Phi X174 Research Articles

A computational model for bacteriophage ϕX174 gene expression.

Bacteriophage ϕX174 has been widely used as a model organism to study fundamental processes in molecular biology. However, several aspects of ϕX174 gene regulation are not fully resolved. Here we construct a computational model for ϕX174 and use the model to study gene regulation during the phage infection cycle. We estimate the relative strengths of transcription regulatory elements (promoters and terminators) by fitting the model to transcriptomics data. We show that the specific arrangement of a promoter followed immediately by a terminator, which occurs naturally in the ϕX174 genome, poses a parameter identifiability problem for the model, since the activity of one element can be partially compensated for by the other. We also simulate ϕX174 gene expression with two additional, putative transcription regulatory elements that have been proposed in prior studies. We find that the activities of these putative elements are estimated to be weak, and that variation in ϕX174 transcript abundances can be adequately explained without them. Overall, our work demonstrates that ϕX174 gene regulation is well described by the canonical set of promoters and terminators widely used in the literature.

Two neglected contributors to water biosafety risks: The impacts of microbial co-existence and light on viral inactivation

Virus-induced risks to water quality persist, with the viability of viruses in complex aquatic environments impacted by various factors, including coexisting bacteria and exposure to light. This study investigated the disinfection efficacy of UV254 irradiation and free chlorine (FC) on Bacteriophage phiX174 in the presence of coexisting bacteria, specifically Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). To elucidate the underlying mechanisms, this study examined the effects of bacterial compounds (i.e., E. coli lipopolysaccharide (LPS) and B. subtilis peptidoglycan (PGN)) on viral inactivation, thermal stability, and environmental persistence. The research results demonstrated that bacterial presence and their associated compounds significantly impaired viral inactivation, with greater concentrations of coexisting bacteria and their compounds exacerbating this effect. Additionally, the photoreactivation of phiX174 was explored following UV254 irradiation, indicating that high-dose UV254, coupled with low nutrient availability and minimal host bacteria levels, effectively suppressed viral photoreactivation. This study is the first to investigate the multifactorial effects on viral photoreactivation. The results offer novel insights into the challenges associated with virus-related pressures in water treatment, with potential implications for optimizing disinfection processes.

PhiX-174 phage concentration in rainwater by skimmed-milk flocculation

ABSTRACT Rainwater, as a sustainable source of drinking water, is becoming increasingly important worldwide. Appropriate microbiological techniques are needed to ensure the acceptability of rainwater for potable uses. Bacteriophage has been widely used as a viral indicator in water studies, and its concentration is an important step when sampling several litres of water. Skimmed-milk flocculation (SMF) is a simple method for viral recovery, and its efficiency has already been assessed for some viruses. However, there are only a few records on bacteriophage recovery using SMF, and none with PhiX-174 coliphage, an important surrogate for human enteroviruses. Thus, this work aimed to evaluate the effectiveness of the SMF technique on the concentration of PhiX-174 coliphage in rainwater. 3 L of rainwater samples were artificially spiked with PhiX-174 and were concentrated to 10 mL volumes by SMF. The phage enumeration on the concentrated samples were evaluated by a standard double layer plaque assay. From the 6 samples tested, the average recovery was 22.8 ± 15.2%, ranging from 10.8 to 52.8%. This is the first time that SMF is applied for PhiX-174 coliphage recovery, and in rainwater. Therefore, SMF is a cost-effective method that can effectively be used to recover bacteriophage PhiX-174 in water samples.

Comparative Analysis of the Impact of Protein on Virus Retention for Different Virus Removal Filters.

