Phage DNA Research Articles

An Actively Homing Insertion Element in a Phage Methylase Contains a Hidden HNH Endonuclease

Background/Objectives: The ShiLan domain was previously identified as an insertion sequence in a phage DNA methylase gene that exhibited similar evolutionary patterns to that of an active intein or self-splicing intron but could not be identified as either. It produces no internal stop codons when read in frame with its host methylase gene, leading to the thought that it may not be an intron and rather be an abnormal type of intein. However, the sequence has no detectable self-splicing domains, which are essential for intein persistence, as preventing an intein from successfully splicing is often detrimental to proper host protein function. Methods: The analysis of alternate open reading frames for the full nucleotide sequence of this insertion element revealed the insertion to be an out-of-frame histidine-asparagine-histidine (HNH) endonuclease. A GTG start codon is located 18 bp into the insertion, and a TAA stop codon within the last four bases of the insertion (TAAC). When this frame is read, an HNH endonuclease is revealed. In-depth computational analysis could not retrieve support for this element being any known type of self-splicing element, neither intein nor intron. When read in-frame with the methylase gene, this insertion is predicted to take on a looping structure that may be able to avoid interference with the DNA methylase activity. We performed searches for sequences similar in nature to the inserted out-of-frame HNH and found several in other phages and prokaryotes. We present our survey of these out-of-frame endonuclease insertion elements as well as some speculation on how these endonucleases are getting translated to facilitate their homing activity. Conclusions: These findings expand our understanding of the possible arrangements for and prevalence of unorthodox mobile genetic elements and overlapping open reading frames in phages.

The activity of indigo carmine against bacteriophages: an edible antiphage agent

Bacteriophage infections in bacterial cultures pose a significant challenge to industrial bioprocesses, necessitating the development of innovative antiphage solutions. This study explores the antiphage potential of indigo carmine (IC), a common FDA-approved food additive. IC demonstrated selective inactivation of DNA phages (P001, T4, T1, T7, λ) with the EC50 values ranging from 0.105 to 0.006 mg/mL while showing no activity against the RNA phage MS2. Fluorescence correlation spectroscopy (FCS) revealed that IC selectively binds to dsDNA, demonstrated by a significant reduction in the diffusion coefficient, whereas no binding was observed with ssDNA or RNA. Mechanistically, IC permeates the phage capsid, leading to genome ejection and capsid deformation, as confirmed by TEM imaging. Under optimal conditions (50 °C, 220 rpm), IC achieved up to a 7-log reduction in phage titer, with kinetic theory supporting the enhanced collision frequency induced by agitation. Additionally, IC protected E. coli cultures from phage-induced lysis without affecting bacterial growth or protein production, as demonstrated by GFP expression assays. IC’s effectiveness and environmental safety, combined with its FDA approval and cost-effectiveness, make it a promising antiphage agent for industrial applications.Key points• Indigo carmine effectively inactivates a broad spectrum of bacteriophages, offering protection to bacteria in industrial cultures.• A novel application of indigo carmine as a food-grade, environmentally safe, and FDA-approved antiphage agent protecting bacterial cultures.• Antiphage activity arises from indigo carmine’s interaction with DNA within the phage capsid without harming bacterial cells or compromising protein production in bacterial cultures.Graphical abstract

Kiwa is a bacterial membrane-embedded defence supercomplex activated by phage-induced membrane changes.

Bacteria and archaea deploy diverse, sophisticated defence systems to counter virus infection, yet many immunity mechanisms remain poorly understood. Here, we characterise the Kiwa defence system as a membrane-associated supercomplex that senses changes in the membrane induced by phage infection and plasmid conjugation. This supercomplex, comprising KwaA tetramers bound to KwaB dimers, as its basic repeating unit, detects structural stress via KwaA, activating KwaB, which binds ejected phage DNA through its DUF4868 domain, stalling phage DNA replication forks and thus disrupting replication and late transcription. We show that phage-encoded DNA mimic protein Gam, which inhibits RecBCD, also targets Kiwa through KwaB recognition. However, Gam binding to one defence system precludes its inhibition of the other. These findings reveal a distinct mechanism of bacterial immune coordination, where sensing of membrane disruptions and inhibitor partitioning enhance protection against phages and plasmids.

