Search Results (11)

Bacterial viruses (phages) are abundant and ecologically impactful, but laboratory-based experimental model systems vastly under-represent known phage diversity, particularly for ssDNA phages. Here, we characterize the genomes and infection properties of two unrelated marine flavophages—ssDNA generalist phage phi18:4 (6.5 Kbp) and dsDNA specialist phage phi18:1 (39.2 Kbp)—when infecting the same Cellulophaga baltica strain #18 (Cba18), of the class Flavobacteriia. Phage phi18:4 belongs to a new family of ssDNA phages, has an internal lipid membrane, and its genome encodes primarily structural proteins, as well as a DNA replication protein common to ssDNA phages and a unique lysis protein. Phage phi18:1 is a siphovirus that encodes several virulence genes, despite not having a known temperate lifestyle, a CAZy enzyme likely for regulatory purposes, and four DNA methyltransferases dispersed throughout the genome that suggest both host modulation and phage DNA protection against host restriction. Physiologically, ssDNA phage phi18:4 has a shorter latent period and smaller burst size than dsDNA phage phi18:1, and both phages efficiently infect this host. These results help augment the diversity of characterized environmental phage–host model systems by studying infections of genomically diverse phages (ssDNA vs. dsDNA) on the same host. Full article

Spiroplasma virus 4 (SpV4) is a bacteriophage of the Microviridae, which packages circular ssDNA within non-enveloped T = 1 icosahedral capsids. It infects spiroplasmas, which are known pathogens of honeybees. Here, the structure of the SpV4 virion is determined using cryo-electron microscopy to a resolution of 2.5 Å. A striking feature of the SpV4 capsid is the mushroom-like protrusions at the 3-fold axes, which is common among all members of the subfamily Gokushovirinae. While the function of the protrusion is currently unknown, this feature varies widely in this subfamily and is therefore possibly an adaptation for host recognition. Furthermore, on the interior of the SpV4 capsid, the location of DNA-binding protein VP8 was identified and shown to have low structural conservation to the capsids of other viruses in the family. The structural characterization of SpV4 will aid future studies analyzing the virus–host interaction, to understand disease mechanisms at a molecular level. Furthermore, the structural comparisons in this study, including a low-resolution structure of the chlamydia phage 2, provide an overview of the structural repertoire of the viruses in this family that infect various bacterial hosts, which in turn infect a wide range of animals and plants. Full article

RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine. Full article

The genome packaging motor of bacteriophages and herpesviruses is built by two terminase subunits, known as large (TerL) and small (TerS), both essential for viral genome packaging. TerL structure, composition, and assembly to an empty capsid, as well as the mechanisms of ATP-dependent DNA packaging, have been studied in depth, shedding light on the chemo-mechanical coupling between ATP hydrolysis and DNA translocation. Instead, significantly less is known about the small terminase subunit, TerS, which is dispensable or even inhibitory in vitro, but essential in vivo. By taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of phage TerSs, in this review, we take an inventory of known TerSs studied to date. Our analysis suggests that TerS evolved and diversified into a flexible molecular framework that can conserve biological function with minimal sequence and quaternary structure conservation to fit different packaging strategies and environmental conditions. Full article

Gene V protein (gVp) of the bacteriophages of the Ff family is a non-specific single-stranded DNA (ssDNA) binding protein. gVp binds to viral DNA during phage replication inside host Escherichia coli cells, thereby blocking further replication and signaling the assembly of new phage particles. gVp is a dimer in solution and in crystal form. A structural model of the complex between gVp and ssDNA was obtained via docking the free gVp to structures of short ssDNA segments and via the detection of residues involved in DNA binding in solution. Using solid-state NMR, we characterized structural features of the gVp in complex with full-length viral ssDNA. We show that gVp binds ssDNA with an average distance of 5.5 Å between the amino acid residues of the protein and the phosphate backbone of the DNA. Torsion angle predictions and chemical shift perturbations indicate that there were considerable structural changes throughout the protein upon complexation with ssDNA, with the most significant variations occurring at the ssDNA binding loop and the C-terminus. Our data suggests that the structure of gVp in complex with ssDNA differs significantly from the structure of gVp in the free form, presumably to allow for cooperative binding of dimers to form the filamentous phage particle. Full article