The performance of virus filters is often determined by the extent of protein fouling, which can affect both filtrate flux and virus retention. However, the mechanisms governing changes in virus retention in the presence of proteins are still not well understood. The objective of this work was to examine the effect of proteins on virus retention by both asymmetric (Viresolve® NFP and Viresolve® Pro) and relatively homogeneous (Ultipor® DV20 and PegasusTM SV4) virus filtration membranes. Experiments were performed with bacteriophage ϕX174 as a model parvovirus and human serum immunoglobulin G (hIgG) as a model protein. The virus retention in 1 g/L hIgG solutions was consistently less than that in a protein-free buffer solution by between 1 to 3 logs for the different virus filters. The virus retention profiles for the two homogeneous membranes were very similar, with the virus retention being highly correlated with the extent of flux decline. Membranes prefouled with hIgG and then challenged with phages also showed much lower virus retention, demonstrating the importance of membrane fouling; the one exception was the Viresolve® Pro membrane, which showed a similar virus retention for the prefouled and pristine membranes. Experiments in which the protein was filtered after the virus challenge demonstrated that hIgG can displace previously captured viruses from within a filter. The magnitude of these effects significantly varied for the different virus filters, likely due to differences in membrane morphology, pore size distribution, and chemistry, providing important insights into the development/application of virus filtration in bioprocessing.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

Resistance differences of representative model microorganisms in different disinfection processes: ZIF-67, UVC and ozone

Due to their inherent nature, each type of microorganism exhibits unique resistance to various disinfection processes. Disinfection with nanomaterials represents a promising technology that has garnered significant interest. Among them, ZIF material has antibacterial and antiviral activities, and ZIF-67 material is a new type of nanomaterial that can effectively kill bacteria and viruses, with the potential for large-scale use. Therefore, in this paper, we investigate the differences between nanomaterial disinfection techniques (employing ZIF-67) and traditional inactivation techniques (e.g., ultraviolet-C and ozone). Phage MS2 and PhiX174 and their respective host E. coli were chosen as the representative microorganisms. Our findings indicate that phages exhibit greater resistance to ZIF-67 and traditional disinfection techniques than bacteria, but different techniques show specific disinfection effects on the three disinfection microorganisms. Considering the differences, choosing appropriate disinfection techniques based on the characteristics of the disinfected substances in practical disinfection technology selection is essential.

Synthesis and characterization of antimicrobial colloidal polyanilines

The potential application of colloidal polyaniline (PANI) as an antimicrobial is limited by challenges related to solubility in common organic solvents, scalability, and antimicrobial potency. To address these limitations, we introduced a functionalized PANI (fPANI) with carboxyl groups through the polymerisation of aniline and 3-aminobenzoic acid in a 1:1 molar ratio. fPANI is more soluble than PANI which was determined using a qualitative study. We further enhanced the solubility and antimicrobial activity of fPANI by incorporating Ag nanoparticles onto the synthesized fPANI colloid via direct addition of 10 mM AgNO3. The improved solubility can be attributed to an approximately 3-fold reduction in size of particles. Mean particle sizes are measured at 1322 nm for fPANI colloid and 473 nm for fPANI-Ag colloid, showing a high dispersion and deagglomeration effect from Ag nanoparticles. Antimicrobial tests demonstrated that fPANI-Ag colloids exhibited superior potency against Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and Bacteriophage PhiX 174 when compared to fPANI alone. The minimum bactericidal concentration (MBC) and minimum virucidal concentration (MVC) values were halved for fPANI-Ag compared to fPANI colloid and attributed to the combination of Ag nanoparticles with the fPANI polymer. The antimicrobial fPANI-Ag colloid presented in this study shows promising results, and further exploration into scale-up can be pursued for potential biomedical applications.

Comparison of the immune effects of the Chlamydia abortus MOMP antigen displayed in different parts of bacterial ghosts.

