T4 Phage Research Articles

Arabidopsis thaliana organelles mimic the T7 phage DNA replisome with specific interactions between Twinkle protein and DNA polymerases Pol1A and Pol1B

BackgroundPlant chloroplasts and mitochondria utilize nuclear encoded proteins to replicate their DNA. These proteins are purposely built for replication in the organelle environment and are distinct from those involved in replication of the nuclear genome. These organelle-localized proteins have ancestral roots in bacterial and bacteriophage genes, supporting the endosymbiotic theory of their origin. We examined the interactions between three of these proteins from Arabidopsis thaliana: a DNA helicase-primase similar to bacteriophage T7 gp4 protein and animal mitochondrial Twinkle, and two DNA polymerases, Pol1A and Pol1B. We used a three-pronged approach to analyze the interactions, including Yeast-two-hybrid analysis, Direct Coupling Analysis (DCA), and thermophoresis.ResultsYeast-two-hybrid analysis reveals residues 120–295 of Twinkle as the minimal region that can still interact with Pol1A or Pol1B. This region is a part of the primase domain of the protein and slightly overlaps the zinc-finger and RNA polymerase subdomains located within. Additionally, we observed that Arabidopsis Twinkle interacts much more strongly with Pol1A versus Pol1B. Thermophoresis also confirms that the primase domain of Twinkle has higher binding affinity than any other region of the protein. Direct-Coupling-Analysis identified specific residues in Twinkle and the DNA polymerases critical to positive interaction between the two proteins.ConclusionsThe interaction of Twinkle with Pol1A or Pol1B mimics the minimal DNA replisomes of T7 phage and those present in mammalian mitochondria. However, while T7 and mammals absolutely require their homolog of Twinkle DNA helicase-primase, Arabidopsis Twinkle mutants are seemingly unaffected by this loss. This implies that while Arabidopsis mitochondria mimic minimal replisomes from T7 and mammalian mitochondria, there is an extra level of redundancy specific to loss of Twinkle function.

Identification of Alzheimer's Disease Autoantibodies and Their Target Biomarkers by Phage Microarrays.

The characterization of the humoral response in Alzheimer's disease (AD) patients might aid in detecting the disease at early stages. We have combined phage display and protein microarrays to identify AD autoantibodies and their target biomarkers. After enrichment of the T7 phage display libraries from AD and healthy brain tissue mRNA in AD-specific phages, 1536 monoclonal phages were printed on microarrays to probe them with 8 AD and 8 healthy control sera. A total of 57 phages showed higher seroreactivity in AD. In total, 13 out of the 44 unique sequences displayed on the phages were selected for validation using 68 AD and 52 healthy control sera. Peptides from Anthrax toxin receptor 1, Nuclear protein 1, Glycogen phosphorylase, and Olfactory receptor 8J1 expressed in bacteria as HaloTag fusion proteins showed a statistically significant ability to discriminate between AD patients and controls. The identified panel of AD autoantibodies might provide new insights into the blood-based diagnosis of the disease.

Construction of a genetically modified T7Select phage system to express the antimicrobial peptide 1018.

Bacteriophage therapy was an ascendant technology for combating bacterial infections before the golden age of antibiotics, but the therapeutic potential of phages was largely ignored after the discovery of penicillin. Recently, with antibiotic-resistant infections on the rise, these phages are receiving renewed attention to combat problematic bacterial infections. Our approach is to enhance bacteriophages with antimicrobial peptides, short peptides with broad-spectrum antibiotic or antibiofilm effects. We inserted coding sequences for 1018, an antimicrobial peptide previously shown to be an effective broad-spectrum antimicrobial and antibiofilm agent, or the fluorescent marker mCherry, into the T7Select phage genome. Transcription and production of 1018 or mCherry began rapidly alter E. coli cultures were infected with genetically modified phages. mCherry fluorescence, which requires a 90 min initial maturation period, was observed in infected cultures after 2 h of infection. Finally, we tested phages expressing 1018 (1018 T7) against bacterial planktonic cultures and biofilms, and found the 1018 T7 phage was more effective than the unmodified T7Select phage at both killing planktonic cells and eradicating established biofilms, validating our phage-driven antimicrobial peptide expression system. The combination of narrow-spectrum phages delivering relatively high local doses of broad-spectrum antimicrobials could be a powerful method to combat resistant infections. The experiments we describe prove this combination is feasible in vitro, but further testing and optimization are required before genetically modified phages are ready for use in vivo.

