Bacteriophage F1 Research Articles

Preparation of VCSM13 Helper Phage for Display Library Reamplification and Bio-Panning.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a "helper" phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.

Effect of humic acid on visible light photocatalytic inactivation of bacteriophage f2 with electrospinning Cu-TiO2 nanofibers: insight into the mechanisms.

Photocatalytic disinfection is a promising technology with low cost and high efficiency. However, most of the current studies on photocatalytic disinfection ignore the widespread presence of natural organic matter (NOM) in water bodies, so the incomplete conclusions obtained may not be applicable. Herein, this paper systematically studied the influence of humic acid (HA), one of the most important components of NOM, on the photocatalytic inactivation of bacteriophage f2 with electrospinning Cu-TiO2 nanofibers. We found that with the addition of HA, the light transmittance of the solution at 550 nm decreased from 94 to 60%, and the band gap of the photocatalyst was increased from 2.96 to 3.05 eV. Compared with reacting without HA, the degradation amount of RNA of f2 decreased by 88.7% after HA was added, and the RNA concentration increased from 1.95 to 4.38 ng·μL-1 after the reaction. Hence, we propose mechanisms of the effect of HA on photocatalytic disinfection: photo-shielding, passivation of photocatalysts, quenching of free radicals, and virus protection. Photo-shielding and photocatalyst passivation lead to the decrease of photocatalyst activity, and the reactive oxygen species (ROSs) (·OH, ·O2-, 1O2, H2O2) are further trapped by HA. The HA in water also can protect the shape of phage f2 and reduce the leakage of protein and the destruction of ribonucleic acid (RNA). This work provides an insight into the mechanisms for the influence of HA in photocatalytic disinfection process and a theoretical basis for its practical application.

Transcriptional Response of Salmonella enterica to Bacteriophage Treatments with Differential Multiplicities of Infection

Salmonella enterica (S. enterica) is a causative agent of numerous foodborne outbreaks, as current industrial measures may be <90% effective. Therefore, bacteriophages have been suggested as an antimicrobial treatment against S. enterica, but it is currently unclear if there is an optimal bacteriophage multiplicity of infection (MOI) against S. enterica. Two bacteriophage cocktails at MOIs 1, 10, 100, 1000 and 10,000 were co-inoculated against four S. enterica strains (S. Enteritidis, S. Newport, S. Muenchen and S. Typhimurium), and populations were estimated on days 0–3. The transcriptional profiles of 20 genes previously indicated to be differentially expressed after bacteriophage treatment were studied by extracting RNA from all four S. enterica strains after bacteriophage SE14, SF5 and SF6 treatment on days 0, 1 and 3, and RT-qPCR was conducted to determine the expression of the 20 selected genes. The results showed that an MOI of 1000 was the most optimal in reducing S. Enteritidis populations to undetectable levels from day 0 to 3. The cas1 (SOS response) and mod (DNA modification and recombination) genes were highly upregulated between 2.5- and 5-fold on day 0 for S. Enteritidis S5-483 and S. Typhimurium S5-536 at MOIs of 1000 and 10,000. On day 3, hsdS (DNA modification and recombination) was upregulated by ~1-fold for S. enteritidis S5-483 after an MOI of 1000. Understanding an optimal bacteriophage MOI can be beneficial to implementing effective and optimal bacteriophage treatments in the industry. Knowledge of S. enterica’s transcriptional response after bacteriophage treatment provides further insight into how S. enterica can survive bacteriophage infection.

Interaction of a Dimeric Single-Stranded DNA-Binding Protein (G5P) with DNA Hairpins. A Molecular Beacon Study.

