Structural basis of bacteriophage T5 infection trigger and E. coli cell wall perforation.

Most bacteriophages present a tail allowing host recognition, cell wall perforation, and viral DNA channeling from the capsid to the infected bacterium cytoplasm. The majority of tailed phages bear a long flexible tail (Siphoviridae) at the tip of which receptor binding proteins (RBPs) specifically interact with their host, triggering infection. In siphophage T5, the unique RBP is located at the extremity of a central fiber. We present the structures of T5 tail tip, determined by cryo-electron microscopy before and after interaction with its E. coli receptor, FhuA, reconstituted into nanodisc. These structures bring out the important conformational changes undergone by T5 tail tip upon infection, which include bending of T5 central fiber on the side of the tail tip, tail anchoring to the membrane, tail tube opening, and formation of a transmembrane channel. The data allow to detail the first steps of an otherwise undescribed infection mechanism.

Tall tails: cryo-electron microscopy of phage tail DNA ejection conduits.

The majority of phages, viruses that infect prokaryotes, inject their genomic material into their host through a tubular assembly known as a tail. Despite the genomic diversity of tailed phages, only three morphological archetypes have been described: contractile tails of Myoviridae-like phages; short non-contractile tails of Podoviridae-like phages; and long and flexible non-contractile tails of Siphoviridae-like phages. While early cryo-electron microscopy (cryo-EM) work elucidated the organisation of the syringe-like injection mechanism of contractile tails, the intrinsic flexibility of the long non-contractile tails prevented high-resolution structural determination. In 2020, four cryo-EM structures of Siphoviridae-like tail tubes were solved and revealed common themes and divergences. The central tube is structurally conserved and homologous to the hexameric rings of the tail tube protein (TTP) also found in contractile tails, bacterial pyocins, and type VI secretion systems. The interior surface of the tube presents analogous motifs of negatively charged amino acids proposed to facilitate ratcheting of the DNA during genome ejection. The lack of a conformational change upon genome ejection implicates the tape measure protein in triggering genome release. A distinctive feature of Siphoviridae-like tails is their flexibility. This results from loose inter-ring connections that can asymmetrically stretch on one side to allow bending and flexing of the tube without breaking. The outer surface of the tube differs greatly and may be smooth or rugged due to additional Ig-like domains in TTP. Some of these variable domains may contribute to adsorption of the phage to prokaryotic and eukaryotic cell surfaces affecting tropism and virulence.

Characterization of a Novel Phage ΦAb1656-2 and Its Endolysin with Higher Antimicrobial Activity against Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.

Biogenesis of a Bacteriophage Long Non-Contractile Tail.

Siphoviruses are main killers of bacteria. They use a long non-contractile tail to recognize the host cell and to deliver the genome from the viral capsid to the bacterial cytoplasm. Here, we define the molecular organization of the Bacillus subtilis bacteriophage SPP1 ~ 6.8 MDa tail and uncover its biogenesis mechanisms. A complex between gp21 and the tail distal protein (Dit) gp19.1 is assembled first to build the tail cap (gp19.1-gp21Nter) connected by a flexible hinge to the tail fiber (gp21Cter). The tip of the gp21Cter fiber is loosely associated to gp22. The cap provides a platform where tail tube proteins (TTPs) initiate polymerization around the tape measure protein gp18 (TMP), a reaction dependent on the non-structural tail assembly chaperones gp17.5 and gp17.5* (TACs). Gp17.5 is essential for stability of gp18 in the cell. Helical polymerization stops at a precise tube length followed by binding of proteins gp16.1 (TCP) and gp17 (THJP) to build the tail interface for attachment to the capsid portal system. This finding uncovers the function of the extensively conserved gp16.1-homologs in assembly of long tails. All SPP1 tail components, apart from gp22, share homology to conserved proteins whose coding genes' synteny is broadly maintained in siphoviruses. They conceivably represent the minimal essential protein set necessary to build functional long tails. Proteins homologous to SPP1 tail building blocks feature a variety of add-on modules that diversify extensively the tail core structure, expanding its capability to bind host cells and to deliver the viral genome to the bacterial cytoplasm.

