Biology subjects

Tsourkas, P.

Publications and source records attributed to Tsourkas, P..

3 recordsLinked to original sources

bioRxiv2023

In Vitro Evolution to Increase the Titers of Difficult Bacteriophages: Rapid Appelmans Protocol

Bacteriophages are becoming increasingly important in the race to find alternatives to antibiotics. Unfortunately, bacteriophages that might otherwise be useful are sometimes discarded due to low titers making them unsuitable for downstream applications. Here, we present two distinct approaches to experimentally evolve novel New Zealand Paenibacillus larvae bacteriophages. The first approach uses the traditional agar-overlay method, whereas the other was a Rapid Appelmans Protocol (RAP) modelled after the established Appelmans Method. Both approaches resulted in an increase in plaque-forming units (PFU/mL). The RAP approach was significantly faster and simpler, and allowed us to evolve a set of bacteriophages in as little as four days, increasing titers 100-1000-fold relative to their ancestors. The resultant titers were sufficient to extract and sequence DNA from these bacteriophages. An analysis of these phage genomes is provided. We also propose a model that describes the parameters that allow the RAP approach to select improvement of bacteriophage titer. The RAP approach is an effective method for experimentally evolving previously intractable bacteriophages in a high-throughput and expeditious manner.

microbiology↗

bioRxiv2020

Phage Commander, a software tool for rapid annotation of bacteriophage genomes using multiple programs

The number of sequenced phage genomes is growing at an exponential rate. The majority of newly sequenced phage genomes are annotated by one or more of several freely-available auto-annotation programs. No program has been shown to consistently outperform the others; thus the choice of which program to use is not obvious. We present the software Phage Commander for rapid annotation of phage genomes using multiple auto-annotation programs. Phage Commander runs a phage genome sequence through nine auto-annotation programs and integrates the results within a single output. Phage Commander generates properly formatted output files for direct export to NCBI GenBank. Users can select the threshold for determining which genes should be exported. Phage Commander was benchmarked using eight high-quality phage genomes whose genes have been identified through experiments. Results show that the best results are obtained by exporting genes identified by at least two or three of the nine auto-annotation programs.

bioinformatics↗

bioRxiv2020

Diversity and Distribution of a Novel Genus of Hyperthermophilic Aquificae Viruses Encoding a Proof-reading Family-A DNA Polymerase

Despite the high abundance of Aquificae in many geothermal systems, these bacteria are difficult to culture and no viruses infecting members of this phylum have been isolated. Here, we describe the complete, circular dsDNA Uncultivated Virus Genome (UViG) of Thermocrinis Octopus Spring virus (TOSV), derived from metagenomic data, along with eight related UViGs representing three additional species, Thermocrinis Great Boiling Spring virus (TGBSV), Aquificae Josephs Coat Spring Virus (AJCSV), and Aquificae Conch Spring Virus (ACSV). Four near-complete UViGs, ranged from 37,256 bp to 41,208 bp and encoded 48 to 53 open reading frames. Despite low overall similarity between viruses from different hot springs, the genomes shared a high degree of synteny, and encoded numerous genes for nucleotide metabolism, including a polyprotein PolA-type polymerase with likely accessory functions, a DNA Pol III beta subunit (sliding clamp), a thymidylate kinase, a DNA gyrase, a helicase, and a DNA methylase. Also present were conserved genes predicted to code for phage capsids, large and small terminases, portal protein, holin, and lytic transglycosylase, all consistent with a distant relatedness to cultivated Caudovirales. TOSV and TGBSV had the highest coverage in their respective metagenomes and are predicted to infect Thermocrinis ruber and Thermocrinis jamiesonii, respectively, as multiple CRISPR spacers matching the viral genomes were identified within Thermocrinis ruber OC1/4T and Thermocrinis jamiesonii GBS1T. Based on the predicted, unusual bi-directional replication strategy, low sequence similarity to known viral genomes, and a unique position in gene-sharing networks, we propose a new putative genus, Pyrovirus, in the order Caudovirales.

microbiology↗