Gene-V protein (G5P/GVP) is a single-stranded (ss)DNA-binding protein (SBP) of bacteriophage f1 that is required for DNA synthesis and repair. In solution, it exists as a dimer that binds two antiparallel ssDNA strands with high affinity in a cooperative manner, forming a left-handed helical protein-DNA filament. Here, we report on fluorescence studies of the interaction of G5P with different DNA oligonucleotides having a hairpin structure (molecular beacon, MB) with a seven base-pair stem (dT24-stem7, dT18-stem7), as well as with DNA oligonucleotides (dT38, dT24) without a defined secondary structure. All oligonucleotides were end-labeled with a Cy3-fluorophore and a BHQ2-quencher. In the case of DNA oligonucleotides without a secondary structure, an almost complete quenching of their strong fluorescence (with about 5% residual intensity) was observed upon the binding of G5P. This implies an exact alignment of the ends of the DNA strand(s) in the saturated complex. The interaction of the DNA hairpins with G5P led to the unzipping of the base-paired stem, as revealed by fluorescence measurements, fluorescence microfluidic mixing experiments, and electrophoretic mobility shift assay data. Importantly, the disruption of ssDNA's secondary structure agrees with the behavior of other single-stranded DNA-binding proteins (SBPs). In addition, substantial protein-induced fluorescence enhancement (PIFE) of the Cy3-fluorescence was observed.

Host Range Expansion of Shigella Phage Sf6 Evolves through Point Mutations in the Tailspike.

ABSTRACTThe first critical step in a virus’s infection cycle is attachment to its host. This interaction is precise enough to ensure the virus will be able to productively infect the cell, but some flexibility can be beneficial to enable coevolution and host range switching or expansion. Bacteriophage Sf6 utilizes a two-step process to recognize and attach to its host Shigella flexneri. Sf6 first recognizes the lipopolysaccharide (LPS) of S. flexneri and then binds outer membrane protein (Omp) A or OmpC. This phage infects serotype Y strains but can also form small, turbid plaques on serotype 2a2; turbid plaques appear translucent rather than transparent, indicating greater survival of bacteria. Reduced plating efficiency further suggested inefficient infection. To examine the interactions between Sf6 and this alternate host, phages were experimentally evolved using mixed populations of S. flexneri serotypes Y and 2a2. The recovered mutants could infect serotype 2a2 with greater efficiency than the ancestral Sf6, forming clear plaques on both serotypes. All mutations mapped to two distinct regions of the receptor-binding tailspike protein: (i) adjacent to the LPS binding site near the N terminus; and (ii) at the distal, C-terminal tip of the protein. Although we anticipated interactions between the Sf6 tailspike and 2a2 O-antigen to be weak, LPS of this serotype appears to inhibit infection through strong binding of particles, effectively removing them from the environment. The mutations of the evolved strains reduce the inhibitory effect by either reducing electrostatic interactions with the O-antigen or increasing reliance on the Omp secondary receptors.IMPORTANCE Viruses depend on host cells to propagate themselves. In mixed populations and communities of host cells, finding these susceptible host cells may have to be balanced with avoiding nonhost cells. Alternatively, being able to infect new cell types can increase the fitness of the virus. Many bacterial viruses use a two-step process to identify their hosts, binding first to an LPS receptor and then to a host protein. For Shigella virus Sf6, the tailspike protein was previously known to bind the LPS receptor. Genetic data from this work imply the tailspike also binds to the protein receptor. By experimentally evolving Sf6, we also show that point mutations in this protein can dramatically affect the binding of one or both receptors. This may provide Sf6 flexibility in identifying host cells and the ability to rapidly alter its host range under selective pressure.

Bacteriophage Sf6 host range mutant that infects Shigella flexneri serotype 2a2 strains.

