The Bacteriophage Pf-10—A Component of the Biopesticide “Multiphage” Used to Control Agricultural Crop Diseases Caused by Pseudomonas syringae

Phytopathogenic pseudomonads are widespread in the world and cause a wide range of plant diseases. In this work, we describe the Pseudomonas phage Pf-10, which is a part of the biopesticide “Multiphage” used for bacterial diseases of agricultural crops caused by Pseudomonas syringae. The Pf-10 chromosome is a dsDNA molecule with two direct terminal repeats (DTRs). The phage genomic DNA is 39,424 bp long with a GC-content of 56.5%. The Pf-10 phage uses a packaging mechanism based on T7-like short DTRs, and the length of each terminal repeat is 257 bp. Electron microscopic analysis has shown that phage Pf-10 has the podovirus morphotype. Phage Pf-10 is highly stable at pH values from 5 to 10 and temperatures from 4 to 60 °C and has a lytic activity against Pseudomonas strains. Phage Pf-10 is characterized by fast adsorption rate (80% of virions attach to the host cells in 10 min), but has a relatively small number of progeny (37 ± 8.5 phage particles per infected cell). According to the phylogenetic analysis, phage Pf-10 can be classified as a new phage species belonging to the genus Pifdecavirus, subfamily Studiervirinae, family Autographiviridae, order Caudovirales.

Complete genome analysis of enterotoxigenic Escherichia coli K88 phage vB_EcoP_E212, a novel genus Lederbergvirus

The features and genome annotation of a newly bacteriophage v B_EcoP_E212 (referred to as E212) which isolated from farm sewage collected in Jilin, China was describes in this study. Bacteriophage E212 belongs to the family Podoviridae, order Caudovirales through transmission electron microscopy. This phage specifically infects enterotoxigenic Escherichia coli K88. The dsDNA molecule of phage E212 was 38252 bp in length and contained 46.98% G + C content. It has been predicted to contain 53 ORFs, and no tRNAs. Phage E212 carried the integrase gene, and no homologues of virulence factors or antimicrobial resistance genes were found in this phage. Phage E212 was assigned to the genus Lederbergvirus in accordance with nucleotide sequence alignment and phylogenetic analysis.

The HIV-1 nucleocapsid chaperone protein forms locally compacted globules on long double-stranded DNA

The nucleocapsid (NC) protein plays key roles in Human Immunodeficiency Virus 1 (HIV-1) replication, notably by condensing and protecting the viral RNA genome and by chaperoning its reverse transcription into double-stranded DNA (dsDNA). Recent findings suggest that integration of viral dsDNA into the host genome, and hence productive infection, is linked to a small subpopulation of viral complexes where reverse transcription was completed within the intact capsid. Therefore, the synthesized dsDNA has to be tightly compacted, most likely by NC, to prevent breaking of the capsid in these complexes. To investigate NC’s ability to compact viral dsDNA, we here characterize the compaction of single dsDNA molecules under unsaturated NC binding conditions using nanofluidic channels. Compaction is shown to result from accumulation of NC at one or few compaction sites, which leads to small dsDNA condensates. NC preferentially initiates compaction at flexible regions along the dsDNA, such as AT-rich regions and DNA ends. Upon further NC binding, these condensates develop into a globular state containing the whole dsDNA molecule. These findings support NC’s role in viral dsDNA compaction within the mature HIV-1 capsid and suggest a possible scenario for the gradual dsDNA decondensation upon capsid uncoating and NC loss.

Complete genome analysis of a newly isolated Shigella sonnei phage vB_SsoM_Z31

This work describes the characterization and genome annotation of a newly isolated lytic phage vB_SsoM_Z31 (referred to as Z31), isolated from wastewater samples collected in Dalian, China. Transmission electron microscope revealed that phage Z31 belongs to the family Myoviridae, order Caudovirales. This phage specifically infects the Shigella sonnei, Shigella dysenteriae and Escherichia coli. The genome of the phage Z31 is an 89,355 bp length dsDNA molecule with a G + C content of 38.87%. It has been predicted to contain 133 ORFs, and 24 tRNAs. No homologs of virulence factors or antimicrobial resistance genes were found in this phage. Based on the results of nucleotide sequence alignment and phylogenetic analysis, phage Z31 was assigned to the genus Felixounavirus, subfamily Ounavirnae.

Analysis of a Novel Bacteriophage vB_AchrS_AchV4 Highlights the Diversity of Achromobacter Viruses

Achromobacter spp. are ubiquitous in nature and are increasingly being recognized as emerging nosocomial pathogens. Nevertheless, to date, only 30 complete genome sequences of Achromobacter phages are available in GenBank, and nearly all of those phages were isolated on Achromobacter xylosoxidans. Here, we report the isolation and characterization of bacteriophage vB_AchrS_AchV4. To the best of our knowledge, vB_AchrS_AchV4 is the first virus isolated from Achromobacter spanius. Both vB_AchrS_AchV4 and its host, Achromobacter spanius RL_4, were isolated in Lithuania. VB_AchrS_AchV4 is a siphovirus, since it has an isometric head (64 ± 3.2 nm in diameter) and a non-contractile flexible tail (232 ± 5.4). The genome of vB_AchrS_AchV4 is a linear dsDNA molecule of 59,489 bp with a G+C content of 62.8%. It contains no tRNA genes, yet it includes 82 protein-coding genes, of which 27 have no homologues in phages. Using bioinformatics approaches, 36 vB_AchrS_AchV4 genes were given a putative function. A further four were annotated based on the results of LC–MS/MS. Comparative analyses revealed that vB_AchrS_AchV4 is a singleton siphovirus with no close relatives among known tailed phages. In summary, this work not only describes a novel and unique phage, but also advances our knowledge of genetic diversity and evolution of Achromobacter bacteriophages.

