No
bacterial species tested formed plaques in the absence of ϕE202. For ϕE202 UV induction studies, one hundred microliters of saturated B. thailandensis E202 culture was used to inoculate two LB broth (3 ml) subcultures. One set of subcultures was incubated for 5 h without interruption. The other set of subcultures was incubated for 3 h, poured into sterile petri dishes in a class II biological safety cabinet, subjected to a hand-held UV light source (254 nm) for 20 sec (25 cm above the sample), pipetted back into culture tubes, and incubated for an additional 2 h. The titer of the filter-sterilized supernatants were determined by performing quantitative plaque assays on serially diluted samples. Negative staining To determine morphotypes, bacteriophages were prepared from 20 ml of a plate culture lysate,
incubated PRI-724 manufacturer at 37°C for 15 min with Nuclease Mixture (Promega), precipitated with Phage Precipitant (Promega), and resuspended in 1 ml of Phage Buffer (Promega). The bacteriophage solution (~100 μl) was added to a strip of parafilm M (Sigma), and a formvar-coated nickel grid (400 mesh) was floated on the bacteriophage solution for 30 min at 25°C. Excess fluid was removed, and the grid was placed on a drop of 1% phosphotungstic acid, pH 6.6 (PTA) for 2 min at 25°C. Excess fluid was removed, and the specimen was examined on a Philips mTOR inhibitor CM100 transmission electron microscope. Nickel grids were glow discharged on the day of use. Bacteriophage sequencing and annotation Libraries were constructed from the genomic DNA from the bacteriophage isolates. Since the phage genomes were estimated to be 50 kb in size, sequencing,
closure, and annotation was performed similar to that of a BAC sequence [23]. Each of the five isolated bacteriophages were completely sequenced to 10× coverage, closed, and annotated, MycoClean Mycoplasma Removal Kit and the sequences deposited in GenBank (Table 1A). Identification of putative prophages and prophage-like elements within strains Presence of prophage sequence within sequenced genomes of nine B. pseudomallei strains, six B. mallei strains [8], three B. multivorans strains, B. thailandensis E264 [24], and B. xenovorans LB400 [25] (Additional file 1, Table S2) was inferred using a number of similarity measures previously described [26, 27]. First, the protein set of each genome was searched against a non-redundant database of viral proteins using BLASTP [28] with a cutoff of e-10. Secondly, the annotation of each strain was searched for several virus-related keywords such as integrase, tail, capsid, portal, terminase, etc. Clustering of such proteins with proteins containing similarity to known phage proteins as identified by BLASTP, as well as the orientation of proteins within clusters was considered strong evidence for prophage presence. Finally, tRNA genes and attachment sites were examined.