Shigella flexneri serotype 2a2 (II:9;10) is the most prevalent strain in causing bacillary dysentery in developing countries. Chemical modifications such as glucosylation, O-acetylation, and phosphoethanolamine modifications of lipopolysaccharide (LPS) O antigen (Oag) contribute to the emergence of various serotypes. Sf6 is a Shigella-specific bacteriophage that infects only a limited range of S. flexneri serotypes [X, Y]. LPS Oag is the primary receptor for bacteriophage Sf6 where it uses its tailspike protein (TSP) in binding and hydrolysing LPS Oags. Sf6TSP has recently been shown to be capable of hydrolysing the LPS Oag of Type II strains, albeit modestly. Phage therapy has regained attention in recent years as an alternative therapeutic approach. Therefore, this study aimed to expand the host range of Sf6 to the prevalent S. flexneri serotype 2a2 strain. We discovered a new lytic Sf6 host range mutant that is capable of infecting S. flexneri serotype 2a2 and identified residues in Sf6TSP that may potentially be involved in binding and hydrolysing serotype 2a2 LPS Oag. This work increased the limited Shigella-specific bacteriophage collection and may be useful in the future for phage therapy and/or biocontrolling of S. flexneri in contaminated food and water.

Influence of Shigella flexneri 2a O Antigen Acetylation on Its Bacteriophage Sf6 Receptor Activity and Bacterial Interaction with Human Cells.

Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a2 is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a2]) with deletions of different Oag modification genes (oacB, oacD, and gtrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen.IMPORTANCE The emergence of antibiotic-resistant strains and lack of efficient vaccines have made Shigella a priority organism for the World Health Organization (1). Therefore, bacteriophage therapy has received increasing attention as an alternative therapeutic approach. LPS Oag is the most variable part of LPS due to chemical modifications and is the target of bacteriophage Sf6 (S. flexneri specific). We dissected the evolution of S. flexneri serotype Y to 2a2, which revealed a new role for a gene acquired during serotype conversion and furthermore identified new specific forms of LPS receptor for Sf6. Collectively, these results unfold the importance of the acquisition of those Oag modification genes and further our understanding of the relationship between Sf6 and S. flexneri.

Structural and functional characterization of the deep-sea thermophilic bacteriophage GVE2 tailspike protein

The genome of the thermophilic bacteriophage GVE2 encodes a putative tailspike protein (GVE2 TSP). Here we report the crystal structure of the truncated GVE2 TSP at 2.0-Å resolution lacking 204 amino acid residues at its N-terminus (ΔnGVE2 TSP), possessing a “vase” outline similar to other TSP's structures. However, ΔnGVE2 TSP displays structural characteristics distinct from other TSPs. Despite lacking 204 amino acid residues, the head domain forms an asymmetric trimer compared to symmetric in other TSPs, suggesting that its long N-terminus may be unique to the long-tailed bacteriophages. Furthermore, the α-helix of the neck is 5–7 amino acids longer than that of other TSPs. The most striking feature is that its binding domain consists of a β-helix with 10 turns, whereas other TSPs have 13 turns, even including the phage Sf6 TSP, which is the closest homologue of GVE2 TSP. The C-terminal structure is also quite different with those of other TSPs. Furthermore, we observed that ΔnGVE2 TSP can slow down growth of its host, demonstrating that this TSP is essential for the phage GVE2 to infect its host. Overall, the structural characteristics suggest that GVE2 TSP may be more primitive than other phage TSPs.

Increasing the Affinity of an O-Antigen Polysaccharide Binding Site in Shigella flexneri Bacteriophage Sf6 Tailspike Protein.

Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen‐binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O‐polysaccharide of dysentery‐causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D 1H,1H‐trNOESY NMR utilizing methine–methine and methine–methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O‐polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP‐based pathogen sensor design.

Complete Genome Sequence of the Cluster F1 Mycobacteriophage KingMidas.

Subcluster F1 bacteriophage KingMidas was isolated from soil collected in Providence, Rhode Island, using Mycobacterium smegmatis mc2155 as the host. The genome is 57,386 bp and contains 105 predicted protein-coding genes but no transfer-messenger RNAs or tRNAs. This siphovirus has an icosahedral head, with a genome 99.1% identical to that of F1 mycobacteriophage Scottish.