A DNA phosphorothioation pathway via adenylated intermediate modulates Tdp machinery.

In prokaryotes, the non-bridging oxygen in the DNA sugar-phosphate backbone can be enzymatically replaced by a sulfur atom, resulting in phosphorothioate (PT) modification. However, the mechanism underlying the oxygen-to-sulfur substitution remains enigmatic. In this study, we discovered a hypercompact DNA phosphorothioation system, TdpABC, in extreme thermophiles. This DNA sulfuration process occurs through two sequential steps: an initial activation step by ATP to form an adenylated intermediate, followed by a substitution step where the adenyl group is replaced with a sulfur atom. Together with the TdpA-TdpB, the TdpABC system provides anti-phage defense by degrading PT-free phage DNA. Cryogenic electron microscopy structural analysis revealed that the TdpA hexamer binds one strand of encircled duplex DNA via hydrogen bonds arranged in a spiral staircase conformation. Nevertheless, the TdpAB-DNA interaction was sensitive to the hydrophobicity of the PT sulfur. PTs inhibit ATP-driven translocation and nuclease activity of TdpAB on self-DNA, thereby preventing autoimmunity.

Erwinia phage Asesino is a nucleus-forming phage that lacks PhuZ

As nucleus-forming phages become better characterized, understanding their unifying similarities and unique differences will help us understand how they occupy varied niches and infect diverse hosts. All identified nucleus-forming phages fall within the Chimalliviridae family and share a core genome of 68 unique genes including chimallin, the major nuclear shell protein. A well-studied but non-essential protein encoded by many nucleus-forming phages is PhuZ, a tubulin homolog which aids in capsid migration, nucleus rotation, and nucleus positioning. One clade that represents 24% of all currently known chimalliviruses lacks a PhuZ homolog. Here we show that Erwinia phage Asesino, one member of this PhuZ-less clade, shares a common overall replication mechanism with other characterized nucleus-forming phages despite lacking PhuZ. We show that Asesino replicates via a phage nucleus that encloses phage DNA and partitions proteins in the nuclear compartment and cytoplasm in a manner similar to previously characterized nucleus-forming phages. Consistent with a lack of PhuZ, however, we did not observe active positioning or rotation of the phage nucleus within infected cells. These data show that some nucleus-forming phages have evolved to replicate efficiently without PhuZ, providing an example of a unique variation in the nucleus-based replication pathway.

VClean: assessing virus sequence contamination in viral genomes.

Recent advancements in viral metagenomics and single-virus genomics have improved our ability to obtain the draft genomes of environmental viruses. However, these methods can introduce virus sequence contaminations into viral genomes when short, fragmented partial sequences are present in the assembled contigs. These contaminations can lead to incorrect analyses;however, practical detection tools are lacking. In this study, we introduce vClean, a novel automated tool that detects contaminations in viral genomes. By applying machine learning to the nucleotide sequence features and gene patterns of the input viral genome, vClean could identify contaminations. Specifically, for tailed double-stranded DNA phages, we attempted accurate predictions by defining single-copy-like genes and counting their duplications. We evaluated the performance of vClean usingsimulated datasets derived from complete reference genomes, achieving a binary accuracy of 0.932. When vClean was applied to 4693 genomes of medium or higher quality derived from public ocean metagenomic data, 1604 genomes (34.2%) were identified as contaminated. We also demonstrated that vClean can detect contamination in single-virus genome data obtained from river water. vClean provides a new benchmark for quality control of environmental viral genomes and has the potential to become an essential tool for environmental viral genome analysis.

Unravelling viral ecology and evolution over 20 years in a freshwater lake.

As freshwater lakes undergo rapid anthropogenic change, long-term studies reveal key microbial dynamics, evolutionary shifts and biogeochemical interactions, yet the vital role of viruses remains overlooked. Here, leveraging a 20 year time series from Lake Mendota, WI, USA, we characterized 1.3 million viral genomes across time, seasonality and environmental factors. Double-stranded DNA phages from the class Caudoviricetes dominated the community. We identified 574 auxiliary metabolic gene families representing over 140,000 auxiliary metabolic genes, including important genes such as psbA (photosynthesis), pmoC (methane oxidation) and katG (hydrogen peroxide decomposition), which were consistently present and active across decades and seasons. Positive associations and niche differentiation between virus-host pairs, including keystone Cyanobacteria, methanotrophs and Nanopelagicales, emerged during seasonal changes. Inorganic carbon and ammonium influenced viral abundances, underscoring viral roles in both 'top-down' and 'bottom-up' interactions. Evolutionary processes favoured fitness genes, reduced genomic heterogeneity and dominant sub-populations. This study transforms understanding of viral ecology and evolution in Earth's microbiomes.

Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis.

Phage engineering is an emerging technology due to the promising potential application of phages in medical and biotechnological settings. Targeted phage mutagenesis tools are required to customize the phages for a specific application and generate, in addition to that, so-called designer phages. CRISPR-Cas technique is used in various organisms to perform targeted mutagenesis. Yet, its efficacy is notably limited for phage mutagenesis due to the highly abundant phage DNA modifications. Addressing this challenge, we have developed a novel approach that involves the temporal removal of phage DNA cytosine modifications, allowing for effective CRISPR-Cas targeting and subsequent introduction of mutations into the phage genome. The removal of cytosine modification relies on the catalytic activity of a eukaryotic ten-eleven translocation methylcytosine (TET) dioxygenase. TET enzymes iteratively de-modify methylated or hydroxymethylated cytosines on phage DNA. The temporal removal of cytosine modification ultimately enables efficient DNA cleavage by Cas enzymes and facilitates mutagenesis. To streamline the application of the coupled TET-CRISPR-Cas system, we use Golden Gate cloning for fast and efficient assembly of a vector that comprises a TET oxidase and a donor DNA required for scarless site-specific phage mutagenesis. Our approach significantly advances the engineering of modified phage genomes, enabling the efficient generation of customized phages for specific applications.

A phage amplification-assisted SEA-CRISPR/Cas12a system for viable bacteria detection.

Rapid and accurate detection of viable bacteria is essential for the clinical diagnosis of urinary tract infections (UTIs) and for making effective therapeutic decisions. However, most current molecular diagnostic techniques are unable to differentiate between viable and non-viable bacteria. In this study, we introduce a novel isothermal platform that integrates strand exchange amplification (SEA) with the CRISPR/Cas12a system, thereby enhancing both the sensitivity and specificity of the assay and achieving detection of phage DNA at concentrations as low as 4 × 102 copies per μL. Moreover, the incorporation of phages facilitates the specific recognition of viable bacteria and amplifies the initial signal through the inherent specificity and propagation properties of these phages. By employing the phage-assisted SEA-Cas12a approach, we successfully detected viable bacteria in human urine samples without the necessity of DNA extraction within 3.5 hours, achieving a detection limit of 103 CFU per mL. Considering its speed, accuracy, and independence from specialized equipment, this platform demonstrates significant potential as a robust tool for the rapid detection of various pathogens in resource-limited settings, thereby facilitating timely clinical management of UTI patients.

The comparison of the antioxidant, antibacterial and antiviral potential of Polish fir honeydew and Manuka honeys

The aim of the present study was to compare the antioxidant, antibacterial and antiviral activities of Podkarpackie coniferous honeydew honey and Manuka honey. The quality of tested honey samples (honeydew-12 and Manuka-4) regarding honey standard was evaluated as well as additional indicators (methylglyoxal, total phenolics and HPTLC phenolic profile, antioxidant potential, glucose oxidase activity, and hydrogen peroxide) were compared. Antibacterial potential was analyzed against Gram-positive (S. aureus and B. cereus) and Gram-negative (E. coli and S. enterica) bacteria. Antiviral activity against different RNA (phi6, MS2) and DNA (T7, phiX174) bacteriophages considered as “viral surrogates” was determined. Based on the determined physicochemical parameters the good quality of tested honeys was confirmed, excluding two samples. The content of polyphenolic compounds in honeydew honey ranged from 583.87 to 1102.42 mg of gallic acid/kg and was strongly correlated with the antioxidant properties. Moreover, for samples with the strongest activity these parameters were comparable to Manuka honey. However, the obtained HPTLC polyphenolic profiles were completely different for honeydew than for Manuka honey which exhibited additional bands (Rf = 0.74 and 0.52). Honeydew honeys were characterized by a strong antiviral and antibacterial properties most of all against Gram-positive bacteria. The MICs (minimal inhibitory concentrations) for S. aureus and B. cereus ranged 15–35% and 8–15% for honeydew and Manuka honeys, respectively. The strongest antiviral properties of honeydew honey were demonstrated mainly against RNA bacteriophages (phi6, MS2) which was even higher than for Manuka honey, especially against MS2 virus. The obtained results suggest that Podkarpackie honeydew honey with the controlled glucose oxidase activity may be a natural substance used to combat viral and bacterial diseases.