Clostridium perfringens is an important pathogen for both humans and animals, causing human foodborne disease and necrotic enteritis in poultry. In the present study, a C. perfringens-specific phage, vB_CpeS_BG3P (designated as BG3P hereafter), was isolated from chicken farm sewage. Both electron microscopy and phylogenetic analysis suggested that phage BG3P is a novel phage belonging to Siphoviridae family. Phage BG3P exhibited a broad host range against different C. perfringens isolates (90.63% of strains were infected). Sequencing of the complete genome revealed a linear double-stranded DNA (43,528 bp) with 28.65% GC content. After sequence analysis, 73 open reading frames (orfs) were predicted, of which only 13 were annotated with known functions. No tRNA and virulence encoding genes were detected. It should be noted that the protein of orf 15 has 97.92% homology to C. perfringens-specific chloramphenicol resistance protein, which has not been reported for any C. perfringens phage. Phylogenetic analysis of the ssDNA binding protein demonstrated that this phage is closely related to C. perfringens phages phiSM101 and phi3626. In considering future use as an antimicrobial agent, some biological characteristics were observed, such as a good pH (3–11) stability and moderate temperature tolerance (<60 °C). Moreover, bacteriophage BG3P showed a good antimicrobial effect against C. perfringens liquid cultures. Thus, phage treatment with MOI ≥ 100 completely inhibited bacterial growth compared to untreated cultures. Although phage BG3P shows good lytic efficiency and broad host range in vitro, future development and application may need to consider removal of the chloramphenicol-like resistance gene or exploring its lysin for future antibacterial applications. Full article

During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts’ defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and—importantly—transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes. Full article

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection. Full article

Tectiviruses infecting the Bacillus cereus group represent part of the bacterial “plasmid repertoire” as they behave as linear plasmids during their lysogenic cycle. Several novel tectiviruses have been recently found infecting diverse strains belonging the B. cereus lineage. Here, we report and analyze the complete genome sequences of phages Sato and Sole. The linear dsDNA genome of Sato spans 14,852 bp with 32 coding DNA sequences (CDSs), whereas the one of Sole has 14,444 bp comprising 30 CDSs. Both phage genomes contain inverted terminal repeats and no tRNAs. Genomic comparisons and phylogenetic analyses placed these two phages within the genus Betatectivirus in the family Tectiviridae. Additional comparative genomic analyses indicated that the “gene regulation-genome replication” module of phages Sato and Sole is more diverse than previously observed among other fully sequenced betatectiviruses, displaying very low sequence similarities and containing some ORFans. Interestingly, the ssDNA binding protein encoded in this genomic module in phages Sato and Sole has very little amino acid similarity with those of reference betatectiviruses. Phylogenetic analyses showed that both Sato and Sole represent novel tectivirus species, thus we propose to include them as two novel species in the genus Betatectivirus. Full article

Phages (viruses that infect bacteria) play important roles in the gut ecosystem through infection of bacterial hosts, yet the gut virome remains poorly characterized. Mammalian gut viromes are dominated by double-stranded DNA (dsDNA) phages belonging to the order Caudovirales and single-stranded DNA (ssDNA) phages belonging to the family Microviridae. Since the relative proportion of each of these phage groups appears to correlate with age and health status in humans, it is critical to understand both ssDNA and dsDNA phages in the gut. Building upon prior research describing dsDNA viruses in the gut of Ciona robusta, a marine invertebrate model system used to study gut microbial interactions, this study investigated ssDNA phages found in the Ciona gut. We identified 258 Microviridae genomes, which were dominated by novel members of the Gokushovirinae subfamily, but also represented several proposed phylogenetic groups (Alpavirinae, Aravirinae, Group D, Parabacteroides prophages, and Pequeñovirus) and a novel group. Comparative analyses between Ciona specimens with full and cleared guts, as well as the surrounding water, indicated that Ciona retains a distinct and highly diverse community of ssDNA phages. This study significantly expands the known diversity within the Microviridae family and demonstrates the promise of Ciona as a model system for investigating their role in animal health. Full article

As one of the most common forms of oxidative DNA damage, 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) generally leads to G:C to T:A mutagenesis. To study DNA replication encountering 8-oxoG by the sole DNA polymerase (Gp90) of Pseudomonas aeruginosa phage PaP1, we performed steady-state and pre-steady-state kinetic analyses of nucleotide incorporation opposite 8-oxoG by Gp90 D234A that lacks exonuclease activities on ssDNA and dsDNA substrates. Gp90 D234A could bypass 8-oxoG in an error-free manner, preferentially incorporate dCTP opposite 8-oxoG, and yield similar misincorporation frequency to unmodified G. Gp90 D234A could extend beyond C:8-oxoG or A:8-oxoG base pairs with the same efficiency. dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG showed fast burst phases. The burst of incorporation efficiency (k_pol/K_d,_dNTP) is decreased as dCTP:G > dCTP:8-oxoG > dATP:8-oxoG. The presence of 8-oxoG in DNA does not affect its binding to Gp90 D234A in a binary complex but it does affect it in a ternary complex with dNTP and Mg²⁺, and dATP misincorporation opposite 8-oxoG further weakens the binding of Gp90 D234A to DNA. This study reveals Gp90 D234A can bypass 8-oxoG in an error-free manner, providing further understanding in DNA replication encountering oxidation lesion for P.aeruginosa phage PaP1. Full article