Ribokinase screened from T7 phage displayed Mycobacterium tuberculosis genomic DNA library had good potential for the serodiagnosis of tuberculosis

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is the leading cause of death among infectious diseases in the worldwide. Lack of more sensitive and effective diagnostic reagents has increased the awareness of rapid diagnosis for tuberculosis. In this study, T7 phage displayed genomic DNA library of M. tuberculosis was constructed to screen the antigens that specially bind with TB-positive serum from the whole genome of M. tuberculosis and to improve the sensitivity and specificity of tuberculosis serological diagnosis. After three rounds of biopanning, results of DNA sequencing and BLAST analysis showed that 19 positive phages displayed four different proteins and the occurrence frequency of the phage which displayed ribokinase was the highest. The results of indirect ELISA and dot immunoblotting indicated that representative phages could specifically bind to tuberculosis-positive serum. The prokaryotic expression vector containing the DNA sequence of ribokinase gene was then constructed and the recombinant protein was expressed and purified to evaluate the serodiagnosis value of ribokinase. The reactivity of the recombinant ribokinase with different clinical serum was detected and the sensitivities and specificities in tuberculosis serodiagnosis were 90% and 86%, respectively by screening serum from tuberculosis patients (n = 90) and uninfected individuals (n = 90) based on ELISA. Therefore, this study demonstrated that ribokinase had good potential for the serodiagnosis of tuberculosis.

Cas3-stimulated runaway replication of modified ColE1 plasmids in Escherichia coli is temperature dependent.

The clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a CRISPR RNA (crRNA)-mediated interference mechanism. In Type I CRISPR-Cas systems, crRNA guided by a Cascade complex recognises the matching target DNA and promotes an R-loop formation, RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complex where it nicks and degrades DNA. The Cas3 activity in CRISPR-Cas immunity is reduced in Δhns cells at 37°C for unknown reasons. Cas3 can also influence regulation of plasmid replication and promote uncontrolled ('runaway') replication of ColE1 plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. In this work we wanted to test whether Cas3-stimulated uncontrolled plasmid replication is affected by the temperature in Δhns and/or ΔhtpG mutants. We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37°C, irrespective of the genotype of the analysed mutants, and dependent on Cas3 helicase function. We also found that plasmid replication was strongly reduced by the hns mutation at 30°C and that Cas3 could interfere with T4 phage replication at both incubation temperatures.

Bacteriophages engineered to display foreign peptides may become short-circulating phages.

SummaryBacteriophages draw scientific attention in medicine and biotechnology, including phage engineering, widely used to shape biological properties of bacteriophages. We developed engineered T4‐derived bacteriophages presenting seven types of tissue‐homing peptides. We evaluated phage accumulation in targeted tissues, spleen, liver and phage circulation in blood (in mice). Contrary to expectations, accumulation of engineered bacteriophages in targeted organs was not observed, but instead, three engineered phages achieved tissue titres up to 2 orders of magnitude lower than unmodified T4. This correlated with impaired survival of these phages in the circulation. Thus, engineering of T4 phage resulted in the short‐circulating phage phenotype. We found that the complement system inactivated engineered phages significantly more strongly than unmodified T4, while no significant differences in phages’ susceptibility to phagocytosis or immunogenicity were found. The short‐circulating phage phenotype of the engineered phages suggests that natural phages, at least those propagating on commensal bacteria of animals and humans, are naturally optimized to escape rapid neutralization by the immune system. In this way, phages remain active for longer when inside mammalian bodies, thus increasing their chance of propagating on commensal bacteria. The effect of phage engineering on phage pharmacokinetics should be considered in phage design for medical purposes.