Comparison of the effects of bacteriophage-derived dsRNA and poly(I:C) on ex vivo cultivated peripheral blood mononuclear cells

Double-stranded RNA (dsRNA), regardless of the origin and nucleotide sequence, exhibits multiple biological activities, including the establishment of an antiviral state and modulation of the immune response. Both involve the stimulation of innate immunity primarily via the release of pro-inflammatory cytokines, which in turn shapes the adaptive immune response. In this study, we compared the immune response triggered by two different dsRNAs: 1) a well-known synthetic dsRNA–poly (I:C); and 2) bacteriophage-derived dsRNA (bf-dsRNA) that is a replicative form of ssRNA bacteriophage f2. Human peripheral blood mononuclear cells (PBMCs) from 61 heathy volunteers were stimulated ex vivo with both dsRNAs. Subsequently, activation markers on the main lymphocyte subpopulations were analysed by flow cytometry and the production of 29 different cytokines and chemokines was measured by Luminex xMAP technology. The effect of bf-dsRNA on ex vivo cultivated PBMCs is similar to that induced by poly(I:C), albeit with subtle dissimilarities. Both treatments increased expression of the lymphocyte CD38 marker and intracellular IFN-γ in CD8+ T and natural killer (NK) cells, as well as the CD95 marker on the main lymphocyte subpopulations. Poly(I:C) was a stronger inducer of IL-6, IL-1β, and CCL4, whereas bf-dsRNA induced higher levels of IFN-α2, CXCL10, and CCL17. These differences might contribute to a distinct clinical manifestation when used as vaccine adjuvants, and bf-dsRNA may have more profound activity against several types of bacteria.

Kinetic analysis of bacteriophage Sf6 binding to outer membrane protein a using whole virions.

For successful infection, viruses must recognize their respective host cells. A common mechanism of host recognition by viruses is to utilize a portion of the host cell as a receptor. Bacteriophage Sf6, which infects Shigella flexneri, uses lipopolysaccharide as a primary receptor and then requires interaction with a secondary receptor, a role that can be fulfilled by either outer membrane proteins (Omp) A or C. Our previous work showed that specific residues in the loops of OmpA mediate Sf6 infection. To better understand Sf6 interactions with OmpA loop variants, we determined the kinetics of these interactions through the use of biolayer interferometry, an optical biosensing technique that yields data similar to surface plasmon resonance. Here, we successfully tethered whole Sf6 virions, determined the binding constant of Sf6 to OmpA to be 36 nM. Additionally, we showed that Sf6 bound to five variant OmpAs and the resulting kinetic parameters varied only slightly. Based on these data, we propose a model in which Sf6: Omp receptor recognition is not solely based on kinetics, but likely also on the ability of an Omp to induce a conformational change that results in productive infection. Keywords: Sf6; Shigella flexneri; OmpA; biolayer interferometry.

Visible-light-driven photocatalytic inactivation of bacteriophage f2 by Cu-TiO2 nanofibers in the presence of humic acid

Pathogenic viruses in drinking water are great threats to public health. Visible-light-driven photocatalysis is a promising technology for virus inactivation. However, the existing photocatalytic antiviral research studies have mostly been carried out in single-component systems, neglecting the effect of natural organic matter, which exists widely in actual water bodies. In this paper, electrospun Cu-TiO2 nanofibers were prepared as photocatalysts, and their photocatalytic antiviral performance in the presence of humic acid (HA) was comprehensively studied for the first time. The properties of the reaction mixture were measured during the reaction. In addition, the safety, reliability and stability of photocatalytic disinfection in the mixed system were evaluated. The results showed that the virus removal efficiency decreased with the increase of the HA concentration. The type of reaction solution, such as PBS buffer solution or water, did not affect the removal efficiency noticeably. Under acidic conditions, the electrostatic forces between photocatalysts and viruses were strengthened, leading to higher virus removal efficiency. As the reaction time went on, the pH value in the solution increased first and then tended to be stable, the conductivity remained stable, and the dissolved oxygen increased first and then decreased. The safety test showed that the concentration of Cu ions released into the solution was lower than specified by the international standards. No photoreactivation was observed, and the addition of HA significantly reduced the reutilization efficiency of the photocatalysts.

