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Nucleic Acids Research Sep 2023Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA...
Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA through interprotein interactions between its core and N-terminus. However, detailed understanding of gp32 filament structure and organization remains incomplete, particularly for longer, biologically-relevant DNA lengths. Moreover, it is unclear how these tightly-bound filaments dissociate from ssDNA during complementary strand synthesis. We use optical tweezers and atomic force microscopy to probe the structure and binding dynamics of gp32 on long (∼8 knt) ssDNA substrates. We find that cooperative binding of gp32 rigidifies ssDNA while also reducing its contour length, consistent with the ssDNA helically winding around the gp32 filament. While measured rates of gp32 binding and dissociation indicate nM binding affinity, at ∼1000-fold higher protein concentrations gp32 continues to bind into and restructure the gp32-ssDNA filament, leading to an increase in its helical pitch and elongation of the substrate. Furthermore, the oversaturated gp32-ssDNA filament becomes progressively unwound and unstable as observed by the appearance of a rapid, noncooperative protein dissociation phase not seen at lower complex saturation, suggesting a possible mechanism for prompt removal of gp32 from the overcrowded ssDNA in front of the polymerase during replication.
Topics: Bacteriophage T4; DNA Helicases; DNA Replication; DNA, Single-Stranded; DNA, Viral; DNA-Binding Proteins; Protein Binding; Viral Proteins
PubMed: 37449435
DOI: 10.1093/nar/gkad595 -
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 -
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; Cell Division; Escherichia coli; Escherichia coli Proteins; Viral Proteins
PubMed: 38675830
DOI: 10.3390/v16040487 -
Environmental Science & Technology Sep 2023Virus concentrations measured in municipal wastewater help inform both the water treatment necessary to protect human health and wastewater-based epidemiology....
Virus concentrations measured in municipal wastewater help inform both the water treatment necessary to protect human health and wastewater-based epidemiology. Wastewater measurements are typically PCR-based, and interpreting gene copy concentrations requires an understanding of the form and stability of the nucleic acids. Here, we study the persistence of model virus genomes in wastewater, the protective effects provided by the virus capsids, and the relative decay rates of the genome and infectious viruses. In benchtop batch experiments in wastewater influent at 25 °C, extraviral (+)ssRNA and dsDNA amplicons degraded by 90% within 15-19 min and 1.6-1.9 h, respectively. When encapsidated, the for MS2 (+)ssRNA increased by 424× and the for T4 dsDNA increased by 52×. The (+)ssRNA decay rates were similar for a range of amplicon sizes. For our model phages MS2 and T4, the nucleic acid signal in untreated wastewater disappeared shortly after the viruses lost infectivity. Combined, these results suggest that most viral genome copies measured in wastewater are encapsidated, that measured concentrations are independent of assay amplicon sizes, and that the virus genome decay rates of nonenveloped (i.e., naked) viruses are similar to inactivation rates. These findings are valuable for the interpretation of wastewater virus measurements.
Topics: Humans; RNA; Wastewater; Capsid; Genome, Viral; Biological Assay
PubMed: 37656816
DOI: 10.1021/acs.est.3c03814 -
Pharmaceutics Dec 2023Therapeutic application of bacterial viruses (phage therapy) has in recent years been rediscovered by many scientists, as a method which may potentially replace...
Therapeutic application of bacterial viruses (phage therapy) has in recent years been rediscovered by many scientists, as a method which may potentially replace conventional antibacterial strategies. However, one of the main problems related to phage application is the stability of bacterial viruses. Though many techniques have been used to sustain phage activity, novel tools are needed to allow long-term phage storage and application in versatile forms. In this study, we combined two well-known methods for bacteriophage immobilization. First, encapsulated phages were obtained by means of extrusion-ionic gelation, and then alginate microspheres were dried using the lyophilization process (freeze-drying). To overcome the risk of phage instability upon dehydration, the microspheres were prepared with the addition of 0.3 M mannitol. Bacteriophage-loaded microspheres were stored at room temperature for 30 days and subsequently exposed to simulated gastric fluid (SGF). The survival of encapsulated phages after drying was significantly higher in the presence of mannitol. The highest number of viable bacteriophages exceeding 4.8 log pfu/mL in SGF were recovered from encapsulated and freeze-dried microspheres, while phages in lyophilized lysate were completely inactivated. Although the method requires optimization, it may be a promising approach for the immobilization of bacteriophages in terms of practical application.
PubMed: 38140132
DOI: 10.3390/pharmaceutics15122792 -
PHAGE (New Rochelle, N.Y.) Sep 2023In the light of the worldwide antimicrobial resistance crisis, new substitutes to antibiotics are urgently needed. Tailocins or phage tail-like bacteriocin particles,...
In the light of the worldwide antimicrobial resistance crisis, new substitutes to antibiotics are urgently needed. Tailocins or phage tail-like bacteriocin particles, produced by bacteria for environmental competition, are a potential antimicrobial alternative to antibiotic treatment. Yet, the availability of characterized Tailocins is limited. We explored the possibility to produce new Tailocins from phage particles, using osmotic shock or chemical treatment by the ammonium quaternary compound benzalkonium chloride on phage S117 and using phage T4 as control. We report that phage S117 was resistant to such treatment, while successful production of Tailocins by osmotic shock was achieved for phage T4. Finally, chemical treatment with benzalkonium chloride was inefficient on phage S117 but successfully inactivated phage T4 without production of Tailocins. Further studies are needed to implement such treatments of phages for producing Tailocins with killing activity.
PubMed: 37841391
DOI: 10.1089/phage.2023.0014 -
Microbiology Resource Announcements Jan 2024is a rarely isolated opportunistic pathogen in animals and humans. Here, we present the annotated genome sequence of phage Mangalyan, a T4-like bacteriophage infecting...
is a rarely isolated opportunistic pathogen in animals and humans. Here, we present the annotated genome sequence of phage Mangalyan, a T4-like bacteriophage infecting isolated from chickens. Phage Mangalyan has a genome length of 140,513 bp and belongs to the family.
PubMed: 38088570
DOI: 10.1128/mra.00963-23 -
Nanomaterials (Basel, Switzerland) Jul 2023Titanium (Ti) is widely recognized for its exceptional properties and compatibility with medical applications. In our study, we successfully formed laser-induced...
Titanium (Ti) is widely recognized for its exceptional properties and compatibility with medical applications. In our study, we successfully formed laser-induced periodic surface structures (LIPSS) on Ti plates with a periodicity of 520-740 nm and a height range of 150-250 nm. To investigate the morphology and chemical composition of these surfaces, we employed various techniques, including field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. Additionally, we utilized a drop-shape analyzer to determine the wetting properties of the surfaces. To evaluate the antibacterial activity, we followed the ISO 22196:2011 standard, utilizing reference bacterial cultures of Gram-positive (ATCC 25923) and Gram-negative (ATCC 25922). The results revealed enhanced antibacterial properties against by more than 99% and by more than 80% in comparison with non-irradiated Ti. Furthermore, we conducted experiments using the bacteriophage T4 (ATCC 11303-B4) and the bacterial host (ATCC 11303) to investigate the impact of Ti plates on the stability of the bacteriophage. Overall, our findings highlight the potential of LIPSS on Ti plates for achieving enhanced antibacterial activity against common bacterial strains while maintaining the stability of bacteriophages.
PubMed: 37513043
DOI: 10.3390/nano13142032 -
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 -
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