Amino Acid Sequence of Oligopeptide Causes Marked Difference in DNA Compaction and Transcription

Compaction of T4 phage DNA (166 kbp) by short oligopeptide octamers composed of two types of amino acids, four cationic lysine (K), and four polar nonionic serine (S) having different sequence order was studied by single-molecule fluorescent microscopy. We found that efficient DNA compaction by oligopeptide octamers depends on the geometrical match between phosphate groups of DNA and cationic amines. The amino acid sequence order in octamers dramatically affects the mechanism of DNA compaction, which changes from a discrete all-or-nothing coil-globule transition induced by a less efficient (K4S4) octamer to a continuous compaction transition induced by a (KS)4 octamer with a stronger DNA-binding character. This difference in the DNA compaction mechanism dramatically changes the packaging density, and the morphology of T4 DNA condensates: DNA is folded into ordered toroidal or rod morphologies during all-or-nothing compaction, whereas disordered DNA condensates are formed as a result of the continuous DNA compaction. Furthermore, the difference in DNA compaction mechanism has a certain effect on the inhibition scenario of the DNA transcription activity, which is gradual for the continuous DNA compaction and abrupt for the all-or-nothing DNA collapse.

Non-host class II ribonucleotide reductase in Thermus viruses: sequence adaptation and host interaction

Ribonucleotide reductases (RNR) are essential enzymes for all known life forms. Their current taxonomic distribution suggests extensive horizontal gene transfer e.g., by processes involving viruses. To improve our understanding of the underlying processes, we characterized a monomeric class II RNR (NrdJm) enzyme from a Thermus virus, a subclass not present in any sequenced Thermus spp. genome. Phylogenetic analysis revealed a distant origin of the nrdJm gene with the most closely related sequences found in mesophiles or moderate thermophiles from the Firmicutes phylum. GC-content, codon usage and the ratio of coding to non-coding substitutions (dN/dS) suggest extensive adaptation of the gene in the virus in terms of nucleotide composition and amino acid sequence. The NrdJm enzyme is a monomeric B12-dependent RNR with nucleoside triphosphate specificity. It exhibits a temperature optimum at 60–70 °C, which is in the range of the growth optimum of Thermus spp. Experiments in combination with the Thermus thermophilus thioredoxin system show that the enzyme is able to retrieve electrons from the host NADPH pool via host thioredoxin and thioredoxin reductases. This is different from other characterized viral RNRs such as T4 phage RNR, where a viral thioredoxin is present. We hence show that the monomeric class II RNR, present in Thermus viruses, was likely transferred from an organism phylogenetically distant from the one they were isolated from, and adapted to the new host in genetic signature and amino acids sequence.

Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System

Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC) represents one group of extracellular CISs that are present in both bacteria and archaea. Here, we report the cryo-EM structure of an intact PVC from P.asymbiotica. This over 10-MDa device resembles a simplified T4 phage tail, containing ahexagonal baseplate complex with six fibers and a capped 117-nanometer sheath-tube trunk. One distinct feature of the PVC is the presence of three variants for both tube and sheath proteins, indicating a functional specialization of them during evolution. The terminal hexameric cap docks onto the topmost layer of the inner tube and locks the outer sheath in pre-contraction state with six stretching arms. Our results on the PVC provide a framework for understanding the general mechanism of widespread CISs and pave the way for using them as delivery tools in biological or therapeutic applications.

Functional recombinant single-chain variable fragment antibody against Agkistrodon acutus venom.

Agkistrodon acutus bites are conventionally treated with animal-derived antivenom, the use of which is limited due to allergic reactions and serum sickness. Thus in the present study, the genes of humanized antibodies produced in response to A. acutus venom were extracted from lymphocytes from patients bitten by A. acutus. A single-chain variable fragment (scFv) library against venom was constructed using a T7 phage display system. ScFv genes that exhibited high affinity to venom were selected by library biopanning. An expression system was constructed for antivenom scFv fused with 6×His tag at its N- and C-terminus using pET-28a (+) vector. The scFv proteins could achieve functional and soluble expression in Escherichia coli via the auto-induction method. The purity and activity of the scFv genes and proteins were confirmed by SDS-PAGE, western blotting and ELISA. The results indicated that three soluble scFv proteins exhibited specific affinity to A. acutus venom and were harvested via the auto-induction method.

1H, 13C and 15N NMR assignments of Bacillus subtilis bacteriophage SPO1 protein Gp46.

