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Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.

The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures.Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required.This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the Virus BioResistor (VBR). The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.

DJ-1, a 20.7 kDa protein, is overexpressed in people who have bladder cancer (BC). Its elevated concentration in urine allows it to serve as a marker for BC. However, no biosensor for the detection of DJ-1 has been demonstrated. Here, we describe a virus bioresistor (VBR) capable of detecting DJ-1 in urine at a concentration of 10 pM in 1 min. The VBR consists of a pair of millimeter-scale gold electrodes that measure the electrical impedance of an ultrathin (≈ 150-200 nm), two-layer polymeric channel. The top layer of this channel (90-105 nm in thickness) consists of an electrodeposited virus-PEDOT (PEDOT is poly(3,4-ethylenedioxythiophene)) composite containing embedded M13 virus particles that are engineered to recognize and bind to the target protein of interest, DJ-1. The bottom layer consists of spin-coated PEDOT-PSS (poly(styrenesulfonate)). Together, these two layers constitute a current divider. We demonstrate here that reducing the thickness of the bottom PEDOT-PSS layer increases its resistance and concentrates the resistance drop of the channel in the top virus-PEDOT layer, thereby increasing the sensitivity of the VBR and enabling the detection of DJ-1. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise (S/N > 100) and excellent sensor-to-sensor reproducibility characterized by coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a concentration of 30 pM, near the 10 pM limit of detection (LOD), encompassing four orders of magnitude in concentration.

Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage's minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells.

Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencingbased reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

The virus bioresistor (VBR) is a chemiresistor that directly transfers information from virus particles to an electrical circuit. Specifically, the VBR enables the label-free detection of a target protein that is recognized and bound by filamentous M13 virus particles, each with dimensions of 6 nm ( w) × 1 μm ( l), entrained in an ultrathin (∼250 nm) composite virus-polymer resistor. Signal produced by the specific binding of virus to target molecules is monitored using the electrical impedance of the VBR: The VBR presents a complex impedance that is modeled by an equivalent circuit containing just three circuit elements: a solution resistance ( Rsoln), a channel resistance ( RVBR), and an interfacial capacitance ( CVBR). The value of RVBR, measured across 5 orders of magnitude in frequency, is increased by the specific recognition and binding of a target protein to the virus particles in the resistor, producing a signal Δ RVBR. The VBR concept is demonstrated using a model system in which human serum albumin (HSA, 66 kDa) is detected in a phosphate buffer solution. The VBR cleanly discriminates between a change in the electrical resistance of the buffer, measured by Rsoln, and selective binding of HSA to virus particles, measured by RVBR. The Δ RVBR induced by HSA binding is as high as 200 Ω, contributing to low sensor-to-sensor coefficients-of-variation (

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two-dimensional (2D) cell, three-dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60-80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein's initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

Adenosine deaminases that act on RNA (ADARs) carry out adenosine (A) to inosine (I) editing reactions with a known requirement for duplex RNA. Here, we show that ADARs also react with DNA/RNA hybrid duplexes. Hybrid substrates are deaminated efficiently by ADAR deaminase domains at dA-C mismatches and with E to Q mutations in the base flipping loop of the enzyme. For a long, perfectly matched hybrid, deamination is more efficient with full length ADAR2 than its isolated deaminase domain. Guide RNA strands for directed DNA editing by ADAR were used to target six different 2΄-deoxyadenosines in the M13 bacteriophage ssDNA genome. DNA editing efficiencies varied depending on the sequence context of the editing site consistent with known sequence preferences for ADARs. These observations suggest the reaction within DNA/RNA hybrids may be a natural function of human ADARs. In addition, this work sets the stage for development of a new class of genome editing tools based on directed deamination of 2΄-deoxyadenosines in DNA/RNA hybrids.