Noncanonical DNA polymerization by aminoadenine-based siphoviruses.

Bacteriophage genomes harbor the broadest chemical diversity of nucleobases across all life forms. Certain DNA viruses that infect hosts as diverse as cyanobacteria, proteobacteria, and actinobacteria exhibit wholesale substitution of aminoadenine for adenine, thereby forming three hydrogen bonds with thymine and violating Watson-Crick pairing rules. Aminoadenine-encoded DNA polymerases, homologous to the Klenow fragment of bacterial DNA polymerase I that includes 3'-exonuclease but lacks 5'-exonuclease, were found to preferentially select for aminoadenine instead of adenine in deoxynucleoside triphosphate incorporation templated by thymine. Polymerase genes occur in synteny with genes for a biosynthesis enzyme that produces aminoadenine deoxynucleotides in a wide array of Siphoviridae bacteriophages. Congruent phylogenetic clustering of the polymerases and biosynthesis enzymes suggests that aminoadenine has propagated in DNA alongside adenine since archaic stages of evolution.

Architecture of the flexible tail tube of bacteriophage SPP1.

Bacteriophage SPP1 is a double-stranded DNA virus of the Siphoviridae family that infects the bacterium Bacillus subtilis. This family of phages features a long, flexible, non-contractile tail that has been difficult to characterize structurally. Here, we present the atomic structure of the tail tube of phage SPP1. Our hybrid structure is based on the integration of structural restraints from solid-state nuclear magnetic resonance (NMR) and a density map from cryo-EM. We show that the tail tube protein gp17.1 organizes into hexameric rings that are stacked by flexible linker domains and, thus, form a hollow flexible tube with a negatively charged lumen suitable for the transport of DNA. Additionally, we assess the dynamics of the system by combining relaxation measurements with variances in density maps.

Accurate and cost-effective NMR chemical shift predictions for proteins using a molecules-in-molecules fragmentation-based method.

We have developed an efficient protocol using our two-layer Molecules-in-Molecules (MIM2) fragmentation-based quantum chemical method for the prediction of NMR chemical shifts of large biomolecules. To investigate the performance of our fragmentation approach and demonstrate its applicability, MIM-NMR calculations are first calibrated on a test set of six proteins. The MIM2-NMR method yields a mean absolute deviation (MAD) from unfragmented full molecule calculations of 0.01 ppm for 1H and 0.06 ppm for 13C chemical shifts. Thus, the errors from fragmentation are only about 3% of our target accuracy of ∼0.3 ppm for 1H and 2-3 ppm for 13C chemical shifts. To compare with experimental chemical shifts, a standard protocol is first derived using two smaller proteins 2LHY (176 atoms) and 2LI1 (146 atoms) for obtaining an appropriate protein structure for NMR chemical shift calculations. The effect of the solvent environment on the calculated NMR chemical shifts is incorporated through implicit, explicit, or explicit-implicit solvation models. The expensive first solvation shell calculations are replaced by a micro-solvation model in which only the immediate interaction between the protein and the explicit solvation environment is considered. A single explicit water molecule for each amine and amide proton is found to be sufficient to yield accurate results for 1H chemical shifts. The 1H and 13C NMR chemical shifts calculated using our protocol give excellent agreement with experiments for two larger proteins, 2MC5 (the helical part with 265 atoms) and 3UMK (33 residue slice with 547 atoms). Overall, our target accuracy of ∼0.3 ppm for 1H and ∼2-3 ppm for 13C has been achieved for the larger proteins. The proposed MIM-NMR method is accurate and computationally cost-effective and should be applicable to study a wide range of large proteins.

Conserved and Diverse Traits of Adhesion Devices from Siphoviridae Recognizing Proteinaceous or Saccharidic Receptors.