Bacterial ghosts (BGs) are promising vaccine platforms owing to their high adjuvant properties and delivery efficiency. Heterologous antigens can be anchored to different parts of BGs using genetic engineering strategies to prepare vaccines. However, several key issues need to be resolved, including the efficient preparation of BGs and determining the optimal anchoring position of exogenous antigens in the BGs. Here, we prepared an efficient temperature-controlled lysis system using lysis gene E of phage PhiX174 and used the major outer membrane protein (MOMP) of Chlamydia abortus (C. abortus) as a model antigen to explore the optimal display location of exogenous antigens in BGs. We demonstrated that the constructed recombinant temperature-controlled lysis plasmid can still stably inhibit E gene expression at 37°C, and the lysis efficiency of E. coli can reach above 99.9%. Four recombinant MOMP Escherichia coli (E. coli) ghost vaccines were constructed using different anchor sequences. These vaccines all induced strong specific antibody responses and secrete high levels of IFN-γ in immunized mice and significantly increased the clearance of C. abortus in a mouse infection model. Notably, the strongest immune effect was observed when MOMP was displayed on the surface of E. coli ghosts (rECG-InpN-M), which resulted in the clearance of C. abortus in mice 6 days earlier than that with the recombinant MOMP vaccine. Altogether, we constructed an efficient BG temperature-controlled lysis system and provided a feasible strategy for developing a BG delivery platform with enhanced immune effects.

Photoinactivation of the bacteriophage PhiX174 by UVA radiation and visible light in SM buffer and DMEM-F12

ObjectiveIt has been observed that viruses can be inactivated by UVA radiation and visible light. The aim of this study is to investigate whether a medium that contains a photosensitizer might have an influence on viral reduction under irradiation by UVA, violet or blue light. Test virus is the bacteriophage PhiX174 in the photosensitizer-free SM buffer and DMEM-F12, which contains the known photosensitizer riboflavin.ResultsThe determined PhiX174 D90 doses in SM buffer and DMEM were 36.8 J/cm² and 13.6 J/cm² at 366 nm, 153.6 J/cm² and 129.1 J/cm² at 408 nm and 4988 J/cm² and 2477.1 J/cm² at 455 nm, respectively. It can be concluded that the medium has a large influence on the results. This might be caused by the photosensitizer riboflavin in DMEM-F12. As riboflavin is a key component in many cell culture media, irradiation experiments with viruses in cell culture media should be avoided if the investigation of intrinsical photoinactivation properties of viruses is aimed for.

Comprehensive performance evaluation of six bioaerosol samplers based on an aerosol wind tunnel

Choosing a suitable bioaerosol sampler for atmospheric microbial monitoring has been a challenge to researchers interested in environmental microbiology, especially during a pandemic. However, a comprehensive and integrated evaluation method to fully assess bioaerosol sampler performance is still lacking. Herein, we constructed a customized wind tunnel operated at 2–20 km/h wind speed to systematically and efficiently evaluate the performance of six frequently used samplers, where various aerosols, including Arizona test dust, bacterial spores, gram-positive and gram-negative bacteria, phages, and viruses, were generated. After 10 or 60 min of sampling, the physical and biological sampling efficiency and short or long-term sampling capabilities were determined by performing aerodynamic particle size analysis, live microbial culturing, and a qPCR assay. The results showed that AGI-30 and BioSampler impingers have good physical and biological sampling efficiencies for short-term sampling. However, their ability to capture aerosols at low concentrations is restricted. SASS 2300 and BSA-350 wet-wall cyclones had excellent enrichment ratios and high microbial cultivability in both short-term and long-term sampling; however, they were not suitable for quantitative studies of aerosols. Polycarbonate filter samplers showed outstanding performance in physical and long-term sampling but lacked the ability to maintain microbial activity, which can be improved by gelatin filter samplers. However, limitations remain for some fragile microorganisms, such as E. coli phage PhiX174 and coronavirus GX_P2V. In addition, the effects of wind speed and direction should be considered when sampling particles larger than 4 µm. This study provides an improved strategy and guidance for the characterization and selection of a bioaerosol sampler for better measurement and interpretation of collected ambient bioaerosols.