The label-free detection of human serum albumin (HSA) in aqueous buffer is demonstrated using a simple, monolithic, two-electrode electrochemical biosensor. In this device, both millimeter-scale electrodes are coated with a thin layer of a composite containing M13 virus particles and the electronically conductive polymer poly(3,4-ethylenedioxy thiophene) or PEDOT. These virus particles, engineered to selectively bind HSA, serve as receptors in this biosensor. The resistance component of the electrical impedance, Zre, measured between these two electrodes provides electrical transduction of HSA binding to the virus-PEDOT film. The analysis of sample volumes as small as 50 μL is made possible using a microfluidic cell. Upon exposure to HSA, virus-PEDOT films show a prompt increase in Zre within 5 s and a stable Zre signal within 15 min. HSA concentrations in the range from 100 nM to 5 μM are detectable. Sensor-to-sensor reproducibility of the HSA measurement is characterized by a coefficient-of-variance (COV) ranging from 2% to 8% across this entire concentration range. In addition, virus-PEDOT sensors successfully detected HSA in synthetic urine solutions.

Bacteriophage M13 assembles its progeny particles in the inner membrane of the host. The major component of the assembly machine is G1p and together with G11p it generates an oligomeric structure with a pore-like inner cavity and an ATP hydrolysing domain. This allows the formation of the phage filament, which assembles multiple copies of the membrane-inserted major coat protein G8p around the extruding single-stranded circular DNA. The phage filament then passes through the G4p secretin that is localized in the outer membrane. Presumably, the inner membrane G1p/G11p and the outer G4p form a common complex. To unravel the structural details of the M13 assembly machine, we purified G1p from infected E. coli cells. The protein was overproduced together with G11p and solubilized from the membrane as a multimeric complex with a size of about 320 kDa. The complex revealed a pore-like structure with an outer diameter of about 12 nm, matching the dimensions of the outer membrane G4p secretin. The function of the M13 assembly machine for phage generation and secretion is discussed. [ABSTRACT FROM AUTHOR]

Advances in self-assembly over the past decade have demonstrated that nano- and microscale particles can be organized into a large diversity of ordered three-dimensional (3D) lattices. However, the ability to generate different desired lattice types from the same set of particles remains challenging. Here, we show that nanoparticles can be assembled into crystalline and open 3D frameworks by connecting them through designed DNA-based polyhedral frames. The geometrical shapes of the frames, combined with the DNA-assisted binding properties of their vertices, facilitate the well-defined topological connections between particles in accordance with frame geometry. With this strategy, different crystallographic lattices using the same particles can be assembled by introduction of the corresponding DNA polyhedral frames. This approach should facilitate the rational assembly of nanoscale lattices through the design of the unit cell.

We describe high resolution MAS solid-state NMR experiments that utilize (1)H detection with 60kHz magic angle spinning; simultaneous cross-polarization from (1)H to (15)N and (13)C nuclei; bidirectional cross-polarization between (13)C and (15)N nuclei; detection of both amide nitrogen and aliphatic carbon (1)H; and measurement of both (13)C and (15)N chemical shifts through multi-dimensional correlation experiments. Three-dimensional experiments correlate amide (1)H and alpha (1)H selectively with (13)C or (15)N nuclei in a polypeptide chain. Two separate three-dimensional spectra correlating (1)Hα/(13)Cα/(1)H(N) and (1)H(N)/(15)N/(1)Hα are recorded simultaneously in a single experiment, demonstrating that a twofold savings in experimental time is potentially achievable. Spectral editing using bidirectional coherence transfer pathways enables simultaneous magnetization transfers between (15)N, (13)Cα(()(i)()) and (13)C'(()(i)(-1)), facilitating intra- and inter-residue correlations for sequential resonance assignment. Non-uniform sampling is integrated into the experiments, further reducing the length of experimental time.

Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-a) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencingbased reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations. [ABSTRACT FROM AUTHOR]

Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ∼1 μm and a diameter of ∼7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6-16.7 Å and a tilt of 36.1-36.6° between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.