Bacteriophages can play beneficial roles in phage therapy and destruction of food pathogens. Conversely, they play negative roles as they infect bacteria involved in fermentation, resulting in serious industrial losses. Siphoviridae phages possess a long non-contractile tail and use a mechanism of infection whose first step is host recognition and binding. They have evolved adhesion devices at their tails' distal end, tuned to recognize specific proteinaceous or saccharidic receptors on the host's surface that span a large spectrum of shapes. In this review, we aimed to identify common patterns beyond this apparent diversity. To this end, we analyzed siphophage tail tips or baseplates, evaluating their known structures, where available, and uncovering patterns with bioinformatics tools when they were not. It was thereby identified that a triad formed by three proteins in complex, i.e., the tape measure protein (TMP), the distal tail protein (Dit), and the tail-associated lysozyme (Tal), is conserved in all phages. This common scaffold may harbor various functional extensions internally while it also serves as a platform for plug-in ancillary or receptor-binding proteins (RBPs). Finally, a group of siphophage baseplates involved in saccharidic receptor recognition exhibits an activation mechanism reminiscent of that observed in Myoviridae.

Characterization and Genome Analysis of a Novel Salmonella Phage vB_SenS_SE1.

Salmonella is a significant food-borne pathogen that infects a large number of people worldwide. In this study, a lytic bacteriophage vB_SenS_SE1 capable of infecting Salmonella is isolated from municipal wastewater in Beijing, and its biological and genomic features are analyzed. Transmission electron micrograph shows that vB_SenS_SE1 is likely a Siphoviridae virus, with an icosahedral head and a long non-contracted tail. The stability test in vitro reveals that it is stable at 4-50 °C and pH 4-12. Based on the one-step growth curve, vB_SenS_SE1 has a 60-min exponential phase and a low burst size (19 PFU per cell). Bioinformatics analysis reveals that vB_SenS_SE1 consists of a circular, double-stranded DNA molecule of 40,987 bp with a GC content of 51.2%. Its genome carries 63 predicted open reading frames (orfs), with 22 orfs encoding known proteins. Phylogenetic analysis of the large terminase subunit shows that vB_SenS_SE1 exhibits strong homology to Salmonella phage St161, St162, VSiP, and FSL SP-031. The CoreGenes analysis shows that it is a member of the virus genus Cornellvirus. The features of phage vB_SenS_SE1 suggest that it has the potential to be an agent to control Salmonella.

Capsid Structure of a Freshwater Cyanophage Siphoviridae Mic1.

Cyanobacteria are the most abundant photosynthetic microorganisms, the global distribution of which is mainly regulated by the corresponding cyanophages. A systematic screening of water samples in the Lake Chaohu enabled us to isolate a freshwater siphocyanophage that infects Microcystis wesenbergii, thus termed Mic1. Using cryoelectron microscopy, we solved the 3.5-Å structure of Mic1 capsid. The major capsid protein gp40 of an HK97-like fold forms two types of capsomers, hexons and pentons. The capsomers interact with each other via the interweaved N-terminal arms of gp40 in addition to a tail-in-mouth joint along the three-fold symmetric axis, resulting in the assembly of capsid in a mortise-and-tenon pattern. The novel-fold cement protein gp47 sticks at the two-fold symmetric axis and further fixes the capsid. These findings provide structural insights into the assembly of cyanophages, and set up a platform to explore the mechanism of specific interactions and co-evolution with cyanobacteria.

Ubiquitous Carbohydrate Binding Modules Decorate 936 Lactococcal Siphophage Virions.

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural "bricks" to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA.

DNA structural transitions facilitate genomic processes, mediate drug-DNA interactions, and inform the development of emerging DNA-based biotechnology such as programmable materials and DNA origami. While some features of DNA conformational changes are well characterized, fundamental information such as the orientations of the DNA base pairs is unknown. Here, we use concurrent fluorescence polarization imaging and DNA manipulation experiments to probe the structure of S-DNA, an elusive, elongated conformation that can be accessed by mechanical overstretching. To this end, we directly quantify the orientations and rotational dynamics of fluorescent DNA-intercalated dyes. At extensions beyond the DNA overstretching transition, intercalators adopt a tilted (θ ~ 54°) orientation relative to the DNA axis, distinct from the nearly perpendicular orientation (θ ~ 90°) normally assumed at lower extensions. These results provide the first experimental evidence that S-DNA has substantially inclined base pairs relative to those of the standard (Watson-Crick) B-DNA conformation.