Genome-wide identification of bacterial genes contributing to nucleus-forming jumbo phage infection.

The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.

Structure of the Staphylococcus aureus bacteriophage 80α neck shows the interactions between DNA, tail completion protein and tape measure protein.

Tailed bacteriophages with double-stranded DNA genomes (class Caudoviricetes ) play an important role in the evolution of bacterial pathogenicity, both as carriers of genes encoding virulence factors and as the main means of horizontal transfer of mobile genetic elements (MGEs) in many bacteria, such as Staphylococcus aureus . The S. aureus pathogenicity islands (SaPIs), including SaPI1, are a type of MGEs are that carry a variable complement of genes encoding virulence factors. SaPI1 is mobilized at high frequency by "helper" bacteriophages, such as 80α, leading to packaging of the SaPI1 genome into virions made from structural proteins supplied by the helper. 80α and SaPI1 virions consist of an icosahedral head (capsid) connected via a unique vertex to a long, non-contractile tail. At one end of the tail, proteins associated with the baseplate recognize and bind to the host. At the other end, a connector or "neck" forms the interface between the tail and the head. The neck consists of several specialized proteins with specific roles in DNA packaging, phage assembly, and DNA ejection. Using cryo-electron microscopy and three-dimensional reconstruction, we have determined the high-resolution structure of the neck section of SaPI1 virions made in the presence of phage 80α, including the dodecameric portal (80α gene product (gp) 42) and head-tail-connector (gp49) proteins, the hexameric head-tail joining (gp50) and tail terminator (gp52) proteins, and the major tail protein (gp53) itself. We were also able to resolve the DNA, the tail completion protein (gp51) and the tape measure protein (gp56) inside the tail. This is the first detailed structural description of these features in a bacteriophage, providing insights into the assembly and infection process in this important group of MGEs and their helper bacteriophages.

Structure and mechanism of the Zorya anti-phage defense system.

Zorya is a recently identified and widely distributed bacterial immune system that protects bacteria from viral (phage) infections. Three Zorya subtypes have been discovered, each containing predicted membrane-embedded ZorAB complexes paired with soluble subunits that differ among Zorya subtypes, notably ZorC and ZorD in type I Zorya systems1,2. Here, we investigate the molecular basis of Zorya defense using cryo-electron microscopy, mutagenesis, fluorescence microscopy, proteomics, and functional studies. We present cryo-EM structures of ZorAB and show that it shares stoichiometry and features of other 5:2 inner membrane ion-driven rotary motors. The ZorA5B2 complex contains a dimeric ZorB peptidoglycan binding domain and a pentameric α-helical coiled-coil tail made of ZorA that projects approximately 70 nm into the cytoplasm. We also characterize the structure and function of the soluble Zorya components, ZorC and ZorD, finding that they harbour DNA binding and nuclease activity, respectively. Comprehensive functional and mutational analyses demonstrate that all Zorya components work in concert to protect bacterial cells against invading phages. We provide evidence that ZorAB operates as a proton-driven motor that becomes activated upon sensing of phage invasion. Subsequently, ZorAB transfers the phage invasion signal through the ZorA cytoplasmic tail to recruit and activate the soluble ZorC and ZorD effectors, which facilitate degradation of the phage DNA. In summary, our study elucidates the foundational mechanisms of Zorya function as an anti-phage defense system.

Dysbiosis of gut microbiota in COVID-19 is associated with intestinal DNA phage dynamics of lysogenic and lytic infection.