Contact-dependent growth inhibition (CDI) is a widespread form of inter-bacterial competition that requires direct cell-to-cell contact. CDI(+) inhibitor cells express CdiA effector proteins on their surface. CdiA binds to specific receptors on susceptible target bacteria and delivers a toxin derived from its C-terminal region (CdiA-CT). Here, we show that purified CdiA-CT(536) toxin from uropathogenic Escherichia coli 536 translocates into bacteria, thereby by-passing the requirement for cell-to-cell contact during toxin delivery. Genetic analyses demonstrate that the N-terminal domain of CdiA-CT(536) is necessary and sufficient for toxin import. The CdiA receptor plays no role in this import pathway; nor do the Tol and Ton systems, which are exploited to internalize colicin toxins. Instead, CdiA-CT(536) import requires conjugative F pili. We provide evidence that the N-terminal domain of CdiA-CT(536) interacts with F pilin, and that pilus retraction is critical for toxin import. This pathway is reminiscent of the strategy used by small RNA leviviruses to infect F(+) cells. We propose that CdiA-CT(536) mimics the pilin-binding maturation proteins of leviviruses, allowing the toxin to bind F pili and become internalized during pilus retraction.

M13 bacteriophage display presents polypeptides as fusions to phage coat proteins. Such phage-displayed ligands offer useful reagents for biosensors. Here, we report a modified phage propagation protocol for the consistent and robust display of two different genetically encoded ligands on the major coat protein, P8. The results demonstrate that the phage surface reaches a saturation point for maximum peptide display.

In the stationary, aligned samples used in oriented sample (OS) solid-state NMR, (1)H-(1)H homonuclear dipolar couplings are not attenuated as they are in magic angle spinning solid-state NMR; consequently, they are available for participation in dipolar coupling-based spin-exchange processes. Here we describe analytically the pathways of (15)N-(15)N spin-exchange mediated by (1)H-(1)H homonuclear dipolar couplings. The mixed-order proton-relay mechanism can be differentiated from the third spin assisted recoupling mechanism by setting the (1)H to an off-resonance frequency so that it is at the "magic angle" during the spin-exchange interval in the experiment, since the "magic angle" irradiation nearly quenches the former but only slightly attenuates the latter. Experimental spectra from a single crystal of N-acetyl leucine confirm that this proton-relay mechanism plays the dominant role in (15)N-(15)N dilute-spin-exchange in OS solid-state NMR in crystalline samples. Remarkably, the "forbidden" spin-exchange condition under "magic angle" irradiation results in (15)N-(15)N cross-peaks intensities that are comparable to those observed with on-resonance irradiation in applications to proteins. The mechanism of the proton relay in dilute-spin-exchange is crucial for the design of polarization transfer experiments.

Bacteriophage M13 assembles its progeny particles in the inner membrane of the host. The major component of the assembly machine is G1p and together with G11p it generates an oligomeric structure with a pore-like inner cavity and an ATP hydrolysing domain. This allows the formation of the phage filament, which assembles multiple copies of the membrane-inserted major coat protein G8p around the extruding single-stranded circular DNA. The phage filament then passes through the G4p secretin that is localized in the outer membrane. Presumably, the inner membrane G1p/G11p and the outer G4p form a common complex. To unravel the structural details of the M13 assembly machine, we purified G1p from infected E. coli cells. The protein was overproduced together with G11p and solubilized from the membrane as a multimeric complex with a size of about 320 kDa. The complex revealed a pore-like structure with an outer diameter of about 12 nm, matching the dimensions of the outer membrane G4p secretin. The function of the M13 assembly machine for phage generation and secretion is discussed.

Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low μM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Lymphoid organs are the main structural components of the immune system. In the current research, the mixture of poly lactic-co-glycolic acid (PLGA), polycaprolactone (PCL), and M13 phage or its RGD-modified form was used in the construction of a fibrillar scaffold using the electrospinning method. The constructs were transplanted intra-abdominally and examined for the formation of lymphoid-like tissues at different time intervals. The confocal and scanning electron microscopy demonstrate that M13 phage-containing scaffolds provide a suitable environment for lymph node-isolated fibroblasts. Morphological analysis demonstrate the formation of lymph node-like tissues in the M13 phage-containing scaffolds after transplantation. Histological analysis confirm both blood and lymph angiogenesis in the implanted construct and migration of inflammatory cells to the M13 phage-containing scaffolds. In addition, flow cytometry and immunohistochemistry analysis showed the homing and compartmentalization of dendritic cells (DCs), B and T lymphocytes within the PLGA/PCL/M13 phage-RGD based scaffolds and similar to what is seen in the mouse lymphoid tissues. It seems that the application of M13 phage could improve the generation of functional lymphoid tissues in the electrospun scaffolds and could be used for lymphoid tissue regeneration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Developing materials that are effective against sexually transmitted pathogens such as Chlamydia trachomatis (Ct) and HIV-1 is challenging both in terms of material selection and improving bio-membrane and cellular permeability at desired mucosal sites. Here, we engineered the prokaryotic bacterial virus (M13 phage) carrying two functional peptides, integrin binding peptide (RGD) and a segment of the polymorphic membrane protein D (PmpD) from Ct, as a phage-based material that can ameliorate Ct infection. Ct is a globally prevalent human pathogen for which there are no effective vaccines or microbicides. We show that engineered phage stably express both RGD motifs and Ct peptides and traffic intracellularly and into the lumen of the inclusion in which the organism resides within the host cell. Engineered phage were able to significantly reduce Ct infection in both HeLa and primary endocervical cells compared with Ct infection alone. Polyclonal antibodies raised against PmpD and co-incubated with constructs prior to infection did not alter the course of infection, indicating that PmpD is responsible for the observed decrease in Ct infection. Our results suggest that phage-based design approaches to vector delivery that overcome mucosal cellular barriers may be effective in preventing Ct and other sexually transmitted pathogens.

Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins (MPs). The scope and limitations of MP display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral MP neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic MP Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7(+) helper phage. The modified phage coat of KO7(+) can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively α-helical, integral MPs failed to express as fusions to an anchoring phage coat protein and therefore did not display on the phage surface. However, the β-barrel MPs ShuA (Shigella heme uptake A) and MOMP (major outer membrane protein), which pass through the membrane 22 and 16 times, respectively, can display surprisingly well on the surfaces of both conventional and KO7(+) phages. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of MPs.

Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

The separate fields of conducting polymer-based electrochemical sensors and virus-based molecular recognition offer numerous advantages for biosensing. Grafting M13 bacteriophage into an array of poly (3,4-ethylenedioxythiophene) (PEDOT) nanowires generated hybrids of conducting polymers and viruses. The virus incorporation into the polymeric backbone of PEDOT occurs during electropolymerization via lithographically patterned nanowire electrodeposition. The resultant arrays of virus-PEDOT nanowires enable real-time, reagent-free electrochemical biosensing of analytes in physiologically relevant buffers.

BACKGROUND Bacteriophages are bionanoparticles with several applications in different biotechnology‐based products. Among them, vaccines have the potential to treat antibiotic‐resistant bacteria and parasitic infections. Traditional methods for their recovery and purification rely on precipitation with polyethylene glycol (PEG) and NaCl. However, the applicability of such an approach is limited, due to large‐scale technical constrains. Recently, our research group developed a bacteriophage M13 recovery and purification strategy using Aqueous Two‐Phase Systems (ATPS), simplifying the methodology and, potentially, reducing costs. This work aims to develop an economic contrast between ATPS and the traditional PEG precipitation method at different operation scales (10 to 1000 L bioreactor volume) to determine the applicability of the ATPS methodology at large scale. For this, the effect of ATPS volume ratio (VR), sample loading, and materials discount over production cost were analyzed. RESULTS: Results indicate that as the discount on material costs increases, ATPS becomes a more affordable unit operation, from a bioreactor scale of 10 L at 0% discount to 410 L at 90% discount (US$ 52.2 to $1.72 per gram, respectively). Material cost contribution is a parameter that needs attention when working with ATPS, as it is the core for the system construction. Methods for reducing their contribution are highly relevant and should be further investigated. CONCLUSION: Employment of economic analyses help to discover critical parameters for a bioprocess, such as material costs for ATPS. This economic analysis work serves as a platform for new strategies for the recovery of bacteriophage and bacteriophage‐like particles using ATPS‐based technologies. © 2020 Society of Chemical Industry (SCI) [ABSTRACT FROM AUTHOR]