Sensitivity of the C-Terminal Nuclease Domain of Kaposi's Sarcoma-Associated Herpesvirus ORF29 to Two Classes of Active-Site Ligands.

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to the Herpesviridae family, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, we have expressed, purified, and characterized the wild-type pORF29C and variants with substitutions at the proposed active-site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.

Photocatalytic Inactivation of Bacteriophage f2 with Ag3PO4/g-C3N4 Composite under Visible Light Irradiation: Performance and Mechanism

Water-borne virus pollution has caused great harm and attracted widespread attention in many countries. Visible-light-driven photocatalysis is considered as a promising process for disinfection. In this study, Ag3PO4/g-C3N4 (AgCN) composites were synthesized by hydrothermal method. The photocatalytic disinfection was investigated using bacteriophage f2 as the model virus. Moreover, the effects of pH and humic acid on photocatalytic disinfection were studied. Meanwhile, the mechanism of enhanced disinfection by Ag3PO4/g-C3N4 was systematically investigated by radical scavenger experiments. The results show that Ag3PO4 particles were uniformly distributed on g-C3N4 sheets. By means of photoluminescence spectrometer analysis, it is confirmed that a lower carrier recombination rate for Ag3PO4/g-C3N4 was achieved compared with Ag3PO3 and g-C3N4. Meanwhile, complete inactivation of f2 with concentration of 3 × 106 PFU/mL was reached within 80 min in the presence of Ag3PO4/g-C3N4 composite. The pH had little effect on removal efficiency overall, while the existence of humic acid resulted in a significant negative effect on the inactivation of f2 due to the optical shielding and absorption of humic acid. Recycling tests of Ag3PO4/g-C3N4 confirmed that Ag3PO4/g-C3N4 presented superior stability. The results from radical scavenger experiments indicated that holes (h+) and hydroxyl radicals (·OH) played important roles in photocatalytic disinfection process.

Removal of bacteriophage f2 in water by Fe/Ni nanoparticles: Optimization of Fe/Ni ratio and influencing factors

Pathogenic viruses in water are seriously harmful to human health and highly resistant to conventional disinfections. As a kind of promising attempts for pollution remediation, Fe-based nanoparticles show excellent performance in removing contaminants. In this study, the Fe/Ni nanoparticles (Fe/Ni NPs) were synthesized using the self-designed device and used for bacteriophage inactivation in water. Scanning electron microscope (SEM) and X-ray diffraction (XRD) showed that the as-prepared Fe/Ni particles were spherical and the average particle size was 93 nm. The synthesized Fe/Ni NPs achieved much higher removal efficiency of bacteriophage f2 than nanoscale zero-valent iron (nZVI), while Ni nanoparticles (Ni NPs) showed no removal effect on the bacteriophage f2. The highest removal efficiency of bacteriophage f2 by Fe/Ni NPs was obtained when the primary ratio of Fe:Ni was 5:1. In addition, the removal efficiency of phage f2 under aerobic condition was significantly higher than that under anaerobic condition with Fe/Ni NPs, and the role of Ni was proved as a catalyst in the system. Besides, the effect of initial pH, initial concentration of bacteriophage f2, particles dose, rotation rate, and temperature on the removal efficiency of bacteriophage f2 were studied. The result showed that the removal efficiency of bacteriophage f2 did not change obviously in the test pH range (5–8), and was positively related with the rotation rate and negatively related with the initial concentration of bacteriophage f2. The particles dose could increase the removal efficiency of phage f2, but the removal efficiency would decrease when the dose was too much due to the aggregation of nanoparticles. The increase of temperature could increase the removal efficiency initially, but decrease the removal efficiency finally due to the accelerated corrosion of iron.

Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens.

Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.