Genetic and functional basis of the reduction effect in bacteriophage ΦX174

The ΦX174 reduction effect describes a plasmid-based inhibitory phenomenon that mimics the superinfection inhibition found in wild phage populations. In this effect, when a portion of the ΦX174 genome – the 3′ end of the pilot protein gene (H), the 5’ end of the replication gene (A), and the H-A intergenic region – is present on a plasmid in the host cell, almost complete protection from phage infection occurs. Here we demonstrate that only the phage pilot protein H portion of the plasmid is sufficient for the observed inhibition, that protein synthesis is necessary for inhibition to occur, that inserting the entire H gene in the plasmid may also impart a blocking effect, and that partial to complete recovery from this inhibition is possible with minimal viral evolution.

Phage single-stranded DNA-binding protein or host DNA damage triggers the activation of the AbpAB phage defense system.

Although numerous phage defense systems have recently been discovered in bacteria, how these systems defend against phage propagation or sense phage infections remains unclear. The Escherichia coli AbpAB defense system targets several lytic and lysogenic phages harboring DNA genomes. A phage-encoded single-stranded DNA-binding protein, Gp32, activates this system similar to other phage defense systems such as Retron-Eco8, Hachiman, ShosTA, Nhi, and Hna. DNA replication inhibitors or defects in DNA repair factors activate the AbpAB system, even without phage infection. This is one of the few examples of activating phage defense systems without phage infection or proteins. The AbpAB defense system may be activated by sensing specific DNA-protein complexes.

Who touched that? Interconnection of high-touch surfaces drives pathogen spread in public spaces

Abstract Background Surface contamination via hands plays an important role in pathogen spread in public spaces. This study aimed to identify frequently touched surfaces and evaluate the spread of a surrogate virus in a hotel lobby. Methods In a working hotel lobby, observation was performed (30 hours) to identify the surfaces and objects touched. An entry doorknob and first floor elevator button were seeded with a bacteriophage (Phi-X174) tracer; 4 hours later 25 surfaces were swabbed to determine tracer distribution and contamination levels. Results A total of 324 individuals performed 627 touches over 13 different fomites in the hotel lobby. The elevator button and front desk counter were the most frequently touched (32 and 22% of all touches respectively), with 55% of individuals touching the elevator button and 79% touching either the elevator button, the front desk counter, or both. More than half (56%) touched 2 or more surfaces; there were 314 interactions between surfaces. Touches from the elevator button to other surfaces (92 interactions) and from other surfaces to the elevator button (41) made up 42% of all interactions and connected the elevator button to 9 other fomites including doors, countertops, seating, a credit card reader and a hand sanitizer pump. From two seeded sites, the tracer spread to 13 surfaces over 4 hours. The most contaminated surfaces were tables, counter tops and door handles. Other contaminated objects were the luggage cart handle, sanitizer pump, and computer equipment. Conclusions Surfaces in the hotel lobby were frequently touched and highly interconnected, resulting in extensive spread of surface contamination in as little as 4 hours. This study demonstrates the importance of hands in distributing contamination between shared surfaces and highlights the need for hand and surface hygiene interventions to disrupt the journey of the germ in public settings. Key messages • Frequently touched surfaces in public spaces are interconnected. • Interconnection of people via touched surfaces drives the spread of pathogens in a public setting.

Targeted Hygiene reduces surface transmission and infection risk for respiratory viruses