Cattle tick (Rhipicephalus microplus) represents a severe problem causing substantial economic losses, estimated in billions of dollars annually. Currently, chemical acaricides represent the most widely used control method. However, several problems such as resistance have been described. Phage-based vaccines represent a fast and low-cost tool for antigen delivery. In this regard, the objective of the present work was to develop a candidate phage-based vaccine displaying a cattle tick antigen (Bm86-derived Sbm7462 antigen) on the surface of bacteriophage M13. Phage ELISA and dot blotting analysis confirmed the display of the antigen. Vaccine immunogenicity was evaluated using a bovine monocyte-derived dendritic cell-based ex vivo assay and a murine in vivo assay. The ex vivo model showed the maturation of dendritic cells after being pulsed with the phage-based vaccine. The humoral response was confirmed in the in vivo assay. These results demonstrated the capacity of the phage-based vaccine to induce both humoral and cellular immune-specific responses. Importantly, this is the first report describing a control method for cattle ticks using a candidate phage-based vaccine. Further studies to evaluate the immunogenicity in a bovine model are needed. The current approach represents a promising alternative to control cattle tick infestations.

The minor coat protein G3p of bacteriophage M13 is the key component for the host interaction of this virus and binds to Escherichia coli at the tip of the F pili. As we show here, during the biosynthesis of G3p as a preprotein, the signal sequence interacts primarily with SecY, whereas the hydrophobic anchor sequence at the C-terminus interacts with YidC. Using arrested nascent chains and thiol crosslinking, we show here that the ribosome-exposed signal sequence is first contacted by SecY but not by YidC, suggesting that only SecYEG is involved at this early stage. The protein has a large periplasmic domain, a hydrophobic anchor sequence of 21 residues and a short C-terminal tail that remains in the cytoplasm. During the later synthesis of the entire G3p, the residues 387, 389 and 392 in anchor domain contact YidC in its hydrophobic slide to hold translocation of the C-terminal tail. Finally, the protein is processed by leader peptidase and assembled into new progeny phage particles that are extruded out of the cell.

Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the alpha(v)beta3 and alpha(v)beta5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C-D(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.

Cervical cancer is a major global health concern, characterized by its high incidence and mortality rates. The detection of tumor markers is crucial for managing cancer, making treatment decisions, and monitoring disease progression. Vascular endothelial growth factor (VEGF) and programmed death-ligand 1 (PDL-1) are key targets in cervical cancer therapy and valuable biomarkers in predicting treatment response and prognosis. In this study, we found that combining the measurement of VEGF and soluble PDL-1 can be used for diagnosing and evaluating the progression of cervical cancer. To explore a more convenient approach for detecting and assessing cervical cancer, we designed and prepared an engineered fd bacteriophage, a human-safe viral nanofiber, equipped with two peptides targeting VEGF and PD-L1. The dual-display phage nanofiber specifically recognizes and binds to both proteins. Utilizing this nanofiber as a novel capture agent, we developed a new enzyme-linked immunosorbent assay (ELISA) method. This method shows significantly enhanced detection sensitivity compared to conventional ELISA methods, which use either anti-VEGF or anti-PD-L1 antibodies as capture agents. Therefore, the phage dual-display nanofiber presents significant potential in detecting cancer markers, evaluating medication efficacy, and advancing immunotherapy drug development. KEY POINTS: • The combined measurement of VEGF and soluble Programmed Death-Ligand 1(sPD-L1) demonstrates an additive effect in the diagnosis of cervical cancer. Fd phage nanofibers have been ingeniously engineered to display peptides that bind to VEGF and PD-L1, enabling the simultaneous detection of both proteins within a single assay • Genetically engineered phage nanofibers, adorned with two distinct peptides, can be utilized for the diagnosis and prognosis of cancer and can be mass-produced cost-effectively through bacterial infections • Employing dual-display fd phage nanofibers as capture probes, the phage ELISA method exhibited significantly enhanced detection sensitivity compared to traditional sandwich ELISA. Furthermore, phage ELISA facilitates the detection of a single protein or the simultaneous detection of multiple proteins, rendering them powerful tools for protein analysis and diagnosis across various fields, including cancer research., (© 2024. The Author(s).)

