Abstract Shiga toxin-converting bacteriophages play a critical role in the emergence and virulence of pathogenic Escherichia coli strains. Despite their significance, detailed structural information on these phages remains scarce. Here we present a high-resolution cryo-electron microscopy and proteomic analysis of the phi24B bacteriophage, revealing an icosahedral capsid with T=9 symmetry, decorated by a processed esterase protein (gp84) and stabilized by cementing proteins. The tail assembly comprises a dodecameric portal, two rings of adapter proteins sharing a common fold, a hexameric nozzle, six lateral tail fibers, and a flexible central needle fiber. The binding sites of the fibers are described. Comparative analysis indicates conservation of the tail structure with related podoviruses but very different peripheral features.

Pseudomonas aeruginosa poses major public health threats, due to its robust, treatment-resistant biofilms, which contribute to multi-drug resistance. Bacteriophages offer a promising alternative. This study evaluates giant Phikzvirus and conventional Pbunavirus phages against pre-formed biofilms from four multi-drug-resistant P. aeruginosa clinical isolates. We tested six phages (three Phikzvirus, three Pbunavirus) against four clinical strains, isolated from chronic urological and pulmonary infections (Ur1, Ur14, Lu3, Lu9) at MOIs 0.001–0.1, quantifying biofilm biomass by crystal violet and visualizing architecture by SEM. Phage treatment leads to significant disrupted biofilms in three isolates achieving more than 50% reduction—comparable with typical antibiotic efficacy against mature biofilms. All biofilms retained their EPS architecture after the treatment, as revealed by SEM, suggesting that residual eDNA-polysaccharide complexes could maintain structural cohesion even after bacterial lysis. The phage’s ability to reduce biofilm biomass demonstrated a significant dependence on the multiplicity of infection (MOI). The best results of anti-biofilm activity of phages (reduction in biofilm biomass by more than 75%) were observed for the phage phiKZ for two strains—Ur1 at MOI = 0.001 and Lu9 at MOI = 0.01—and for the phage phi14/1 for the Ur1 strain at MOI = 0.001. The biofilm formed by the antibiotic-resistant clinical isolate Ur14 demonstrated exceptional resistance to both types of bacteriophages despite the sensitivity of bacteria of this strain to the studied phages. This highlights the need for personalized phage therapy, where tailored phage cocktails are selected for each specific bacterial strain to achieve optimal destruction of the biofilm.

Background: The majority of bacteriophages stop replicating once the host culture approaches the stationary phase. Only a few bacteriophages are able to replicate in host cells at the transition to, or already in, the early stationary phase of growth (early stationary phase infective, eSPI phages) have been characterized so far. Materials and Methods: The coliphage DH23 was isolated from the river water using an enrichment procedure with a stationary phase culture of the host. Genomic sequencing, epigenetic modifications detection, and morphological and biological characterization were performed. Results: DH23 is a small siphovirus with a genome of 44,682 b.p. Phylogenetic analysis suggests that DH23 may be considered as a new species within the Dhillonvirus genus, closely related to Sodalis phage SO-1 and coliphage TheodorHerzl. Phage DH23 plaques continue to expand for several days of incubation and it is able to form plaques when applied to already mature 24 h-old host lawn. Conclusions: Bacteriophage DH23 features an eSPI phenotype very similar to phage T7. Expanding the number of characterized eSPI coliphages may facilitate deciphering the molecular basis of eSPI phenotype and development of new eSPI platforms for phage therapy or other applications.

Biochemistry has a strong interest in ion channels, a diverse group of membrane proteins. This group has long remained difficult to study at the structural level, but much progress has been made in recent decades with improvements in biochemical methods and computational procedures. Voltage-gated ion channels undergo conformational changes upon functioning, and multiple models of activation mechanisms were proposed based on experimental data. This review focuses on the structural studies of the functioning of voltage-gated ion channels, which are accompanied by conformational changes in proteins, and the methodological advances that allow their observation. Particular attention is paid to the lipid modulation of ion channels, as well as to the use of various lipids and membrane mimetics to stabilize specific conformations of ion channels. Modern experimental methods that allow obtaining ion channel proteins in specific functional states are discussed, including cryoelectron microscopy and cryoelectron tomography. NMR (Nuclear Magnetic Resonance) spectroscopy approaches and molecular modeling studies of conformational dynamics of ion channels and their isolated domains are also discussed. Finally, new algorithms for studying the conformational mobility of proteins have been recently developed, which help to better understand the mechanisms of ion channel domain movements.

