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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.
Highlights
Thermophilic bacteriophages are rarely studied and no life cycles have been deciphered to the extent approaching model Escherichia coli (E. coli) viruses, exemplified by λ, T4, T7 or M13
We describe the complete, annotated TP-84 genome sequence, show that the sequence is unrelated to other characterized bacteriophages and present an in-depth proteomics analysis
The TP-84 bacteriophage was cultivated under modified conditions, purified and subjected to transmission electron microscopy (TEM) imaging, confirming the originally published siphoviral morphology (Fig 1) [14]
Summary
Thermophilic bacteriophages are rarely studied and no life cycles have been deciphered to the extent approaching model Escherichia coli (E. coli) viruses, exemplified by λ, T4, T7 or M13. Older scientific literature includes a number of poorly characterized thermophilic “Bacillus” bacteriophages such as 24 podo- and siphoviruses of [Bacillus] caldotenax and 'B. caldovelox’ [2], temperate Bacillus stearothermophilus bacteriophages φμ-4 [3], and siphovirus Tφ3, which possesses a 125 nm long and 10 nm wide tail [4]. None of these bacteriophages has been sequenced. The temperate Geobacillus bacteriophage GBSV1 has a 34.7 kb genome, a GC-content of 44.4% and encodes 54 proteins [8]
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