Graphical abstract Abstract All known living organisms use at least 20 amino acids as the basic building blocks of life. Efforts to reduce the number of building blocks in a replicating system to below the 20 canonical amino acids have not been successful to date. In this work, we use filamentous phage as a model system to investigate the feasibility of removing methionine (Met) from the proteome. We show that all 24 elongation Met sites in the M13 phage genome can be replaced by other canonical amino acids. Most of these changes involve substitution of methionine by leucine (Leu), but in some cases additional compensatory mutations are required. Combining Met substituted sites in the proteome generally led to lower viability/infectivity of the mutant phages, which remains the major challenge in eliminating all methionines from the phage proteome. To date a total of 15 (out of all 24) elongation Mets have been simultaneously deleted from the M13 proteome, providing a useful foundation for future efforts to minimize the genetic code. [ABSTRACT FROM AUTHOR]

Aqueous two‐phase systems (ATPS) have been widely exploited for the recovery and partial purification of biological compounds. Recently our research group characterized the primary recovery and partial purification of bacteriophage M13 using polymer‐salt and ionic liquid‐salt ATPS. From such study, it was concluded that PEG 400‐potassium phosphate ATPS with a volume ratio (VR) of 1 and 25% w/w TLL were the best suitable for the primary recovery of bacteriophage M13 from a crude extract, achieving a recovery yield of 83.3%. Although such system parameters were proven to be adequate for the recovery of the product of interest, it was concluded that further optimization was desirable and attainable by studying the effect of additional system parameters such as VR, concentration of neutral salt (M) and sample load (% w/w). This research work presents an optimization of a previously reported process for the recovery of bacteriophage M13 directly from a crude extract using ATPS. The increase in VR and sample load showed a positive effect in the recovery of M13 indicating an improved performance of the proposed ATPS. According to the results presented here, a system composed of PEG 400 17.2% (w/w), potassium phosphate 15.5% (w/w) and a sample load of 30% (w/w) allowed the recovery of M13 directly from a crude extract with a top phase recovery of 80.1%, representing an increase of 4.8 times in the final concentration and a reduction of 2.65 times in the processing costs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1177–1184, 2018 [ABSTRACT FROM AUTHOR]

The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV–vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5 × 10 4 pfu/ml red-shifted the UV–vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV–vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10–80 μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. [ABSTRACT FROM AUTHOR]

BACKGROUND Bacteriophage M13 is an Escherichia coli-specific non-lytic filamentous virus commonly used in applications ranging from antibody screening and nanomaterial construction to drug delivery, among others. In this tenor, alternative methods for the fractionation, recovery and partial purification of phage particles are desired. In this work, the use of aqueous two-phase systems (ATPS) was evaluated as an alternative method for the recovery of phage particles. RESULTS The partition behavior of M13 in PEG-salt and ionic liquid (IL)-salt ATPS was characterized using a pre-purified feedstock. In PEG-salt ATPS, M13 was preferentially partitioned to the interface. In IL ATPS, however, M13 showed a high-top phase preference with recovery yields above 65%. Selected systems were tested for the extraction of M13 from a crude fermentation broth. From crude broth, a PEG 400-potassium phosphate system with volume ratio (VR) of 1 and 25% w/w tie line length (TLL) gave the best M13 top phase recovery (83%) and purification fold (18.2) in terms of total protein concentration. CONCLUSIONS The results presented here demonstrate the practical application of ATPS as an efficient process for the primary recovery and partial purification of M13 and represent the first study of the extraction of viral particles directly from a crude broth as well as the use of IL-Salt ATPS. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Steady state studies in a chemostat enable the control of microbial growth rate at defined reaction conditions. The effects of bacteriophage M13 infection on maximum growth rate of Escherichia coli JM109 were studied in parallel operated chemostats on a milliliter-scale to analyze the steady state kinetics of phage production. The bacteriophage infection led to a decrease in maximum specific growth rate of 15% from 0.74 h −1 to 0.63 h −1 . Under steady state conditions, a constant cell specific ssDNA formation rate of 0.15 ± 0.004 mg ssDNA g CDW −1 h −1 was observed, which was independent of the growth rate. Using the estimated kinetic parameters for E. coli infected with bacteriophage M13, the ssDNA concentration in the steady state could be predicted as function of the dilution rate and the glucose concentration in the substrate. Scalability of milliliter-scale data was approved by steady state studies on a liter-scale at a selected dilution rate. An ssDNA space-time yield of 5.7 mg L −1 h −1 was achieved with increased glucose concentration in the feed at a dilution rate of 0.3 h −1 , which is comparable to established fed-batch fermentation with bacteriophage M13 for ssDNA production. [ABSTRACT FROM AUTHOR]

