Fig 1.
Electron microscopy image of the purified TP-84 bacteriophage.
Purified TP-84 sample loaded onto 300 mesh copper grid (Sigma), covered with 2% collodion (Sigma), sprayed with carbon and stained with 2% uranyl acetate (BDH Chemicals). Visualised with a Tecnai G2 Spirit BioTWIN TEM set at 120 kV. Pictures were captured with a Veleta CCD camera.
Fig 2.
The GC-skew distribution over the genome of TP-84.
The bacteriophage genome was analysed using the default settings of GC Content Calculator (Biologics International Corp, Indianapolis, USA; http://www.biologicscorp.com/tools/GCContent/; blue).
Fig 3.
Genome organization of the thermophilic, 47.7-kb bacteriophage TP-84.
Putative genes, encoding proteins with assigned biological function, are marked with red arrows. Genes with assigned function confirmed by proteomic analysis are marked with orange arrows. Genes without assigned biological function are marked with black arrows. P—putative host-dependent promoter, T–Rho-independent terminator, SSB–single-stranded DNA-binding protein. The scheme was created using SnapGene software (http://www.snapgene.com) and further modified.
Fig 4.
Phylogenetic trees for TP-84 bacteriophage.
A) Phylogenetic tree constructed using the large subunit terminase (TerL). B) Phylogenetic tree constructed using thymidylate synthase (Ts) proteins. The trees do not include all Geobacillus phage proteins since the differences between all the sequences renders the trees unreliable.
Fig 5.
Experimental validation of TP-84 SSB and structural proteins.
Panels A-C. Expression, purification and functional assay of TP-84 SSB-His6 protein. Lanes M1, molecular weight protein marker, LMW-SDS Marker (GE Healthcare). Panel A. SDS-PAGE analysis of the recombinant E. coli TOP10 [pBADMycHisA-TP-84_SSB] cells induction time course. Lane 1, E. coli TOP10 [pBADMycHisA-TP-84_SSB] cells prior to arabinose induction; lane 2, 2 h after induction; lane 3, 4 h after induction; lane 4, 16 h after induction. Panel B. Metal-affinity purification of TP-84 SSB-His6 protein. Lane 1, purified TP-84 SSB-His6 protein. Panel C. PCR assay of DNA-binding capabilities of TP-84 SSB-His6 protein. Lane M2, molecular weight DNA marker, 100-bp Plus (Thermo Scientific, USA); lane 1, PCR reaction without addition of TP-84 SSB-His6 protein; lane 2, 0.36 μg of TP-84 SSB-His6 protein added; lane 3, 0.72 μg; lane 4, 1.08 μg; lane 5, 1.44 μg; lane 6, 2.16 μg; lane 7, 2.88 μg; lane 8, 3.6 μg. Panel D. SDS-PAGE analysis of the proteins of purified TP-84 bacteriophage. Protein bands yielding MS results of high credibility are assigned to the matching TP-84 CDSs (S5 File).
Fig 6.
Experimental validation of TP-84 endolysin activity.
Panel A. SDS-PAGE of combined peak fractions no 2–6 from CM-Sephadex chromatography (S4 File). The column was used to purify TP-84-infected, G. stearothermophilus cell lysate proteins. Lane M3, molecular weight protein marker, PM2500 (SMOBIO). Lane 1, cation exchange chromatography peak fractions from purification of the TP-84 lysate proteins. Panel B. Graph showing E. coli cell in vitro wall lysis as a decrease in turbidity measured at 450 nm. Blue line, control reaction buffer. Red line, hen egg lysozyme. Grey line, peak fractions 2–6 from purification of the TP-84 lysate proteins.
Fig 7.
Experimental validation of TP-84 proteins biosynthesized during the infection time-course.
Samples for MS analysis were taken from G. stearothermophilus cultures at time intervals: U, uninfected control prior to infection; 0, sample taken immediately upon TP-84 addition; 5, 5 min after infection; 10, 10 min; 15, 15 min; 20, 20 min; 25, 25 min; 30, 30 min. Panel A-D show graphs for each TP-84 protein detected in the culture samples, grouped according to their function. Panel A) DNA replication, recombination, transcription-related and nucleotide metabolism protein. Panel B). Structural proteins and packaging. Panel C) Cell wall and membrane degrading proteins. Panel D) Unknown function proteins.
Table 1.
Putative and experimentally confirmed CDSs of bacteriophage TP-84 genome and their functions.