Abstract Background In public spaces, the importance of surface contamination in the transmission of respiratory viruses has been debated. This study aimed to compare the spread of a surrogate virus in a hotel lobby before and after a Targeted Hygiene intervention, and to quantify the reduction in risk of infection from respiratory diseases. Methods In a working hotel lobby, 13 fomites were seeded with a bacteriophage (Phi-X174) tracer at 8 am; 4 hours later 25 surfaces were swabbed to determine baseline tracer distribution and contamination levels. This was then repeated with the addition of a pre-determined Targeted Hygiene intervention performed 2 hours after seeding. Four replicate baseline and intervention trials were conducted, and data were compared for statistically significant (p < 0.05) differences. Risk of infection was estimated via Quantitative Microbial Risk Assessment modelling. Results Following the Targeted Hygiene intervention there was a significant reduction in the spread of the tracer (contamination of 13% of sampled surfaces vs. 50% at baseline). Tracer concentrations (PFU/site) were significantly lower overall (9.1E+02 PFU vs. 5.8E+04 PFU, p < 0.0001), including surfaces that had not been disinfected. Our model estimates that the risk of infection from common respiratory viruses via surface transmission was reduced by 97%. Conclusions Extensive spread of surface contamination can occur in a hotel lobby in as little as 4 hours. This study demonstrates how a Targeted Hygiene intervention can significantly disrupt the journey of the germ, even on surfaces not cleaned and disinfected, resulting in a consequent reduction of infection risk via surface transmission of common respiratory infections. This demonstrates the value of carrying out effective hygiene interventions to those managing commercial spaces. Key messages • Targeted Hygiene can significantly reduce the spread of respiratory pathogens via surface transmission, providing a means for facilities managers to achieve high standards using a targeted approach. • The Targeted Hygiene approach can significantly reduce the risk of infection from common respiratory viruses via surface transmission in public spaces.

Preparation of Escherichia coli ghost of anchoring bovine Pasteurella multocida OmpH and its immunoprotective effect

Pasteurella multocida is a pathogen that can infect humans and animals. A ghost is an empty bacterial body devoid of cytoplasm and nucleic acids that can be efficiently presented by antigen-presenting cells. To study a novel ghost vector vaccine with cross-immune protection, we used bacteriophage PhiX174 RF1 and Pasteurella multocida standard strain CVCC393 as templates to amplify the split genes E and OmpH to construct a bidirectional expression vector E’-OmpH-pET28a-ci857-E. This is proposed to prepare a ghost Escherichia coli (engineered bacteria) capable of attaching and producing Pasteurella multocida OmpH on the inner membrane of Escherichia coli (BL21). The aim is to assess the antibody levels and the effectiveness of immune protection by conducting a mouse immunoprotective test. The bidirectional expression vector E’-OmpH-pET28a-ci857-E was successfully constructed. After induction by IPTG, identification by SDS-PAGE, western blot, ghost culture and transmission electron microscope detection, it was proven that the Escherichia coli ghost anchored to Pasteurella multocida OmpH was successfully prepared. The immunoprotective test in mice showed that the antibody levels of Pasteurella multocida inactivated vaccine, OmpH, ghost (aluminum glue adjuvant) and ghost (Freund’s adjuvant) on day 9 after immunization were significantly different from those of the PBS control group (P < 0.01). The immune protection rates were 100%, 80%, 75%, and 65%, respectively, and the PBS negative control was 0%, which proved that they all had specific immune protection effects. Therefore, this study lays the foundation for the further study of ghosts as carriers of novel vaccine-presenting proteins.

Structure of the native chemotaxis core signaling unit from phage E-protein lysed E. coli cells.

Bacterial chemotaxis is a ubiquitous behavior that enables cell movement toward or away from specific chemicals. It serves as an important model for understanding cell sensory signal transduction and motility. Characterization of the molecular mechanisms underlying chemotaxis is of fundamental interest and requires a high-resolution structural picture of the sensing machinery, the chemosensory array. In this study, we combine cryo-electron tomography and molecular simulation to present the complete structure of the core signaling unit, the basic building block of chemosensory arrays, from Escherichia coli. Our results provide new insight into previously poorly-resolved regions of the complex and offer a structural basis for designing new experiments to test mechanistic hypotheses.

Say YES to single-gene lysis!

Viral strategies to lyse host microbes can sometimes involve just a single gene, although the mechanism of action can be hard to discern. In Science, Orta etal. present structures of a protein complex in which protein E of bacteriophage φX174 functions to inhibit host peptidoglycan synthesis, thereby inducing lysis.