This study compared intestinal DNA phage dynamics and gut microbiota changes observed at the onset of coronavirus disease 2019 (COVID-19). The study participants included 19 healthy individuals and 19 patients with severe acute respiratory syndrome coronavirus 2 infection. Significant differences were observed in the diversity of the intestinal DNA virome after the onset of COVID-19 compared with that in healthy individuals. Classification by their tail morphology resulted in the order Caudovirales, a double-stranded DNA phage, accounting for >95% of all participants. In classifying phages based on host bacteria, a decreased number of phages infecting mainly the Clostridia class was observed immediately after the onset of COVID-19 and recovered over time. After the onset of COVID-19, two distinct movement patterns of intestinal phages and their host bacteria were observed: phage- and bacteria-predominant. The abundance of obligate anaerobes, such as Clostridium_sense_strict_1, Fusicatenibacter, and Romboutsia, and the phages hosting these bacteria decreased immediately after the onset of COVID-19, and faster phage recovery was observed compared with bacterial recovery. In contrast, the genus Staphylococcus, a facultative anaerobic bacterium, increased immediately after the onset of COVID-19, whereas the phages infecting Staphylococcus decreased. Furthermore, immediately after the onset of COVID-19, the percentage of lytic phages increased, whereas that of temperate phages decreased. These observations suggest that the gut microbiota dysbiosis observed immediately after the onset of COVID-19 may be linked to phage dynamics that control gut microbiota and may also affect the recovery from dysbiosis.IMPORTANCEBacteriophages infect and replicate with bacteria and archaea and are closely associated with intestinal bacteria. The symbiotic relationship between gut microbiota and bacteriophages is of interest, but it is challenging to study their dynamics in the human body over time. SARS-CoV-2 infection has been reported to alter the gut microbiota, which is involved in gut immune regulation and pathophysiology, although changes in the intestinal phages of patients with SARS-CoV-2 and their dynamic relationship with the gut microbiota remain unclear. SARS-CoV-2 infection, which follows a transient pathological course from disease onset to cure, may provide a reliable model to investigate these interactions in the gut environment. Therefore, this study aimed to elucidate the correlation between gut microbiota and intestinal DNA virome dynamics in COVID-19 pathogenesis. This study found that the dysbiosis observed in SARS-CoV-2 infection involves a growth strategy that depends on the phage or bacterial dominance.

Diverse microviruses circulating in invertebrates within a lake ecosystem.

Microviruses are single-stranded DNA bacteriophages and members of the highly diverse viral family Microviridae. Microviruses have a seemingly ubiquitous presence across animal gut microbiomes and other global environmental ecosystems. Most of the studies on microvirus diversity so far have been associated with vertebrate gut viromes. In this study, we investigate the less explored invertebrate microviruses in a freshwater ecosystem. We analysed microviruses from invertebrates in the Chironomidae, Gastropoda, Odonata, Sphaeriidae, Unionidae clades, as well as from water and benthic sediment sampled from a lake ecosystem in New Zealand. Using gene-sharing networks and an expanded framework of informal and proposed microvirus subfamilies, the 463 distinct microvirus genomes identified in this study were grouped as follows: 382 genomes in the Gokushovirinae subfamily and 47 in the Pichovirinae subfamily clade, 18 belonging to Group D, 3 belonging to the proposed Alpavirinae subfamily clade, 1 belonging to the proposed Occultatumvirinae/Tainavirinae subfamilies clade and 12 belonging to an undefined viral cluster VC 1. Inverse associations of microviruses were noted between environmental benthic sediment samples and the Odonata group, while 'defended' invertebrates in the Gastropoda, Sphaeriidae and Unionidae groups showed correlative associations in the principal coordinate analysis of unique microvirus genomes (each genome sharing <98% genome-wide pairwise identity with each other) across sample types. This study expands the known diversity of microviruses and highlights the diversity of these relatively poorly classified bacteriophages.

Phage-Derived Endolysins Against Resistant Staphylococcus spp.: A Review of Features, Antibacterial Activities, and Recent Applications.