Bacterial antibiotic resistance is a serious threat to public health and bacteriophage therapy is an alternative for antibiotics in the era of multidrug resistance. While phage draws attention in fighting bacterial infection and is used in protein display to study macromolecular interactions, the molecular machinery of the host invasion mechanism remains largely unclear for many bacteriophages. Despite recent studies on T4 and T7 phages of Gram-negative model organism Escherichia coli revealing many interesting features of their invasive strategies, the studies on Gram-positive bacterial phages still lag far behind their counterparts. SPO1 is a lytic phage of model organism Bacillus subtilis and one of the best studied Gram-positive bacterial phages. SPO1 features a unique Host Takeover Module coding for 24 proteins which show little similarity to any previously known proteins. Gp46, located in this module, is an acidic protein that is produced by SPO1 presumably during the host takeover event. Here we describe the complete resonance assignment of Gp46 as the basis for the first structure determination of SPO1 phage protein and further mechanism study.

Utilizing in vitro DNA assembly to engineer a synthetic T7 Nanoluc reporter phage for Escherichia coli detection

Bacteria have major role in regulating human health and disease, therefore, there is a continuing need to develop new detection methods and therapeutics to combat them. Bacteriophages can be used to infect specific bacteria, which make them good candidates for detecting and editing bacterial populations. However, creating phage-based detection assays is somewhat limited by the difficulties in the engineering of phages. We present here a synthetic biology strategy to engineer phages using a simple in vitro method. We used this method to insert a NanoLuc luciferase expression cassette into the T7 phage, in order to construct the NRGp6 reporter phage. The synthetic NRGp6 phage was used to efficiently detect low concentrations of Escherichia coli from liquid culture. We envision that our approach will benefit synthetic biologists for constructing different kinds of engineered phages, and enable new approaches for phage-based therapeutics and diagnostics.

Abstract P1-05-03: Isoform specific targeting of insulin receptor

Abstract The insulin receptor (InsR) exists in both an A and B isoform. InsR-B differs from InsR-A by the inclusion of exon 11, which encodes 12 amino acid residues at the C-terminus of the InsR alpha-subunit. Increased InsR-A expression is associated with mitogenic signaling pathways while InsR-B is linked to insulin-mediated metabolic functions. Predominant InsR-A expression may therefore be important in growth and fetal development of embryos, whereas predominant InsR-B expression has a role in metabolic insulin action in adult life. Increased InsR-A expression is seen in breast cancer. In endocrine resistant breast cancer, InsR-A is expressed at high levels (Gradishar, et al. Clin Cancer Res 22:301 2016 PMID: 26324738). Thus, developing InsR-A specific inhibitors could be a useful therapy for breast cancer. We have previously published InsR specific binders using a T7 phage gene 2 protein (Gp2), a small protein scaffold (Chan, et al. Mol Cancer Ther 16:1324 2017 PMID: 28468775), with the long-term goal of creating effective InsR inhibitors and diagnostics. Using yeast display and directed evolution, we identified three Gp2 variants (Gp2 #1, #5, and #10) with low nanomolar affinity and specific binding to cell surface InsR. We have shown that these Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and resistant breast cancer, but did not downregulate InsR expression. To further characterize the specificity of Gp2 variants, we used two techniques. HEK293T cells were infected with lentiviral vectors expressing either InsR-A tagged with mCherry or InsR-B tagged with eGFP. Using these cells, we performed “mock panning” and showed the Gp2 #5 variant bound both InsR-A and InsR-B, but had higher affinity for InsR-B. We also incorporated Gp2 #5 into the capsid of a tropism-null adeno-associated virus (AAV). Using this Gp2-AAV, we infected HEK293T-InsR-A or InsR-B cells at a number of different multiplicities of infection. These data were consistent with panning data and showed specific Gp2-AAV infection of cells expressing high levels of InsR-B, but not InsR-A. Thus, our data show that Gp2 variants we created have a higher affinity for InsR-B than InsR-A. Despite this preferred affinity, these Gp2 binders have sufficient binding to InsR-A to disrupt the biological effects of insulin in breast cancer cells. Thus, even relatively low affinity binding to InsR-A can disrupt its function. Further development of InsR-A Gp2 binders may be developed and provide more specific targeting of the breast cancer specific isoform of InsR. Citation Format: LaPara K, Chan JY, Zdechlik A, Ljunggren K, Schmidt D, Hackel B, Yee D. Isoform specific targeting of insulin receptor [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-03.