Photocatalytic Membrane Reactor (PMR) for Virus Removal in Drinking Water: Effect of Humic Acid

In the actual water environment, the health risk of waterborne viruses is evaluated to be 101–104 times higher at a similar level of exposure compared with bacteria and has aroused strong concern in many countries in the world. Photocatalytic membrane reactor (PMR), a new process for virus inactivation in water, has gradually become one of the main tools to inactivate pathogenic organisms in water. However, there is relatively little attention to the effect of natural organic matters (NOMs) on the PMR system, which actually exists in the water environment. In this paper, the TiO2-P25, a common type in sales and marketing, was selected as the photocatalyst, and humic acid was regarded as the representative substance of NOMs for investigating thoroughly the influence of humic acid on virus removal by the PMR system. It was found that competitive adsorption between the virus and humic acid occurred, which markedly reduced the amount of virus adsorbed on the surface of the photocatalyst. Moreover, with humic acid, the direct contact behavior between the virus and the photocatalyst was blocked to some extent, and the disinfection of phage f2 by the active free radicals produced by photocatalysis was furthermore badly affected. Meanwhile, the special structure of humic acid, which made humic acid be able to absorb light of 270–500 nm, led to the reduction of photocatalytic efficiency. Further experiments showed that when there was a certain concentration of humic acid in water, intermittent operation mode or higher membrane flux (>40 L/(m2·h)) was selected to partly alleviate the adverse effects of humic acid.

Photocatalytic disinfection performance in virus and virus/bacteria system by Cu-TiO2 nanofibers under visible light

The presence of pathogenic microorganisms in water is a great threat to human health, and photocatalysis is promising for disinfection. However, the research on virus inactivation with visible-light photocatalysis is still limited, especially the coexistence of virus and its host bacteria. In this study, bacteriophage f2 and its host E. coil 285 were used as the model microorganisms, and the disinfection performance of prepared Cu-TiO2 nanofibers under visible light was investigated. The result showed that the prepared Cu-TiO2 nanofibers showed a brilliant ability in terms of removing bacteriophage f2 and E. coil 285 under visible light. Series experiments indicated that the initial pH didn't affect the photocatalytic disinfection performance significantly. In the certain range, the removal efficiency of bacteriophage f2 increased with the increase of catalyst dosage, light intensity and temperature, but decreased with the increase of initial virus concentration. In virus/bacteria mixed system, bacteriophage f2 exhibited stronger resistance to photocatalytic oxidation than E. coil 285, and the removal of bacteriophage f2 was obviously affected by being mixed with E. coil 285, while the removal of E. coil 285 almost remained unchanged after being mixed with bacteriophage f2. Further research proved that competitive adsorption in mixed system played a certain role in E. coli 285 inactivation, while the free reactive oxygen species (ROSs) in the bulk phase played a crucial role in phage f2 inactivation.

High-resolution structure of podovirus tail adaptor suggests repositioning of an octad motif that mediates the sequential tail assembly

The sophisticated tail structures of DNA bacteriophages play essential roles in life cycles. Podoviruses P22 and Sf6 have short tails consisting of multiple proteins, among which is a tail adaptor protein that connects the portal protein to the other tail proteins. Assembly of the tail has been shown to occur in a sequential manner to ensure proper molecular interactions, but the underlying mechanism remains to be understood. Here, we report the high-resolution structure of the tail adaptor protein gp7 from phage Sf6. The structure exhibits distinct distribution of opposite charges on two sides of the molecule. A gp7 dodecameric ring model shows an entirely negatively charged surface, suggesting that the assembly of the dodecamer occurs through head-to-tail interactions of the bipolar monomers. The N-terminal helix-loop structure undergoes rearrangement compared with that of the P22 homolog complexed with the portal, which is achieved by repositioning of two consecutive repeats of a conserved octad sequence motif. We propose that the conformation of the N-terminal helix-loop observed in the Sf6-gp7 and P22 portal:gp4 complex represents the pre- and postassembly state, respectively. Such motif repositioning may serve as a conformational switch that creates the docking site for the tail nozzle only after the assembly of adaptor protein to the portal. In addition, the C-terminal portion of gp7 shows conformational flexibility, indicating an induced fit on binding to the portal. These results provide insight into the mechanistic role of the adaptor protein in mediating the sequential assembly of the phage tail.