Antimicrobial resistance is a significant global public health issue, and the dissemination of antibiotic resistance in Gram-positive bacterial pathogens has significantly increased morbidity, mortality rates, and healthcare costs. Among them, Staphylococcus, especially methicillin-resistant Staphylococcus aureus (MRSA), causes a wide range of diseases due to its diverse pathogenic factors and infection strategies. These bacteria also present significant issues in veterinary medicine and food safety. Effectively managing staphylococci-related problems necessitates a concerted effort to implement preventive measures, rapidly detect the pathogen, and develop new and safe antimicrobial therapies. In recent years, there has been growing interest in using endolysins to combat bacterial infections. These enzymes, which are also referred to as lysins, are a unique class of hydrolytic enzymes synthesized by double-stranded DNA bacteriophages. They possess glycosidase, lytic transglycosylase, amidase, and endopeptidase activities, effectively destroying the peptidoglycan layer and resulting in bacterial lysis. This unique property makes endolysins powerful antimicrobial agents, particularly against Gram-positive organisms with more accessible peptidoglycan layers. Therefore, considering the potential benefits of endolysins compared to conventional antibiotics, we have endeavored to gather and review the characteristics and uses of endolysins derived from staphylococcal bacteriophages, as well as their antibacterial effectiveness against Staphylococcus spp. based on conducted experiments and trials.

The role of bacteriophages in facilitating the horizontal transfer of antibiotic resistance genes in municipal wastewater treatment plants

Bacteriophages play integral roles in the ecosystem; however, their precise involvement in horizontal gene transfer and the spread of antibiotic resistance genes (ARGs) are not fully understood. In this study, a coculture system involving consortia of bacteriophages and multidrug-resistant bacteria from an aerobic tank in a municipal wastewater treatment plant (WWTP) was established to investigate the functions of bacteriophages in ARG transfer and spread. The results of the cocultivation of the MRB and bacteriophage consortia indicated that the bacterial community remained stable throughout the whole process, but the addition of bacteriophages significantly increased ARG abundance, especially in bacteriophage DNA. Nine out of the 11 identified ARGs significantly increased, indicating that more bacteriophage particles carried ARGs in the system after cocultivation. In addition, 686 plasmids were detected during cocultivation, of which only 3.36 % were identified as conjugative plasmids, which is significantly lower than the proportion found among previously published plasmids (25.2 %, totaling 14,029 plasmids). Our findings revealed that bacteriophages may play important roles in the horizontal transfer of ARGs through both bacteriophage-mediated conduction and an increase in extracellular ARGs; however, conjugative transfer may not be the main mechanism by which multidrug-resistant bacteria acquire and spread ARGs. Unlike in most previous reports, a coculture system of diverse bacteria and bacteriophages was established in this study to assess bacteriophage functions in ARG transfer and dissemination in the environment, overcoming the limitations associated with the isolation of bacteria and bacteriophages, as well as the specificity of bacteriophage hosts.

Bacteriophage-related epigenetic natural and non-natural pyrimidine nucleotides and their influence on transcription with T7 RNA polymerase

DNA modifications on pyrimidine nucleobases play diverse roles in biology such as protection of bacteriophage DNA from enzymatic cleavage, however, their role in the regulation of transcription is underexplored. We have designed and synthesized a series of uracil 2ʹ-deoxyribonucleosides and 5ʹ-O-triphosphates (dNTPs) bearing diverse modifications at position 5 of nucleobase, including natural nucleotides occurring in bacteriophages, α-putrescinylthymine, α-glutaminylthymine, 5-dihydroxypentyluracil, and methylated or non-methylated 5-aminomethyluracil, and non-natural 5-sulfanylmethyl- and 5-cyanomethyluracil. The dNTPs bearing basic substituents were moderate to poor substrates for DNA polymerases, but still useful in primer extension synthesis of modified DNA. Together with previously reported epigenetic pyrimidine nucleotides, they were used for the synthesis of diverse DNA templates containing a T7 promoter modified in the sense, antisense or in both strands. A systematic study of the in vitro transcription with T7 RNA polymerase showed a moderate positive effect of most of the uracil modifications in the non-template strand and some either positive or negative influence of modifications in the template strand. The most interesting modification was the non-natural 5-cyanomethyluracil which showed significant positive effect in transcription.

Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.

Variable orthogonality of serine integrase interactions within the ϕC31 family

Serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases (ϕC31, ϕBT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases – RDF pairs. ϕC31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, ϕC31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate ϕC31 integrase. ϕBT1 and TG1 RDFs were noticeably more effective than ϕC31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase – RDF pair. The binding surface on the RDF is much more conserved than the binding surface on the integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase – RDF pairs. Additionally, the ability of a particular integrase’s preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches.