​softberry.​com/​, the GeneMark program [67] and the GLIMMER program [68]. We considered an open reading frame (ORF) prediction to be good when it was identified by each of the three prediction tools. Discrepant ORFs were manually verified by the Artemis selleck inhibitor viewer [69] and by identification

of putative ribosomal binding sites. 3MA Each gene was functionally classified by assigning a cluster of orthologous group (COG) number or a Kyoto encyclopedia of genes and genomes (KEGG) number, and each predicted protein was compared against every protein in the non- redundant (nr) protein databases http://​ncbi.​nlm.​nih.​gov. In order to associate a function with a predicted gene, we used a minimum cut-off of 30% identity and 80% coverage of the gene length, checking at least two best hits among the COG, KEGG, and non- redundant protein databases. The rRNA genes were identified by the FGENESB tool on the basis of sequence conservation, while tRNA genes were detected with the tRNAscan-SE program. The BLASTp algorithm AZD1152 nmr was used to search for protein similarities with other pneumococcal genomes or deposited sequences referred in the present study, following these criteria: >50% similarity at the amino acid level and >50% coverage of protein length. Phage characterization AP200 was grown in BHI broth at 37°C to achieve a turbidity corresponding to OD620 0.2-0.3. Mytomycin C (Sigma-Aldrich, St. Louis, MO) was added to a final concentration

of 0.1 μg/ml and the culture was incubated until lysis occurred, as shown by a decrease in turbidity. Cellular debris was pelleted at 16000 g for 15 min. The induced supernatant was filtered through a 0.44-μm pore size filter (Millipore, Billerica, MA). For Ixazomib mouse negative staining, the filtered supernatant was ultracentrifuged at 100,000 g for 2 h at 4°C. Suspensions

of the pellet were placed on Formvar-carbon coated 400 mesh copper grids for 10 s, wicked with filter paper and placed on a drop of 2% sodium phosphotungstate, pH 7.00, for 10 s, wicked again and air-dried. Negatively stained preparations were observed with a Philips 208 electron microscope at 80 kV. To obtain phage DNA, the phage pellet was lysed with sodium dodecyl sulfate (0.5%), EDTA (10 mM) and proteinase K (500 μg/ml) for 2 h at 37°C. Phage DNA was precipitated with a 10% volume of 3 M NaOAc (pH 5.2) and 2 volumes of ethanol at -70°C for 2 h, washed with 70% ethanol and resuspended in deionized H2O. In order to demonstrate the circularization of the excised prophage, a PCR assay using the phage DNA as template and divergent primers pair (FR9 5′- CTAGACTTGCGATAGCAGTTACC- 3′ and FR10 5′- GCTTGAACAATTAAGCCAAGCG-3′) designed on the opposite ends of the prophage sequence, was carried out. The PCR product was purified and submitted to sequencing analysis using a Perkin-Elmer ABI 377 DNA sequencer (PE Applied Byosystem). To demonstrate phage activity, a plaque assay was performed. Briefly, 0.1 ml of filtered induced supernatant was pre-incubated with 0.