Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus

PLoS One. 2018 Apr 6;13(4):e0195449. doi: 10.1371/journal.pone.0195449. eCollection 2018.

Abstract

Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its' 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host's transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the Siphoviridae family. Thus a taxonomic proposal of a new Tp84virus genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in Escherichia coli, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition
  • Base Sequence
  • Computational Biology
  • DNA, Viral / genetics
  • DNA, Viral / metabolism
  • Genome, Viral*
  • Geobacillus stearothermophilus / virology*
  • Microscopy, Electron, Transmission
  • Molecular Sequence Annotation
  • Phylogeny
  • Proteomics
  • Siphoviridae / classification
  • Siphoviridae / genetics*
  • Siphoviridae / physiology
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / physiology

Substances

  • DNA, Viral
  • Viral Proteins

Grants and funding

The project has been sponsored in part by the National Center for Development (Poland) grant no POIG.01.04.00-22-140/12 issued to BioVentures Institute Ltd., in which the University of Gdansk has been a scientific partner. The BioVentures Institute Ltd. provided support in the form of salaries for the following authors: Piotr M. Skowron, Joanna Zebrowska, Lukasz Janus, Kasjan Szemiako, Edyta Czajkowska, Natalia Maciejewska, Malgorzata Skowron and Agnieszka Zylicz-Stachula. These authors are (or have been) also University of Gdansk employees or students. The specific roles of these authors are articulated in the ‘author contributions’ section. During the grant realisation, BioVentures Institute Ltd. also provided materials, reagents and equipment. BioVentures Institute Ltd. has played a predominant role in this study during all of its stages: the concept development, experiments design and realisation, as well as the decision to publish and the manuscript preparation.