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ACS Biomaterials Science & Engineering Jun 2023The use of bacteriophages, viruses that specifically infect bacteria, as antibiotics has become an area of great interest in recent years as the effectiveness of...
The use of bacteriophages, viruses that specifically infect bacteria, as antibiotics has become an area of great interest in recent years as the effectiveness of conventional antibiotics recedes. The detection of phage interactions with specific bacteria in a rapid and quantitative way is key for identifying phages of interest for novel antimicrobials. Outer membrane vesicles (OMVs) derived from Gram-negative bacteria can be used to make supported lipid bilayers (SLBs) and therefore membrane models that contain naturally occurring components of the bacterial outer membrane. In this study, we employed OMV derived SLBs and use both fluorescent imaging and mechanical sensing techniques to show their interactions with T4 phage. We also integrate these bilayers with microelectrode arrays (MEAs) functionalized with the conducting polymer PEDOT:PSS and show that the pore forming interactions of the phages with the SLBs can be monitored using electrical impedance spectroscopy. To highlight our ability to detect specific phage interactions, we also generate SLBs using OMVs derived from , which is resistant to T4 phage infection, and identify their lack of interaction with the phage. The work presented here shows how interactions occurring between the phages and these complex SLB systems can be monitored using a range of experimental techniques. We believe this approach can be used to identify phages that work against bacterial strains of interest, as well as more generally to monitor any pore forming structure (such as defensins) interacting with bacterial outer membranes, and thus aid in the development of next generation antimicrobials.
Topics: Lipid Bilayers; Bacteriophages; Escherichia coli; Anti-Bacterial Agents
PubMed: 37137156
DOI: 10.1021/acsbiomaterials.3c00021 -
Biochemistry Jun 2023Noble gases have well-established biological effects, yet their molecular mechanisms remain poorly understood. Here, we investigated, both experimentally and...
Noble gases have well-established biological effects, yet their molecular mechanisms remain poorly understood. Here, we investigated, both experimentally and computationally, the molecular modes of xenon (Xe) action in bacteriophage T4 lysozyme (T4L). By combining indirect gassing methods with a colorimetric lysozyme activity assay, a reversible, Xe-specific (20 ± 3)% inhibition effect was observed. Accelerated molecular dynamic simulations revealed that Xe exerts allosteric inhibition on the protein by expanding a C-terminal hydrophobic cavity. Xe-induced cavity expansion results in global conformational changes, with long-range transduction distorting the active site where peptidoglycan binds. Interestingly, the peptide substrate binding site that enables lysozyme specificity does not change conformation. Two T4L mutants designed to reshape the C-terminal Xe cavity established a correlation between cavity expansion and enzyme inhibition. This work also highlights the use of Xe flooding simulations to identify new cryptic binding pockets. These results enrich our understanding of Xe-protein interactions at the molecular level and inspire further biochemical investigations with noble gases.
Topics: Xenon; Muramidase; Noble Gases; Binding Sites; Proteins
PubMed: 37192381
DOI: 10.1021/acs.biochem.3c00046 -
Journal of Veterinary Research Dec 2023In the light of the problem of antibiotic resistance, the use of combined alternative therapies in combatting bacteria-related disorders has gained popularity....
INTRODUCTION
In the light of the problem of antibiotic resistance, the use of combined alternative therapies in combatting bacteria-related disorders has gained popularity. Bacteriophages are one element implemented in new combination therapy. is known to have antimicrobial activity and regarded as potentially having a synergistic effect with bacteriophages. Therefore, possible interactions of lytic bacteriophages (MS2, T4 and Phi6) with acetone and methanol extracts (SRa and SRm) in the bacterial environment were examined.
MATERIAL AND METHODS
The interactions were tested using a microdilution method, phage-extract co-incubation assay, static interaction (synography) and dynamic growth profile experiments in a bioreactor.
RESULTS
The interactions of the tested factors in a static environment differed from those in a dynamic environment. Dynamic conditions altered the effect of the extracts in a concentration-dependent manner. How different the effect of the SRa extract was to that of the SRm extract on bacterial growth in a dynamic environment depended on the species of the phage and bacterial host. The greatest differences were observed for strains and their phages, whereas and the Phi6 phage reacted very similarly to both extracts. Differences also emerged for the same extract in different strains and their phages.
CONCLUSION
Every extract type should be tested on a case-by-case basis and experiment outcomes should not be generalised before gathering data. Moreover, many varied experiments should be performed, especially when examining such multifactorial mixtures. The tested mixtures could potentially be used in multidrug-resistant bacterial infection treatments.