ABSTRACT The bacteriophage M13 has found frequent applications in nanobiotechnology due to its chemically and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Additionally, its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream production methods for phage and scaffold ssDNA are underexamined with respect to future industrial usage. Here, the high-cell-density phage production with Escherichia coli as host organism was studied in respect of medium composition, infection time, multiplicity of infection, and specific growth rate. The specific growth rate and the multiplicity of infection were identified as the crucial state variables that influence phage amplification rate on one hand and the concentration of produced ssDNA on the other hand. Using a growth rate of 0.15 h−1 and a multiplicity of infection of 0.05 pfu cfu−1 in the fed-batch production process, the concentration of pure isolated M13 ssDNA usable for scaffolded DNA origami could be enhanced by 54% to 590 mg L−1. Thus, our results help enabling M13 production for industrial uses in nanobiotechnology. Biotechnol. Bioeng. 2017;114: 777-784. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

One of the primary goals for the researchers is to create a high-quality sensor with a simple structure because of the urgent requirement to identify biomolecules at low concentrations to diagnose diseases and detect hazardous chemicals for health early on. Recently graphene has attracted much interest in the field of improved biosensors. Meanwhile, graphene with new materials such as CaF 2 has been widely used to improve the applications of graphene-based sensors. Using the fantastic features of the graphene/CaF 2 multilayer, this article proposes an improvement sensor in the sensitivity (S), the figure of merit (FOM), and the quality factor (Q). The proposed sensor is based on the five-layers graphene/dielectric grating integrated with a Fabry-Perot cavity. By tuning graphene chemical potential (µ c ), due to the semi-metal features of graphene, the surface plasmon resonance (SPR) waves excited at the graphene/dielectric boundaries. Due to the vertical polarization of the source to the gratings and the symmetry of the electric field, both corners of the grating act as electric dipoles, and this causes the propagation of plasmonic waves on the graphene surface to propagate towards each other. Finally, it causes Fabry-Perot (FP) interference on the surface of graphene in the proposed structure's active medium (the area where the sample is located). In this article, using the inherent nature of FP interference and its S to the environment's refractive index (RI), by changing a minimal amount in the RI of the sample, the resonance wavelength (interferometer order) shifts sharply. The proposed design can detect and sense some cancers, such as Adrenal Gland Cancer, Blood Cancer, Breast Cancer I, Breast Cancer II, Cervical Cancer, and skin cancer precisely. By optimizing the structure, we can achieve an S as high as 9000 nm/RIU and a FOM of about 52.14 for the first resonance order (M 1 ). Likewise, the remarkable S of 38,000 nm/RIU and the FOM of 81 have been obtained for the second mode (M 2 ). In addition, the proposed label-free SPR sensor can detect changes in the concentration of various materials, including gases and biomolecules, hemoglobin, breast cancer, diabetes, leukemia, and most alloys, with an accuracy of 0.001. The proposed sensor can sense urine concentration with a maximum S of 8500 nm/RIU and cancers with high S in the 6000 nm/RIU range to 7000 nm/RIU. Also, four viruses, such as M13 bacteriophage, HIV type one, Herpes simplex type 1, and influenza, have been investigated, showing Maximum S (for second resonance mode of λ R (M 2 ) of 8000 nm/RIU (λ R (M 2 ) = 11.2 µm), 12,000 nm/RIU (λ R (M 2 ) = 10.73 µm), 38,000 nm/RIU (λ R (M 2 ) = 11.78 µm), and 12,000 nm/RIU (λ R (M 2 ) = 10.6 µm), respectively, and the obtained S for first resonance mode (λ R (M 1 )) for mentioned viruses are 4740 nm/RIU (λ R (M 1 ) = 8.7 µm), 8010 nm/RIU (λ R (M 1 ) = 8.44 µm), 8100 nm/RIU (λ R (M 1 ) = 10.15 µm), and 9000 (λ R (M 1 ) = 8.36 µm), respectively., (© 2023. Springer Nature Limited.)