Cobaviruses\xa0\u2013\xa0a new globally distributed phage group infecting Rhodobacteraceae in marine ecosystems

Bacteriophages are widely considered to influence bacterial communities, however most phages are still unknown or not studied well enough to understand their ecological roles. We have isolated two phages infecting Lentibacter sp. SH36, affiliated with the marine Roseobacter group, and retrieved similar phage genomes from publicly available metagenomics databases. Phylogenetic analysis placed the new phages within the Cobavirus group, in the here newly proposed genus Siovirus and subfamily Riovirinae of the Podoviridae. Gene composition and presence of direct terminal repeats in cultivated cobaviruses point toward a genome replication and packaging strategy similar to the T7 phage. Investigation of the genomes suggests that viral lysis of the cell proceeds via the canonical holin-endolysin pathway. Cobaviral hosts include members of the genera Lentibacter, Sulfitobacter and Celeribacter of the Roseobacter group within the family Rhodobacteraceae (Alphaproteobacteria). Screening more than 5,000 marine metagenomes, we found cobaviruses worldwide from temperate to tropical waters, in the euphotic zone, mainly in bays and estuaries, but also in the open ocean. The presence of cobaviruses in protist metagenomes as well as the phylogenetic neighborhood of cobaviruses in glutaredoxin and ribonucleotide reductase trees suggest that cobaviruses could infect bacteria associated with phototrophic or grazing protists. With this study, we expand the understanding of the phylogeny, classification, genomic organization, biogeography and ecology of this phage group infecting marine Rhodobacteraceae.

Modification of T2 phage infectivity toward Escherichia coli O157:H7 via using CRISPR/Cas9.

Phage therapy is getting considerable attention as a method for prophylaxis of food poisoning caused by Escherichia coli O157:H7, an important pathogen causing life-threatening bloody diarrhea. Despite previous studies have shown the feasibility of phage therapy to E. coli O157:H7, promising results have not been obtained in vivo yet. A major drawback of phage therapy is that bacteriophages have high specificity and cannot infect all the sub-strains of a particular pathogenic strain. To overcome this hurdle, we thought to establish a way to artificially expand the host-range of E. coli O157:H7-specific phages. To develop a proof-of-concept for this method, we focused on T2 phage, which cannot infect E. coli O157:H7 strains, and PP01 phage, which displays broad infectivity to them, and attempted to make T2 phage able to infect E. coli O157:H7 as efficiently as PP01. We report the trials of T2 genome editing using the CRISPR/Cas9 system and the modification of both long and short tail fibers of this phage based on comparison with PP01. The resultant recombinant showed the adsorption rate comparable to PP01. Thus, we provided the evidence that the short tail fiber of PP01 plays an important role in adsorption to E. coli O157:H7.

Novel Regulatory Mechanisms Identified in Viral DNA Packaging Proteins using Molecular Dynamics Simulations

Many viruses use large terminase proteins (TerL) to convert chemical energy from ATP hydrolysis into mechanical work in order to package their genome into a viral pro-capsid. TerL proteins belong to the Additional Strand, Conserved Glutamate superfamily of P-loop ATPases, which contain canonical Walker motifs and a catalytic glutamate residue downstream of the Walker B motif. Although several crystal structures of TerL proteins are now available, these structures do not provide sufficient information to form a detailed mechanism of ATP hydrolysis and signal transduction. Through molecular dynamics simulations of three representative TerLs (from T4, Sf6, and P74-26 phages) in apo and ATP-bound states we identify a novel “arginine toggle” mechanism, whereby a conserved Walker A arginine toggles its interaction between two glutamate residues - one located in the lid subdomain and one located in the ATPase active site - in order to couple ATP binding to conformational change. Not only is this one of the first steps in the mechanochemical coupling pathway, but it also promotes catalysis of hydrolysis, both of which are consistent with experimental biochemical and single-molecule data. We go on to simulate mutant TerL proteins modified in silico to provide new insights into the specific binding pocket deformations that correspond with hindered ATP hydrolysis and slowed DNA packaging observed experimentally. Lastly, we present evidence that a “glutamate switch” residue found in other AAA+ motor proteins, which holds the catalytic glutamate residue in an inactive pose to regulate the rate of ATP hydrolysis, might also be conserved in some TerL proteins.

