Complete Nucleotide Sequence and Molecular Characterization of Bacillus Phage TP21 and its Relatedness to Other Phages with the Same Name

Abstract

Three different Bacillus bacteriophages designated TP21 are known from the literature. We have determined the sequence and structure of the TP21-L genome, and compared it to the other phages. The genome is 37.5 kb in size, possesses fixed invariable genome ends and features the typical modular organization of a temperate siphovirus. TP21-L is neither identical to TP21 isolated by Thorne (TP21-T), as shown by a PCR-based approach nor to TP21 isolated by He et al. (TP21-H), as estimated from phage dimensions. For reasons of clarity, we suggest renaming the different TP21 isolates.

Keywords: Bacillus; TP21; bacteriophage.

Figure 1.

Transmission electron micrographs of TP21-L virions negatively stained with 2% uranyl acetate or 2% ammonium molybdate. The bar represents 50 nm. A putative tail fiber structure is indicated by arrows.

Figure 2.

Genome map of TP21-L. The lysogeny control region is shown in blue, the lysis cassette in red. Structural genes (late genes) are depicted in yellow and putative early genes involved in DNA recombination, replication and modification in green. Abbreviations are: TerS: Terminase small subunit. TerL: Terminase large subunit. Cps: Major capsid protein. Tmp: Tail tape measure protein. Hol: Holin. Ply: Endolysin. Numbers to both sides of the graph indicate nt position in the genome sequence. Genes are numbered according to annotation.

Figure 3.

Pulsed-field gel electrophoresis of full length TP21-L genomic DNA prepared by phenolic extraction. a. and b. MidRange PFG Marker I and II (New England Biolabs, Switzerland). c. TP21-L DNA

Figure 4.

Gel electrophoresis of TP21-L DNA predigested with Bal31 nuclease (New England Biolabs), phenolized and subsequently digested with PvuI restriction endonuclease (Fermentas). Marker: 1kb Marker (Fermentas). Other lanes: untreated DNA and PvuI digest of DNA pretreated with Bal31 for indicated time. Arrows indicate fragments disappearing over time.

Figure 5.

SDS-PAGE of TP21-L structural proteins. Left: Size Marker, molecular mass is indicated. Right: TP21-L structural proteins and their assignment as deduced from MALDI-MS/MS analysis. Abbreviations: Cps: Major capsid protein. Tmp: Tail tape measure protein.

Figure 6.

PCR detection of TP21-T. Left block: A colony of HER1399 was used as template. Right block: A colony of DB 4848 / UM 101 was used as template. M: 1kb Marker (Fermentas). 1. Primer combination TP21-L1_Fw and R; 2: Primer combination TP21-L2_Fw and R; 3: Primer combination TP21-T1_Fw and R; 4: Primer combination TP21-T2_Fw and R. 5: Negative control (no primer).