The impact of storage buffer and storage conditions on fecal samples for bacteriophage infectivity and metavirome analyses

BackgroundThere is an increasing interest in investigating the human gut virome for its influence on the gut bacterial community and its putative influence on the trajectory towards health or disease. Most gut virome studies are based on sequencing of stored fecal samples. However, relatively little is known about how conventional storage buffers and storage conditions affect the infectivity of bacteriophages and influence the downstream metavirome sequencing.ResultsWe demonstrate that the infectivity and genome recovery rate of different spiked bacteriophages (T4, c2 and Phi X174) are variable and highly dependent on storage buffers. Regardless of the storage temperature and timespan, all tested phages immediately lost 100% (DNA/RNA Shield) or more than 90% (StayRNA and RNAlater) of their infectivity. Generally, in SM buffer at 4 °C phage infectivity was preserved for up to 30 days and phage DNA integrity was maintained for up to 100 days. While in CANVAX, the most effective buffer, all spiked phage genomes were preserved for at least 100 days. Prolonged storage time (500 days) at – 80 °C impacted viral diversity differently in the different buffers. Samples stored in CANVAX or DNA/RNA Shield buffer had the least shifts in metavirome composition, after prolonged storage, but they yielded more contigs classified as “uncharacterised”. Moreover, in contrast to the SM buffer, these storage buffers yielded a higher fraction of bacterial DNA in metavirome-sequencing libraries. We demonstrated that the latter was due to inactivation of the DNases employed to remove extra-cellular DNA during virome extraction. The latter could be partly avoided by employing additional washing steps prior to virome extraction.ConclusionFecal sample storage buffers and storage conditions (time and temperature) strongly influence bacteriophage infectivity and viral composition as determined by plaque assay and metavirome sequencing. The choice of buffer had a larger effect than storage temperature and storage time on the quality of the viral sequences and analyses. Based on these results, we recommend storage of fecal virome samples at in SM buffer at 4 °C for the isolation of viruses and at – 80 °C for metagenomic applications if practically feasible (i.e., access to cold storage). For fecal samples stored in other buffers, samples should be cleared of these buffers before viral extraction and sequencing.2F_FbCzVMdkP5vWvM-fCtaVideo

In situ generation of cold atmospheric plasma-activated mist and its biocidal activity against surrogate viruses for COVID-19.

To provide an alternative to ultra violet light and vapourized hydrogen peroxide to enhance decontamination of surfaces as part of the response to the COVID-19 pandemic. We developed an indirect method for in situ delivery of cold plasma and evaluated the anti-viral activity of plasma-activated mist (PAM) using bacteriophages phi6, MS2, and phiX174, surrogates for SARS-CoV-2. Exposure to ambient air atmospheric pressure derived PAM caused a 1.71 log10 PFU ml-1 reduction in phi6 titer within 5min and a 7.4 log10 PFU ml-1 reduction after 10min when the the PAM source was at 5 and 10cm. With MS2 and phiX174, a 3.1 and 1.26 log10 PFU ml-1 reduction was achieved, respectively, after 30min. The rate of killing was increased with longer exposure times but decreased when the PAM source was further away. Trace amounts of reactive species, hydrogen peroxide and nitrite were produced in the PAM, and the anti-viral activity was probably attributable to these and their secondary reactive species. PAM exhibits virucidal activity against surrogate viruses for COVID-19, which is time and distance from the plasma source dependent.

The mechanism of the phage-encoded protein antibiotic from ΦX174.

The historically important phage ΦX174 kills its host bacteria by encoding a 91-residue protein antibiotic called protein E. Using single-particle electron cryo-microscopy, we demonstrate that protein E bridges two bacterial proteins to form the transmembrane YES complex [MraY, protein E, sensitivity to lysis D (SlyD)]. Protein E inhibits peptidoglycan biosynthesis by obstructing the MraY active site leading to loss of lipid I production. We experimentally validate this result for two different viral species, providing a clear model for bacterial lysis and unifying previous experimental data. Additionally, we characterize the Escherichia coli MraY structure-revealing features of this essential enzyme-and the structure of the chaperone SlyD bound to a protein. Our structures provide insights into the mechanism of phage-mediated lysis and for structure-based design of phage therapeutics.