Bacteriophage biodistribution and infectivity from honeybee to bee larvae using a T7 phage model

Bacteriophages (phages) or viruses that specifically infect bacteria have widely been studied as biocontrol agents against animal and plant bacterial diseases. They offer many advantages compared to antibiotics. The American Foulbrood (AFB) is a bacterial disease affecting honeybee larvae caused by Paenibacillus larvae. Phages can be very significant in fighting it mostly due to European restrictions to the use of antibiotics in beekeeping. New phages able to control P. larvae in hives have already been reported with satisfactory results. However, the efficacy and feasibility of administering phages indirectly to larvae through their adult workers only by providing phages in bees’ feeders has never been evaluated. This strategy is considered herein the most feasible as far as hive management is concerned. This in vivo study investigated the ability of a phage to reach larvae in an infective state after oral administration to honeybees. The screening (by direct PFU count) and quantification (by quantitative PCR) of the phage in bee organs and in larvae after ingestion allowed us to conclude that despite 104 phages reaching larvae only an average of 32 were available to control the spread of the disease. The fast inactivation of many phages in royal jelly could compromise this therapeutic approach. The protection of phages from hive-derived conditions should be thus considered in further developments for AFB treatment.

The pKa values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme.

T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H-T4L follows a double displacement mechanism with His26 serving as a nucleophile to form a covalent glycosyl-enzyme intermediate (Kuroki et al., PNAS 1999; 96:8949-8954). To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or define the ionization states of the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pKa value of the putative nucleophile His26 is 6.8 ± 0.1, whereas that of the general acid Glu11 is 4.7 ± 0.1. If the proposed mechanism holds true, then T26H-T4L follows a reverse protonation pathway in which only a minor population of the free enzyme is in its catalytically competent ionization state with His26 deprotonated and Glu11 protonated. Our studies also confirm that all arginines in T26H-T4L, including the active site Arg145, are positively charged under neutral pH conditions. BRIEF STATEMENT: The replacement of a single amino acid changes T4 lysozyme from an inverting to a retaining glycoside hydrolase. Using NMR spectroscopy, we measured the pKa values of the ionizable residues in the active site of this mutant enzyme. Along with previously reported data, these results provide important constraints for understanding the catalytic mechanisms by which the wild-type and mutant form of T4 lysozyme cleave bacterial peptidoglycan.

Phages Can Be More Effective and Specific Than Antibiotics in Combating Bacteria

Every year, tens of thousands of people die of infections from antibiotic-resistant bacteria. Phage therapy has been suggested as an alternative because bacteria resistant to antibiotics may still be susceptible to phages. Furthermore, phages have the advantage of being more specific and thus less likely to harm beneficial bacteria. However, phages may have limited effectiveness in combating bacteria since bacteria possess several antiviral defense mechanisms and can quickly develop resistance to phages. The purpose of this study was to compare the effectiveness and specificity of antibiotics and phages in combating bacteria. To this end, we exposed strains of the bacteria Escherichia coli to T4 bacteriophages and antibiotics and assessed effectiveness and specificity of bacterial killing. Fission yeast, a unicellular eukaryotic organism, was used as a control. Cell growth, phage plaque formation, or cell concentrations were observed or measured. It was found that T4 phages are more specific and effective in fighting or inhibiting both antibiotic-resistant and sensitive bacteria than antibiotics. One phage can kill at least 4000 actively growing bacterial cells within two hours. Therefore, the data suggest that phage therapy can be developed as an efficient tool to combat antibiotic-resistant bacteria.

A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides.

Nicking endonucleases (NEases) selectively cleave single DNA strands in double-stranded DNAs at a specific site. They are widely used in bioanalytical applications and in genome editing; however, the peculiarities of DNA–protein interactions for most of them are still poorly studied. Previously, it has been shown that the large subunit of heterodimeric restriction endonuclease BspD6I (Nt.BstD6I) acts as a NEase. Here we present a study of interaction of restriction endonuclease BspD6I with modified DNA containing single non-nucleotide insertion with an azobenzene moiety in the enzyme cleavage sites or in positions of sugar-phosphate backbone nearby. According to these data, we designed a number of effective stimulus-responsive oligonucleotide inhibitors bearing azobenzene or triethylene glycol residues. These modified oligonucleotides modulated the functional activity of Nt.BspD6I after cooling or heating. We were able to block the cleavage of T7 phage DNA by this enzyme in the presence of such inhibitors at 20–25°C, whereas the Nt.BspD6I ability to hydrolyze DNA was completely restored after heating to 45°C. The observed effects can serve as a basis for the development of a platform for regulation of NEase activity in vitro or in vivo by external signals.