-
Viruses Mar 2024The molecular mechanism of how the infecting DNA of bacteriophage T4 passes from the capsid through the bacterial cell wall and enters the cytoplasm is essentially...
The molecular mechanism of how the infecting DNA of bacteriophage T4 passes from the capsid through the bacterial cell wall and enters the cytoplasm is essentially unknown. After adsorption, the short tail fibers of the infecting phage extend from the baseplate and trigger the contraction of the tail sheath, leading to a puncturing of the outer membrane by the tail tip needle composed of the proteins gp5.4, gp5 and gp27. To explore the events that occur in the periplasm and at the inner membrane, we constructed T4 phages that have a modified gp27 in their tail tip with a His-tag. Shortly after infection with these phages, cells were chemically cross-linked and solubilized. The cross-linked products were affinity-purified on a nickel column and the co-purified proteins were identified by mass spectrometry, and we found that predominantly the inner membrane proteins DamX, SdhA and PpiD were cross-linked. The same partner proteins were identified when purified gp27 was added to spheroplasts, suggesting a direct protein-protein interaction.
Topics: Bacteriophage T4; Escherichia coli; Cell Division; Escherichia coli Proteins; Viral Proteins
PubMed: 38675830
DOI: 10.3390/v16040487 -
Nature May 2024Bacteria have adapted to phage predation by evolving a vast assortment of defence systems. Although anti-phage immunity genes can be identified using bioinformatic...
Bacteria have adapted to phage predation by evolving a vast assortment of defence systems. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.
Topics: Escherichia coli; DNA Glycosylases; Bacteriophage T4; Virus Replication; T-Phages; Genome, Viral; Soil Microbiology; Metagenomics; Phylogeny
PubMed: 38632404
DOI: 10.1038/s41586-024-07329-9 -
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 -
Journal of Molecular Biology May 2024Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein essential for DNA replication. gp32 forms stable protein filaments on ssDNA...
Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein essential for DNA replication. gp32 forms stable protein filaments on ssDNA through cooperative interactions between its core and N-terminal domain. gp32's C-terminal domain (CTD) is believed to primarily help coordinate DNA replication via direct interactions with constituents of the replisome. However, the exact mechanisms of these interactions are not known, and it is unclear how tightly-bound gp32 filaments are readily displaced from ssDNA as required for genomic processing. Here, we utilized truncated gp32 variants to demonstrate a key role of the CTD in regulating gp32 dissociation. Using optical tweezers, we probed the binding and dissociation dynamics of CTD-truncated gp32, *I, to an 8.1 knt ssDNA molecule and compared these measurements with those for full-length gp32. The *I-ssDNA helical filament becomes progressively unwound with increased protein concentration but remains significantly more stable than that of full-length, wild-type gp32. Protein oversaturation, concomitant with filament unwinding, facilitates rapid dissociation of full-length gp32 from across the entire ssDNA segment. In contrast, *I primarily unbinds slowly from only the ends of the cooperative clusters, regardless of the protein density and degree of DNA unwinding. Our results suggest that the CTD may constrain the relative twist angle of proteins within the ssDNA filament such that upon critical unwinding the cooperative interprotein interactions largely vanish, facilitating prompt removal of gp32. We propose a model of CTD-mediated gp32 displacement via internal restructuring of its filament, providing a mechanism for rapid ssDNA clearing during genomic processing.
Topics: Bacteriophage T4; DNA Replication; DNA, Single-Stranded; DNA, Viral; DNA-Binding Proteins; Optical Tweezers; Protein Binding; Protein Domains; Viral Proteins
PubMed: 38508303
DOI: 10.1016/j.jmb.2024.168544 -
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 -
Viruses Jan 2024In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive... (Review)
Review
In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring-DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.
Topics: DNA, Viral; Bacteriophage T4; Fluorescence; Virus Assembly; DNA Packaging; Endodeoxyribonucleases
PubMed: 38399968
DOI: 10.3390/v16020192 -
Archives of Biochemistry and Biophysics May 2024Recent research into membrane interactions has uncovered a diverse range of therapeutic opportunities through the bioengineering of human and non-human macromolecules....
Recent research into membrane interactions has uncovered a diverse range of therapeutic opportunities through the bioengineering of human and non-human macromolecules. Although the majority of this research is focussed on fundamental developments, emerging studies are showcasing promising new technologies to combat conditions such as cancer, Alzheimer's and inflammatory and immune-based disease, utilising the alteration of bacteriophage, adenovirus, bacterial toxins, type 6 secretion systems, annexins, mitochondrial antiviral signalling proteins and bacterial nano-syringes. To advance the field further, each of these opportunities need to be better understood, and the therapeutic models need to be further optimised. Here, we summarise the knowledge and insights into several membrane interactions and detail their current and potential uses therapeutically.
PubMed: 38387829
DOI: 10.1016/j.abb.2024.109939 -
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 -
Foods (Basel, Switzerland) Dec 2023Finalyse, a T4 bacteriophage, is a pre-harvest intervention that utilizes a combination of bacteriophages to reduce incoming O157:H7 prevalence by destroying the...
Finalyse, a T4 bacteriophage, is a pre-harvest intervention that utilizes a combination of bacteriophages to reduce incoming O157:H7 prevalence by destroying the bacteria on the hides of harvest-ready cattle entering commercial abattoirs. The objective of this study was to evaluate the efficacy of Finalyse, as a pre-harvest intervention, on the reduction in pathogens, specifically O157:H7, on the cattle hides and lairage environment to overall reduce incoming pathogen loads. Over 5 sampling events, a total of 300 composite hide samples were taken using 25 mL pre-hydrated Buffered Peptone Water (BPW) swabs, collected before and after the hide wash intervention, throughout the beginning, middle, and end of the production day ( = 10 swabs/sampling point/timepoint). A total of 171 boot swab samples were also simultaneously taken at the end of the production day by walking from the front to the back of the pen in a pre-determined 'Z' pattern to monitor the pen floor environment from 3 different locations in the lairage area. The prevalence of pathogens was analyzed using the BAX System Real-Time PCR Assay. There were no significant reductions observed for and/or any Shiga toxin-producing (STEC) on the hides after the bacteriophage application ( > 0.05). O157:H7 and O111 hide prevalence was very low throughout the study; therefore, no further analysis was conducted. However, boot swab monitoring showed a significant reduction in O157:H7, O26, and O45 in the pen floor environment ( < 0.05). While using Finalyse as a pre-harvest intervention in the lairage areas of commercial beef processing facilities, this bacteriophage failed to reduce O157:H7 on the hides of beef cattle, as prevalence was low; however, some STECs were reduced in the lairage environment, where the bacteriophage was applied. Overall, an absolute conclusion was not formed on the effectiveness of Finalyse and its ability to reduce O157:H7 on the hides of beef cattle, as prevalence on the hides was low.
PubMed: 38231835
DOI: 10.3390/foods12234349 -
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