The demand for the ultrasensitive and rapid quantitative analysis of trace target analytes has become increasingly urgent. However, the sensitivity of traditional immunoassay-based detection methods is limited due to the contradiction between molecular recognition and signal amplification caused by the size effect of nanoprobes. To address this dilemma, we describe versatile M13 phage-assisted immunorecognition and signal transduction spatiotemporal separation that enable ultrasensitive light-scattering immunoassay systems for the quantitative detection of low-abundance target analytes. The newly developed immunoassay strategy combines the M13 phage-assisted light scattering signal fluctuations of gold nanoparticles (AuNPs) with gold in situ growth (GISG) technology. Given the synergy of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation, the practical detection capability of this strategy can achieve the ultrasensitive and rapid quantification of ochratoxin A and alpha-fetoprotein in real samples at the subfemtomolar level within 50 min, displaying about 4 orders of magnitude enhancement in sensitivity compared with traditional phage-based ELISA. To further improve the sensitivity of our immunoassay, the biotin-streptavidin amplification scheme is implemented to detect severe acute respiratory syndrome coronavirus 2 spike protein down to the attomolar range. Overall, this study offers a direction for ultrasensitive quantitative detection of target analytes by the synergistic combination of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation.

We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binder─the P3 peptide─was used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0-100 μg mL -1 ) with a detection limit (LOD = 3σ/ S ) of 0.34 μg mL -1 . Moreover, the dissociation constant K d of 4.2 ± 0.144 μg mL -1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.

DNA is an incredibly dense storage medium for digital data. However, computing on the stored information is expensive and slow, requiring rounds of sequencing, in silico computation, and DNA synthesis. Prior work on accessing and modifying data using DNA hybridization or enzymatic reactions had limited computation capabilities. Inspired by the computational power of "DNA strand displacement," we augment DNA storage with "in-memory" molecular computation using strand displacement reactions to algorithmically modify data in a parallel manner. We show programs for binary counting and Turing universal cellular automaton Rule 110, the latter of which is, in principle, capable of implementing any computer algorithm. Information is stored in the nicks of DNA, and a secondary sequence-level encoding allows high-throughput sequencing-based readout. We conducted multiple rounds of computation on 4-bit data registers, as well as random access of data (selective access and erasure). We demonstrate that large strand displacement cascades with 244 distinct strand exchanges (sequential and in parallel) can use naturally occurring DNA sequence from M13 bacteriophage without stringent sequence design, which has the potential to improve the scale of computation and decrease cost. Our work merges DNA storage and DNA computing, setting the foundation of entirely molecular algorithms for parallel manipulation of digital information preserved in DNA.

Filamentous bacteriophages package their circular, single stranded DNA genome with the major coat protein pVIII and the minor coat proteins pIII, pVII, pVI, and pIX. Here, we report the cryo-EM structure of a ~500 Å long bacteriophage M13 mini variant. The distal ends of the mini phage are sealed by two cap-like complexes composed of the minor coat proteins. The top cap complex consists of pVII and pIX, both exhibiting a single helix structure. Arg33 of pVII and Glu29 of pIX, located on the inner surface of the cap, play a key role in recognizing the genome packaging signal. The bottom cap complex is formed by the hook-like structures of pIII and pVI, arranged in helix barrels. Most of the inner ssDNA genome adopts a double helix structure with a similar pitch to that of the A-form double-stranded DNA. These findings provide insights into the assembly of filamentous bacteriophages., (© 2023. Springer Nature Limited.)