Wound infections due to antibiotic resistance pose a global public health problem. Phage therapy is a promising approach to address this issue. To improve localization, phage stability, delivery, and antibacterial performance, we propose polymer mix gel microbeads encapsulated with phages as a model for the delivery of phiKZ bacteriophage to combat Pseudomonas aeruginosa. Phages were loaded into the alginate pre-gel under magnetic stirring, with further cross-linking by chitosan and/or Ca2+ ions. The obtained gel microbeads were characterized using FTIR and Raman spectroscopy, and their cytotoxicity and antimicrobial properties were evaluated. This study demonstrated the efficient loading of high-titer phage lysate, achieving up to 99% encapsulation efficiency for alginate–chitosan microbeads. The key characteristics of the microbeads include stable physicochemical properties, slow but continuous phage release over 48 h in physiological saline, and low cytotoxicity. The phage-loaded microbeads demonstrated strong in vitro antimicrobial activity against P. aeruginosa PAO1, resulting in mean reductions of 6.9 log10 and 4.8 log10 CFU/mL for alginate and alginate–chitosan formulations, respectively. This corresponded to a decrease in bacterial concentration from approximately 1.1 × 1011 CFU/mL in untreated controls to 1.1 × 105 CFU/mL and 7.7 × 106 CFU/mL for alginate and alginate–chitosan formulations after 3 h of incubation.

Jumbo bacteriophages possess exceptionally large capsids accommodating genomes encode additional proteins, which support their infection and replication. A distinctive structural element, known as the inner body, has been observed in a number of phiKZ-like phage particles its proteins are believed to play an essential role in phage genome organization and ejection. However, the precise localization and three-dimensional structure of the inner body have remained elusive. Here, we applied the high-dose cryo-electron microscopy ("bubblegram") approach to localize the inner body within the capsid of phiKZ-like jumbo phage phiK601. The inner body was resolved cylindrical structure approximately 22 nm in diameter, tilted by similar to 20 degrees relative to the tail axis positioned asymmetrically, likely contacting the portal vertex and the opposing capsid edge. surrounded by 17 concentric layers of packaged DNA and exhibits positional flexibility within capsid.

Studying the mechanisms by which Gram-negative heterotrophic bacteria transition from active metabolism to dormancy is an important task, as it is directly related to the problem of bacterial antibiotic resistance and the spread of nosocomial infections. Using electron microscopy, microbiology, and molecular modeling, we investigated the dose-dependent mechanisms of action of 4-hexylresorcinol (4HR), a chemical analog of the anabiosis autoinducer, on the cell membranes of Gram-negative bacteria (using Escherichia coli as an example), leading to the formation of stressed, dormant, and mummified cells. It was shown that 4HR penetrates membranes equally easily both as single molecules and as micelles, distributing itself across the membrane so that the hydrocarbon radicals are aligned parallel to the lipid tails. When micelles penetrate the membrane, uneven distribution of 4HR within and between leaflets occurs, as well as lipid redistribution within the membrane, leading to the appearance of a third peak on the phospholipid electron density profile and a third black band in the membrane region in TEM images of such cells. At 4HR concentrations in solution of 200 µM, its micelles cover the cell membranes in a thick layer, penetrate into the membrane, and completely saturate it. Even higher concentrations create agglomerates or actually micellar arrays within the cell membranes, leading to cell death through mummification.

Nucleosomes are fundamental elements of chromatin organization that participate in compacting genomic DNA and serve as targets for the binding of numerous regulatory proteins. Currently, over 500 different nucleosome structures are known. Despite the large number of nucleosome structures, all of them were formed on only about twenty different DNA sequences. Using cryo-electron microscopy, we determined the structure of the nucleosome formed on a high-affinity Widom 603 DNA sequence at 4 Å resolution; an atomic model was built. We proposed an integrative modeling approach to study the nucleosomal DNA unwrapping based on the cryoelectron microscopy (cryo-EM) data. We also demonstrated the DNA unwrapping of the Widom 603 nucleosome using small angle X-ray scattering and single particle Förster resonance energy transfer measurements. Our results are consistent with the asymmetry of nucleosomal DNA unwrapping. Our data revealed the dependence of nucleosome structure and dynamics on the sequence of nucleosomal DNA.