Self-Assembly of Tail Tube Protein of Bacteriophage vB_EcoS_NBD2 into Extremely Long Polytubes in E. coli and S. cerevisiae.

Nucleotides, peptides and proteins serve as a scaffold material for self-assembling nanostructures. In this study, the production of siphovirus vB_EcoS_NBD2 (NBD2) recombinant tail tube protein gp39 reached approximately 33% and 27% of the total cell protein level in Escherichia coli and Saccharomyces cerevisiae expression systems, respectively. A simple purification protocol allowed us to produce a recombinant gp39 protein with 85%⁻90% purity. The yield of gp39 was 2.9 ± 0.36 mg/g of wet E. coli cells and 0.85 ± 0.33 mg/g for S. cerevisiae cells. The recombinant gp39 self-assembled into well-ordered tubular structures (polytubes) in vivo in the absence of other phage proteins. The diameter of these structures was the same as the diameter of the tail of phage NBD2 (~12 nm). The length of these structures varied from 0.1 µm to >3.95 µm, which is 23-fold the normal NBD2 tail length. Stability analysis demonstrated that the polytubes could withstand various chemical and physical conditions. These polytubes show the potential to be used as a nanomaterial in various fields of science.

Jet nebulization of bacteriophages with different tail morphologies - Structural effects.

It was previously demonstrated that the loss of infectivity of a myovirus PEV44 after jet nebulization was closely related to a change in bacteriophage (phage) structure. In this follow-up study, we further examined the impact of jet nebulization on tailed phages, which constitute 96% of all known phages, from three different families, Podoviridae (PEV2), Myoviridae (PEV40) andSiphoviridae (D29). Transmission electron microscopy (TEM) identified major changes in phage structures after jet nebulization, correlating with their loss of infectivity. For the podovirus PEV2, jet nebulization had a negligible impact on its activity (0.04 log10 pfu/mL loss) and structural change. On the other hand, the proportion of intact phages in the nebulized samples dropped from 50% to ∼27% for PEV40 and from 15% to ∼2% for D29. Phage deactivation of PEV40 measured by the TEM structural damage (0.52 log10 pfu/mL) was lower than that obtained by plaque assay (1.02 log10 pfu/mL), but within the range of variation (±0.5 log10 pfu/mL). However, TEM quantification considerably underestimated the titer reduction of D29 phage, ∼2 log pfu/mL lower than that obtained in plaque assay (3.25 log10 pfu/mL loss). In conclusion, nebulization-induced titre loss was correlated with morphological damage to phages and in particular, the tail length may be an important consideration for selection of phages in inhaled therapy using jet nebulization.

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif.

To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase.

Phage Tail-Like Bacteriocins.

Many dsDNA bacterial viruses (bacteriophages/phages) have long tail structures that serve as organelles for DNA delivery to host targets. These structures, particularly those of Myoviridae and Siphoviridae phages, have an evolutionary relationship with other cellular biological entities that share the common function of penetrating the bacterial envelope. Among these are type VI secretion systems, insecticidal protein complexes, and bacteriocins. Phage tail-like bacteriocins (PTLBs) are widespread in bacteria, comprising different types that likely evolved independently. They can be divided into two major classes: the R-type PTLBs, which are related to contractile Myoviridae phage tails, and the F-type PTLBs, which are related to noncontractile Siphoviridae phage tails. This review provides an overview of the history, biology, and diversity of these entities and also covers recent efforts to utilize these potent bactericidal agents as human therapeutics against bacterial disease.

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1.

The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough mutational analysis of the TMP from lactococcal phage TP901-1 (TMPTP901-1) was undertaken. We generated 56 mutants aimed at defining TMPTP901-1 domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMPTP901-1 as a minimum. Furthermore, the role of TMPTP901-1 in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMPTP901-1-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMPTP901-1 regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection.

Direct observation of DNA threading in flap endonuclease complexes.

Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.

Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid?

We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.