Sequence Analysis of a Jumbo Bacteriophage, Xoo-sp14, That Infects Xanthomonas oryzae pv. oryzae

ABSTRACT A jumbo bacteriophage, Xoo-sp14, infecting Xanthomonas oryzae pv. oryzae was isolated from rice fields in China. Here, we report the complete genome sequence of this phage, revealing that it had a linear double-stranded DNA (dsDNA) molecule 232,104 bp long, with a G+C content of 58%. It has 251 annotated protein-coding sequences.

Base pairing and stacking contributions to double stranded DNA formation

Double-strand (ds)DNA formation and dissociation are of fundamental biological importance. The negatively DNA charge influences the dsDNA stability. However, the base pairing and the stacking between neighboring bases are responsible for the sequence dependent stability of dsDNA. The stability of a dsDNA molecule can be estimated from empirical nearest-neighbor models based on contributions assigned to base pair steps along the DNA and additional parameters due to DNA termini. In efforts to separate contributions it has been concluded that base-stacking dominates dsDNA stability whereas base-pairing contributes negligibly. Using a different model for dsDNA formation we re-analyze dsDNA stability contributions and conclude that base stacking contributes already at the level of separate ssDNAs but that pairing contributions drive the dsDNA formation. The theoretical model also predicts that stability contributions of base pair steps that contain only guanine/cytosine, mixed steps and steps with only adenine/thymine follows the order 6:5:4, respectively, as expected based on the formed hydrogen bonds. The model is fully consistent with available stacking data and nearest-neighbor dsDNA parameters. It allows to assign a narrowly distributed value for the effective free energy contribution per formed hydrogen bond during dsDNA formation of −0.72 kcal·mol-1 based entirely on experimental data.

DNA sequence symmetries from randomness: the origin of the Chargaff’s second parity rule

Most living organisms rely on double-stranded DNA (dsDNA) to store their genetic information and perpetuate themselves. This biological information has been considered as the main target of evolution. However, here we show that symmetries and patterns in the dsDNA sequence can emerge from the physical peculiarities of the dsDNA molecule itself and the maximum entropy principle alone, rather than from biological or environmental evolutionary pressure. The randomness justifies the human codon biases and context-dependent mutation patterns in human populations. Thus, the DNA ‘exceptional symmetries,’ emerged from the randomness, have to be taken into account when looking for the DNA encoded information. Our results suggest that the double helix energy constraints and, more generally, the physical properties of the dsDNA are the hard drivers of the overall DNA sequence architecture, whereas the selective biological processes act as soft drivers, which only under extraordinary circumstances overtake the overall entropy content of the genome.

Ivermectin Inhibits Bovine Herpesvirus 1 DNA Polymerase Nuclear Import and Interferes With Viral Replication

Bovine herpesvirus1 (BoHV-1) is a major bovine pathogen. Despite several vaccines being available to prevent viral infection, outbreaks are frequent and cause important economic consequences worldwide. The development of new antiviral drugs is therefore highly desirable. In this context, viral genome replication represents a potential target for therapeutic intervention. BoHV-1 genome is a dsDNA molecule whose replication takes place in the nuclei of infected cells and is mediated by a viral encoded DNA polymerase holoenzyme. Here, we studied the physical interaction and subcellular localization of BoHV-1 DNA polymerase subunits in cells for the first time. By means of co-immunoprecipitation and confocal laser scanning microscopy (CLSM) experiments, we could show that the processivity factor of the DNA polymerase pUL42 is capable of being autonomously transported into the nucleus, whereas the catalytic subunit pUL30 is not. Accordingly, a putative classic NLS (cNLS) was identified on pUL42 but not on pUL30. Importantly, both proteins could interact in the absence of other viral proteins and their co-expression resulted in accumulation of UL30 to the cell nucleus. Treatment of cells with Ivermectin, an anti-parasitic drug which has been recently identified as an inhibitor of importin α/β-dependent nuclear transport, reduced UL42 nuclear import and specifically reduced BoHV-1 replication in a dose-dependent manner, while virus attachment and entry into cells were not affected. Therefore, this study provides a new option of antiviral therapy for BoHV-1 infection with Ivermectin.

Single-Molecule Mechanics in Ligand Concentration Gradient

Single-molecule experiments provide unique insights into the mechanisms of biomolecular phenomena. However, because varying the concentration of a solute usually requires the exchange of the entire solution around the molecule, ligand-concentration-dependent measurements on the same molecule pose a challenge. In the present work we exploited the fact that a diffusion-dependent concentration gradient arises in a laminar-flow microfluidic device, which may be utilized for controlling the concentration of the ligand that the mechanically manipulated single molecule is exposed to. We tested this experimental approach by exposing a λ-phage dsDNA molecule, held with a double-trap optical tweezers instrument, to diffusionally-controlled concentrations of SYTOX Orange (SxO) and tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP). We demonstrate that the experimental design allows access to transient-kinetic, equilibrium and ligand-concentration-dependent mechanical experiments on the very same single molecule.