TP21-L was propagated using host HER1399 (ATCC 13472) in liquid culture [10
], and purified by poly-ethylene glycol precipitation and density gradient centrifugation as described elsewhere [12
]. Electron microscopy of purified negatively stained phage particles [13
] (), allowed assignment of TP21-L to the Siphoviridae
family, in the order of the Caudovirales
]. The phage features an isometric head of 58.5 nm diameter and a long, non-contractile, flexible tail of 144.8 nm length and 11.0 nm diameter. A putative tail fiber could be observed (, indicated by arrows), corresponding to a putative tail fiber encoding gene in the genome of TP21-L (see below).
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.
Phage genomic DNA was extracted from purified phage as described elsewhere [12
]. The TP21-L genome sequence was determined using a shotgun cloning and sequencing approach, followed by manual gap closing using primer walking. After assembly of the complete genome with 5.6-fold average coverage, TP21-L revealed a unit genome sequence of 37,456 base pairs (), which is in good agreement with pulsed field gel electrophoresis analysis of full length phage DNA [13
] (). The G+C content is 37.8 mol%, slightly higher than that of the host bacteria (35.5 mol%, as determined from Genbank data of published host bacteria genomes (NC_003909, NC_004742, NC_005957, NC_003997, and NC_008600). The sequence of TP21-L has been deposited in the databases under accession number EU887664
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 (more ...)
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
The TP21-L DNA molecule features invariable, fixed ends, as revealed by the typical pattern of single fragments disappearing over time when Bal31-predigested DNA is subjected to restriction digestion [13
] (). Heating of restriction digests prior to electrophoresis did not reveal any change compared to non-heated samples (data not shown), suggesting the absence of cohesive ends [16
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 (more ...)
Fifty-six putative open reading frames could be identified in the TP21-L genome, but only 17 of them revealed homologies to proteins of other bacteriophages (). No indication for modified, hydroxy-methylated bases [17
] was found by restriction profiling. Mass spectrometry based identification (MALDI-MS peptide fingerprinting) of SDS-PAGE-separated structural proteins () [13
] led to the identification of ten distinct protein species: one could be allocated to a minor structural protein (gp17) of 192.6 kDa, and another could be allocated to the putative tape measure protein (gp15) of 99.9 kDa. The dimension of the phage tail is in good correlation with the size of the Tmp protein, as demonstrated for phage Lambda and others [19
] (, ). The putative portal protein (gp3) was identified in a band of approx 67 kDa, not in agreement with the predicted mass, and a tail fiber protein (, ) (gp16) was identified in a band of approx. 58 kDa. A putative capsid protein (gp4) as well as the major capsid protein (gp7) were also identified (39 kDa and 30 kDa, respectively). Yet another band (approximately 21 kDa) apparently contained traces of the major capsid protein, probably due to proteolytic cleavage or posttranslational modification of Cps, a fact also observed in other phages [13
]. The predicted gene products gp6, gp8, and gp11 could also be identified as structural proteins, but do not exhibit amino acid homology to any known protein from the databases. However, the predicted mass of gp6 did not match the observed mass (), suggesting posttranslational processing of this protein.
Table 1. General features, database matches and functional assignments of coding sequences (cds) of the Bacillus phage TP21-L genome for which homologies (e-value < 0.01) to known proteins could be found. Molecular mass (MM) and isoelectric points (pI) (more ...)
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. (more ...)
From the genome sequence (), the endolysin (gp19) [10
], a putative holin (gp18; featuring a TMHMM-predicted transmembrane domain [24
]), as well as the lysogeny control region could be deduced. The latter consists of only two genes (gp20 and gp21) (), whose predicted products share homologies with a transcription repressor and a recombinase. This correlates well with the ability of TP21-L to lysogenize its host (HER1399). In order to demonstrate this, we have isolated clones of HER1399 resistant to infection with TP21-L, sub-cultured the cells, treated them with UV light (254 nm, 120 mJ/cm2
, 2 min), and used the sterile-filtered supernatants for demonstration of lytic activity against HER1399 on pre-inoculated agar plates. As a control, a UV-induced culture of wild-type HER1399 did not release any phage. The lysogenized strains were homoimmune to superinfection with up to 109
Pfu/ml TP21-L. PCR using TP21-L specific primers (see below) confirmed the presence of phage in the lysogenized strains (results not shown).
Unfortunately, the designation TP21 has coincidentally been assigned to a total of three phages in the past. In our attempt to identify the individual TP21 isolates and clarify their nomenclature, the origins and history of the isolates is briefly described. TP21-L was made available to our lab by Hans-Wolfgang Ackermann, from the Félix d’Herelle Reference Center for Bacterial Viruses in 1994; it was described as a phage infecting Bacillus thuringiensis and designated TP21. Unfortunately, it was not possible to further elucidate the exact origin of this phage, and it was also not kept in the Félix d’Herelle Reference Center for Bacterial Viruses collection. However, since the genome sequence is now available, and it is also clear that TP21-L is unique among the Bacillus phages, it should be re-deposited in the collection.
The second Bacillus
phage named TP21 was isolated more than 20 years ago by Ruhfel and Thorne [11
]. We propose that this phage should be renamed to TP21-T. The phage is unusual since it was shown to persist as an autonomously replicating plasmidal prophage in B. thuringiensis
]. In order to determine that TP21-L is different from TP21-T, we designed and used a set of PCR primers to amplify unique sequences of 0.5 to 1.1 kb from the genomes of both phages (the primer sequences were generated based on the unpublished genome sequence of TP21-T (R. Okinaka and P. Jackson, personal communication). Amplification with primers specific for TP21-L phage do not yield detectable products using template DNA from B. thuringiensis kurstaki
HD1-9 or strain DP 4848 / UM 101, a carrier of TP21-T (), although the primer combination TP21-L2_Fw an TP21-L2_R seems to produce an unspecific product both with HER1399 and DB 4848 as template (, lane 2). Using primer combinations specific for TP21-T produced products of the expected size with strain DB 4848 as template but not with HER1399 (). Vice versa, using TP21-L DNA as template did also not yield specific amplification products with TP21-T-specific primers [11
] (data not shown). We conclude that TP21-L is not the same as TP21-T from Ruhfel and Thorne [11
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: (more ...)
The third TP21 isolate was isolated from a Chinese factory producing B. thuringiensis
powder and described by He and coworkers [9
], and thus renamed to TP21-H. This phage apparently features an elongated head of 87 x 55 nm and a flexible tail of 140 x 8 nm in size (B2 morphotype), thus clearly distinguishing it from TP21-L (). Unfortunately, however, further details are not available, and the whereabouts of TP21-H are unclear. It is not kept in the Félix d’Herelle Reference Center for Bacterial Viruses collection or any other collection, and has probably been lost (H.-W. Ackermann, personal communication). Even though, we propose to name this phage TP21-H, should it ever be rediscovered or reisolated.