.

Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the "neck" region, where the DNA-filled capsid is connected to the host-recognizing tail remains poorly understood. We describe cryo-EM structures of the neck, the neck-capsid and neck-tail junctions, and capsid of the Agrobacterium phage Milano.

Function of the bacteriophage P2 baseplate central spike Apex domain in the infection process.

The contractile tail of bacteriophage P2 functions to drive the tail tube across the outer membrane of its host bacterium, a prerequisite event for subsequent translocation of phage genomic DNA into the host cell. The tube is equipped with a spike-shaped protein (product of P2 gene , gpV or Spike) that contains a membrane-attacking Apex domain carrying a centrally positioned Fe ion. The ion is enclosed in a histidine cage that is formed by three symmetry-related copies of a conserved HxH (histidine, any residue, histidine) sequence motif.

Structural basis of template strand deoxyuridine promoter recognition by a viral RNA polymerase.

Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the -10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the -10 element of its deoxyuridine-containing promoter in the template strand.

Computational models in the service of X-ray and cryo-electron microscopy structure determination.

Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP.

Structural basis for recognition of bacterial cell wall teichoic acid by pseudo-symmetric SH3b-like repeats of a viral peptidoglycan hydrolase.

Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands.

Quantitative description of a contractile macromolecular machine.

Contractile injection systems (CISs) [type VI secretion system (T6SS), phage tails, and tailocins] use a contractile sheath-rigid tube machinery to breach cell walls and lipid membranes. The structures of the pre- and postcontraction states of several CISs are known, but the mechanism of contraction remains poorly understood. Combining structural information of the end states of the 12-megadalton R-type pyocin sheath-tube complex with thermodynamic and force spectroscopy analyses and an original modeling procedure, we describe the mechanism of pyocin contraction.

Reprogramming bacteriophage host range: design principles and strategies for engineering receptor binding proteins.

Bacteriophages (phages) use specialized tail machinery to deliver proteins and genetic material into a bacterial cell during infection. Attached at the distal ends of their tails are receptor binding proteins (RBPs) that recognize specific molecules exposed on host bacteria surfaces. Since the therapeutic capacity of naturally occurring phages is often limited by narrow host ranges, there is significant interest in expanding their host range via directed evolution or structure-guided engineering of their RBPs.

Structure and function of virion RNA polymerase of a crAss-like phage.

CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut. The genomes of all crAss-like phages encode a large virion-packaged protein that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs). Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes.

The Central Spike Complex of Bacteriophage T4 Contacts PpiD in the Periplasm of .

Infecting bacteriophage T4 uses a contractile tail structure to breach the envelope of the host cell. During contraction, the tail tube headed with the "central spike complex" is thought to mechanically puncture the outer membrane. We show here that a purified tip fragment of the central spike complex interacts with periplasmic chaperone PpiD, which is anchored to the cytoplasmic membrane.

Structure and Function of the T4 Spackle Protein Gp61.3.

The bacteriophage T4 genome contains two genes that code for proteins with lysozyme activity- and . Gene encodes the well-known T4 lysozyme (commonly called T4L) that functions to break the peptidoglycan layer late in the infection cycle, which is required for liberating newly assembled phage progeny. Gene product (gp5) is the tail-associated lysozyme, a component of the phage particle.

Structure and function of bacteriophage CBA120 ORF211 (TSP2), the determinant of phage specificity towards E. coli O157:H7.

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.

Action of a minimal contractile bactericidal nanomachine.

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage.

Modeling the Architecture of Depolymerase-Containing Receptor Binding Proteins in Phages.

carries a thick polysaccharide capsule. This highly variable chemical structure plays an important role in its virulence. Many bacteriophages recognize this capsule with a receptor binding protein (RBP) that contains a depolymerase domain.

Target highlights in CASP13: Experimental target structures through the eyes of their authors.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120.

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood.

Structure and transformation of bacteriophage A511 baseplate and tail upon infection of cells.

Contractile injection systems (bacteriophage tails, type VI secretions system, R-type pyocins, etc.) utilize a rigid tube/contractile sheath assembly for breaching the envelope of bacterial and eukaryotic cells. Among contractile injection systems, bacteriophages that infect Gram-positive bacteria represent the least understood members.

Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition.

The ability of phages to infect specific bacteria has led to their exploitation as bio-tools for bacterial remediation and detection. Many phages recognize bacterial hosts via adhesin tips of their long tail fibers (LTFs). Adhesin sequence plasticity modulates receptor specificity, and thus primarily defines a phage's host range.

Structure and Analysis of R1 and R2 Pyocin Receptor-Binding Fibers.

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to "simple" contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle.

Contractile injection systems of bacteriophages and related systems.

Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure consists of a contractile sheath enveloping a rigid tube that is sharpened by a spike-shaped protein complex at its tip. The spike complex forms the centerpiece of a baseplate complex that terminates the sheath and the tube.

Stretching the arms of the type VI secretion sheath protein.

The bacterial type VI secretion system (T6SS) is a multicomponent complex responsible for the translocation of effector proteins into the external milieu. The T6SS consists of an external sheath, an internal rigid tube, a baseplate, and a T6SS‐specific membrane complex. Secretion is accomplished by the contraction of the sheath, which expels the effector‐loaded tube.

The O-specific polysaccharide lyase from the phage LKA1 tailspike reduces Pseudomonas virulence.

Pseudomonas phage LKA1 of the subfamily Autographivirinae encodes a tailspike protein (LKA1gp49) which binds and cleaves B-band LPS (O-specific antigen, OSA) of Pseudomonas aeruginosa PAO1. The crystal structure of LKA1gp49 catalytic domain consists of a beta-helix, an insertion domain and a C-terminal discoidin-like domain. The putative substrate binding and processing site is located on the face of the beta-helix whereas the C-terminal domain is likely involved in carbohydrates binding.

Refined Cryo-EM Structure of the T4 Tail Tube: Exploring the Lowest Dose Limit.

The bacteriophage T4 contractile tail (containing a tube and sheath) was the first biological assembly reconstructed in three dimensions by electron microscopy at a resolution of ∼35 Å in 1968. A single-particle reconstruction of the T4 baseplate was able to generate a 4.1 Å resolution map for the first two rings of the tube using the overall baseplate for alignment.