The Dps protein is the major DNA-binding protein of prokaryotes, which protects DNA during starvation by forming a crystalline complex. The structure of such an intracellular DNA-Dps complex is still unknown. However, the phenomenon of a decrease in the size of the Dps protein from 90 Å to 69–75 Å during the formation of a complex with DNA has been repeatedly observed, and no explanation has been given. In this work, we show that during the formation of intracellular DNA–Dps crystals, the protein transitions to another oligomeric form: from a dodecameric (of 12 monomers), which has an almost spherical shape with a diameter of 90 Å, to a trimeric (of three monomers), which has a shape close to a torus-like structure with a diameter of 70 Å and a height of 40 Å. The trimer model was obtained through the molecular dynamic modeling of the interaction of the three monomers of the Dps protein. Placement of the obtained trimer in the electron density of in vitro DNA–Dps crystal allowed for the determination of the lattice parameters of the studied crystal. This crystal model was in good agreement with the SAXS data obtained from intracellular crystals of 2-day-old Escherichia coli cells. The final crystal structure contains a DNA molecule in the through channel of the crystal structure between the Dps trimers. It was discussed that the mechanism of protein transition from one oligomeric form to another in the cell cytoplasm could be regulated by intracellular metabolites and is a simple and flexible mechanism of prokaryotic cell transition from one metabolic state to another.

Eukaryotic transcription involves a complex interplay of protein factors that dynamically engage with chromatin at distinct stages. Among these, the histone chaperone FACT (Facilitates Chromatin Transcription) plays a unique role in nucleosome disassembly and reassembly during transcription, replication, and repair. While its functional importance is well established, the underlying structural mechanisms involved in these activities remain incompletely understood. The remarkable functional versatility of FACT in regulating genetic information processing likely stems from its distinctive structural and mechanical properties. This review focuses on the structural organization of FACT and analysis of the mechanisms involved in chromatin reorganization by this unusual histone chaperone.

Argonaute proteins provide innate immunity in all domains of life through guide-dependent recognition and cleavage of invader nucleic acids. Many short prokaryotic Argonautes (pAgos) lack nuclease activity and are instead co-encoded with tentative nuclease effectors, but their activation mechanisms remain unknown. Here, we characterize SPARHA systems (short prokaryotic argonautes, HNH-associated), containing HNH nuclease effectors. RNA-guided target DNA recognition by pAgo induces formation of SPARHA filaments with a chain of double nuclease sites formed by HNH tetramers, leading to indiscriminate collateral degradation of DNA and protecting bacterial population from invaders. We show that the assembly of filaments proceeds via a universal activation pathway involving a cascade of target-induced conformational changes in SPARHA, conserved in other short pAgo systems containing various types of effectors. pAgos and associated effectors act as modular defense systems that translate recognition of specific DNA into immune response through assembly of supramolecular complexes, deleterious for invaders and potentially useful for biotechnology.

Bacterial infections brought on by biofilms are the most common health concern in injuries, food industries among others, therefore, composite scaffolds that possess antibacterial characteristics are desirable. Herein, we developed a phage functionalized silk fibroin-based scaffold through surface charge modification of the composite scaffold with polyethyleimine (PEI). This was aimed at assessing the antibacterial efficiency of the composite scaffold against the host strain Bacillus subtilis, which would ultimately serve as a model approach for the design of diverse antibacterial biomaterials. The SF scaffold was initially formed through the direct freeze-thaw method, prior to polymerization with PEI and AR9 phage functionalization of the PEI polymerized Scaffolds by incubating with phage lysate. PEI exhibits antibacterial properties against both Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria, although it is significantly cytotoxic. To develop a biocompatible AR9 phage delivery scaffold with effective antibacterial properties against Bacillus subtilis, we modified the surface of a silk fibroin scaffold with PEI, resulting in a highly charged silk fibroin scaffold via use of low molecular weight PEI and concentration-based optimization of scaffold polymerization with PEI. The morphological and physiochemical properties of formed scaffold were assessed through Raman and Fourier infrared spectroscopy, while the antibacterial assays were done through growth inhibition zones/cell viability assays. The polymerized phage scaffold SF20_PEI.AR9 possessed the highest antimicrobial effect with clear inhibition zones of about 7.8 mm compared to about1.8 mm for the PEI polymerized scaffold (SF20_PEI) due to the lytic effect of surface attached phages on the bacterial cells thus underscoring significant effect of PEI polymerization in stabilizing AR9 phage attachment on the scaffolds. This direct polymerization approach achieved significant stabilization of the phages in the biomaterial mainly due to minimal alteration of the PEI architecture and thus could serve as a model for future development of phage functionalized scaffolds.