The fragment was cloned into a pET21a vector at the NdeI/EcoRI sites. The second fragment (bp 377-753) was amplified with forward primer 5′-CCGCCGGgaattcAGTATAAAAGTGAGGGCTTA-3′, containing an EcoRI site, and reverse primer 5′-CCaagcttTTAAAACACTTCTTTCACAATCAATCTCTC-3′, www.selleckchem.com/products/LY294002.html containing a HindIII site. The second fragment was cloned in tandem with the first fragment, thus generating the full-length phage P954 lysin gene with an internal EcoRI site. The cat gene was isolated along with its constitutive promoter from the S. aureus – E. coli shuttle plasmid pSK236 by ClaI digestion. Cohesive ends were filled with the Klenow

fragment of DNA polymerase I and ligated into the blunted EcoRI site of the full-length phage P954 endolysin gene, thereby disrupting it. The S. aureus-specific temperature-sensitive origin of replication from the shuttle vector pCL52.2 was introduced selleck chemicals at the XhoI restriction site of this construct to generate pGMB390. Mitomycin C click here induction of phage P954 lysogens The S. aureus RN4220 lysogen of phage P954 was inoculated in LB medium and incubated at 37°C with shaking at 200 rpm for 16 hr. The cells were then subcultured in LB medium at 2% inoculum and incubated at 37°C with shaking at 200 rpm until the culture attained an absorbance of 1.0 at 600 nm. Mitomycin C was then added to a final concentration

of 1 μg/ml, and the culture was incubated at 37°C with shaking at 200 rpm for 4 hr for prophage induction. Recombination and screening for recombinants S. aureus RN4220 cells were transformed with pGMB390 by electroporation according to the protocol described by Schenk and Laddaga [30] with a BioRad Gene Pulser, plated on LB

agar containing chloramphenicol (10 μg/ml), and incubated at 37°C for 16 hr. Chloramphenicol-resistant colonies were selected and grown in LB at 37°C until the cultures reached an absorbance of 1.0 at 600 nm. Recombination was then initiated by infecting these cells with phage P954 (MOI = 3) for 30 min. Progeny phage were harvested from the lysate as described previously, lysogenized in S. aureus RN4220, and plated on LB agar containing chloramphenicol (10 μg/ml) Astemizole (round I). Ninety-six chloramphenicol-resistant colonies were picked up, grown, and induced with Mitomycin C. Cultures that did not lyse after the 16-hr Mitomycin C induction were treated with 1% chloroform and lysed with glass beads; the released phages were again lysogenized in S. aureus RN4220 (round II). Chloramphenicol-resistant colonies of round II lysogens were similarly grown and subjected to Mitomycin C induction. The chloramphenicol-resistant lysogens that did not release phages upon Mitomycin C induction were selected for PCR analysis. Genomic DNA of the selected lysogens was purified, and PCR was performed with different sets of primers to confirm disruption of the phage P954 endolysin gene.