PubMed: 38130461
DOI: 10.2478/jvetres-2023-0059 -
Nature Structural & Molecular Biology Mar 2024Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between...
Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.
Topics: Adenosine Triphosphatases; Cryoelectron Microscopy; DNA Replication; DNA; ATPases Associated with Diverse Cellular Activities; Mutagenesis; Adenosine Triphosphate
PubMed: 38177685
DOI: 10.1038/s41594-023-01177-3 -
RSC Advances Feb 2024The exploration of single-strand DNA-binding protein (SSB)-ssDNA interactions and their crucial roles in essential biological processes lagged behind other types of...
The exploration of single-strand DNA-binding protein (SSB)-ssDNA interactions and their crucial roles in essential biological processes lagged behind other types of protein-nucleic acid interactions, such as protein-dsDNA and protein-RNA interactions. The ssDNA binding protein gene product 32 (gp32) of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during the DNA synthesis. To gain deeper insights into the electrostatic conditions influencing the stability of the ssDNA-gp32 molecular complex, like the salt concentration or some metal ions proven to specifically bind to gp32, we employed a method that performs rapid measurements of the DNA-protein stability using an α-Hemolysin (α-HL) protein nanopore. We indirectly probed the stability of a protein-nucleic acid complex by monitoring the dissociation process between the gp32 protein and the ssDNA molecular complex in single-molecular electrophysiology experiments, but also through fluorescence spectroscopy techniques. We have shown that the complex is more stable in 0.5 M KCl solution than in 2 M KCl solution and that the presence of Zn ions further increases this stability for any salt used in the present study. This method can be applied to other nucleic acid-protein molecular complexes, as well as for an accurate determination of the drug-protein carrier stability.
PubMed: 38352678
DOI: 10.1039/d3ra07746b -
BioRxiv : the Preprint Server For... Apr 2024A major challenge faced by is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, has...
A major challenge faced by is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, has evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized more were encoded in given the relative paucity of phage that have been isolated which infect this species. Using a genomic library, we identified a Type IV restriction system consisting of two genes within a 16kB region of the pathogenicity island-2 that we name TgvA and TgvB (ype I-embedded mrSD-like system of PI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modification are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We show that additional phage defense genes are encoded in VPI-2 that protect against other phage like T3, secΦ18, secΦ27 and λ. Our study uncovers a novel Type IV restriction system in , increasing our understanding of the evolution and ecology of while highlighting the evolutionary interplay between restriction systems and phage genome modification.
PubMed: 38617239
DOI: 10.1101/2024.04.05.588314 -
Molecular Biology Reports Feb 2024Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence...
BACKGROUND
Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility.
METHODS
Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His)-tagged uvsY.
RESULTS
Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY.
CONCLUSIONS
The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.
Topics: Recombinases; Solubility; Gene Library; Amino Acids; Mutagenesis
PubMed: 38411701
DOI: 10.1007/s11033-024-09367-y -
Scientific Reports Sep 2023Immobilization of bacteriophages onto solid supports such as magnetic particles has demonstrated ultralow detection limits as biosensors for the separation and detection...
Immobilization of bacteriophages onto solid supports such as magnetic particles has demonstrated ultralow detection limits as biosensors for the separation and detection of their host bacteria. While the potential impact of magnetized phages is high, the current methods of immobilization are either weak, costly, inefficient, or laborious making them less viable for commercialization. In order to bridge this gap, we have developed a highly efficient, site-specific, and low-cost method to immobilize bacteriophages onto solid supports. While streptavidin-biotin represents an ideal conjugation method, the functionalization of magnetic particles with streptavidin requires square meters of coverage and therefore is not amenable to a low-cost assay. Here, we genetically engineered bacteriophages to allow synthesis of a monomeric streptavidin during infection of the bacterial host. The monomeric streptavidin was fused to a capsid protein (Hoc) to allow site-specific self-assembly of up to 155 fusion proteins per capsid. Biotin coated magnetic nanoparticles were functionalized with mSA-Hoc T4 phage demonstrated in an E. coli detection assay with a limit of detection of < 10 CFU in 100 mLs of water. This work highlights the creation of genetically modified bacteriophages with a novel capsid modification, expanding the potential for bacteriophage functionalized biotechnologies.
Topics: Bacteriophages; Streptavidin; Biotin; Escherichia coli; Bacteriophage T4; Bacteria; Magnetic Phenomena
PubMed: 37758721
DOI: 10.1038/s41598-023-42626-9