DSR2, a Sir2 domain-containing protein, protects bacteria from phage infection by hydrolyzing NAD+. The enzymatic activity of DSR2 is triggered by the SPR phage tail tube protein (TTP), while suppressed by the SPbeta phage-encoded DSAD1 protein, enabling phages to evade the host defense. However, the molecular mechanisms of activation and inhibition of DSR2 remain elusive. Here, we report the cryo-EM structures of apo DSR2, DSR2-TTP-NAD+ and DSR2-DSAD1 complexes. DSR2 assembles into a head-to-head tetramer mediated by its Sir2 domain. The C-terminal helical regions of DSR2 constitute four partner-binding cavities with opened and closed conformation. Two TTP molecules bind to two of the four C-terminal cavities, inducing conformational change of Sir2 domain to activate DSR2. Furthermore, DSAD1 competes with the activator for binding to the C-terminal cavity of DSR2, effectively suppressing its enzymatic activity. Our results provide the mechanistic insights into the DSR2-mediated anti-phage defense system and DSAD1-dependent phage immune evasion.

Several immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defence1-5. Here we studied an antiphage defence system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fibre, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defence system release a mixture of partially assembled, tailless phage particles and fully assembled phages in which the central tail fibre is obstructed by the covalently attached ubiquitin-like protein. These phages show severely impaired infectivity, explaining how the defence system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.

Bacteria can repurpose their own bacteriophage viruses (phage) to kill competing bacteria. Phage-derived elements are frequently strain specific in their killing activity, although there is limited evidence that this specificity drives bacterial population dynamics. Here, we identified intact phage and their derived elements in a metapopulation of wild plant-associated Pseudomonas genomes. We discovered that the most abundant viral cluster encodes a phage remnant resembling a phage tail called a tailocin, which bacteria have co-opted to kill bacterial competitors. Each pathogenic Pseudomonas strain carries one of a few distinct tailocin variants that target the variable polysaccharides in the outer membrane of co-occurring pathogenic Pseudomonas strains. Analysis of herbarium samples from the past 170 years revealed that the same tailocin and bacterial receptor variants have persisted in Pseudomonas populations. These results suggest that tailocin genetic diversity can be mined to develop targeted \"tailocin cocktails\" for microbial control.

Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense.

The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.

Contractile injection systems (CISs) are prokaryotic phage tail-like nanostructures loading effector proteins that mediate various biological processes. Although CIS functions have been diversified through evolution and hold the great potential as protein delivery systems, the functional characterisation of CISs and their effectors is currently limited to a few CIS lineages. Here, we show that the CISs of Streptomyces davawensis belong to a unique group of bacterial CISs distributed across distant phyla and facilitate sporogenic differentiation of this bacterium. CIS loss results in decreases in extracellular DNA release, biomass accumulation, and spore formation in S. davawensis. CISs load an effector, which is a remote homolog of phage tapemeasure proteins, and its C-terminal domain has endonuclease activity responsible for the CIS-associated phenotypes. Our findings illustrate that CISs can contribute to the reproduction of bacteria through the action of the effector and suggest an evolutionary link between CIS effectors and viral cargos.

The integration of recognition and therapeutic functions in multifunctional biosensors is of great importance in guaranteeing food security and reducing the occurrence of foodborne illness caused by foodborne pathogens. In this study, a biosensor utilizing a \"sense-and-treat\" approach was developed by integrating phage tailspike protein (TSP) with gold nanoparticles (AuNPs@TSP). The synthesized AuNPs@TSP showed strong binding affinity towards Salmonella typhimurium causing color changes and exhibited effective bactericidal activity when exposed to near-infrared (NIR) irradiation. This biosensor facilitated rapid colorimetric detection of S. typhimurium in 50 min, with a LOD (limit of detection) of 2.53 × 103 CFU/mL output on a smartphone APP after analyzing the red-green-blue (RGB) values from color rendering results. Furthermore, the biosensor displayed high selectivity, rapid response time, and broad applicability when tested with real samples. Moreover, the biosensor exhibited a remarkably efficient antibacterial efficacy of 100 % against S. typhimurium under 808 nm light irradiation for 6 min. This study provides a comprehensive investigation into the potential utilization of biosensors for rapid detection and eradication of foodborne pathogens in food industry.

Bacteriophage infection, a pivotal process in microbiology, initiates with the phage\'s tail recognizing and binding to the bacterial cell surface, which then mediates the injection of viral DNA. Although comprehensive studies on the interaction between bacteriophage lambda and its outer membrane receptor, LamB, have provided rich information about the system\'s biochemical properties, the precise molecular mechanism remains undetermined. This study revealed the high-resolution cryo-electron microscopy (cryo-EM) structures of the bacteriophage lambda tail complexed with its irreversible Shigella sonnei 3070 LamB receptor and the closed central tail fiber. These structures reveal the complex processes that trigger infection and demonstrate a substantial conformational change in the phage lambda tail tip upon LamB binding. Providing detailed structures of bacteriophage lambda infection initiation, this study contributes to the expanding knowledge of lambda-bacterial interaction, which holds significance in the fields of microbiology and therapeutic development.

Infection of bacteria by phages is a complex multi-step process that includes specific recognition of the host cell, creation of a temporary breach in the host envelope, and ejection of viral DNA into the bacterial cytoplasm. These steps must be perfectly regulated to ensure efficient infection. Here we report the dual function of the tail completion protein gp16.1 of bacteriophage SPP1. First, gp16.1 has an auxiliary role in assembly of the tail interface that binds to the capsid connector. Second, gp16.1 is necessary to ensure correct routing of phage DNA to the bacterial cytoplasm. Viral particles assembled without gp16.1 are indistinguishable from wild-type virions and eject DNA normally in vitro. However, they release their DNA to the extracellular space upon interaction with the host bacterium. The study shows that a highly conserved tail completion protein has distinct functions at two essential steps of the virus life cycle in long-tailed phages.

Phage therapy, reemerging as a promising approach to counter antimicrobial-resistant infections, relies on a comprehensive understanding of the specificity of individual phages. Yet the significant diversity within phage populations presents a considerable challenge. Currently, there is a notable lack of tools designed for large-scale characterization of phage receptor-binding proteins, which are crucial in determining the phage host range.
In this study, we present SpikeHunter, a deep learning method based on the ESM-2 protein language model. With SpikeHunter, we identified 231,965 diverse phage-encoded tailspike proteins, a crucial determinant of phage specificity that targets bacterial polysaccharide receptors, across 787,566 bacterial genomes from 5 virulent, antibiotic-resistant pathogens. Notably, 86.60% (143,200) of these proteins exhibited strong associations with specific bacterial polysaccharides. We discovered that phages with identical tailspike proteins can infect different bacterial species with similar polysaccharide receptors, underscoring the pivotal role of tailspike proteins in determining host range. The specificity is mainly attributed to the protein\'s C-terminal domain, which strictly correlates with host specificity during domain swapping in tailspike proteins. Importantly, our dataset-driven predictions of phage-host specificity closely match the phage-host pairs observed in real-world phage therapy cases we studied.
Our research provides a rich resource, including both the method and a database derived from a large-scale genomics survey. This substantially enhances understanding of phage specificity determinants at the strain level and offers a valuable framework for guiding phage selection in therapeutic applications.