Amplified DNA was gel-purified

with a QIAEX II gel extraction kit (Qiagen, Santa Clarita, Calif.) and labeled with the Biotin High Prime System (Roche Applied Science). Genomic DNA was purified using a cetyltrimethylammonium bromide miniprep protocol [76], digested with EcoRI and PstI, separated by electrophoresis in a 0.8% agarose gel, and transferred onto a BrightStar-Plus ��-Nicotinamide clinical trial nylon membrane (Ambion, Inc., Austin, TX) in 0.4 M NaOH. The membranes were pre-hybridized and hybridized at 58°C in a solution containing 5 × SSC [80], 4 × Denhardt’s solution [80], 0.1% SDS, and 300 μg per ml of denatured salmon sperm DNA (Sigma). After hybridization, the membranes were washed twice in 2 × SSC, 0.1% SDS at room temperature, twice in 0.2 × SSC, 0.1% SDS at room temperature, and once in 0.2 × SSC, 0.1% SDS at 60°C. Lytic assays Full-length hol genes from strains Pf-5 and Q8r1-96 were amplified by using KOD Hot Start DNA polymerase (Novagen, Inc.) and oligonucleotide primer pairs holupPf5 (5′ AGG GAC CTC TAG AAA CAT CGT

TA 3′) – holowPf5 (5′ TTT TGG ATC CGG TGA GTC AAG GCT G 3′) and hol-xba (5′ GAC CAG TCT AGA CAT GCT CAT CA 3′) – hol-low (5′ TTT TGG ATC CGC GGT ATC GCT T 3′), respectively. Full-length lys genes from Pf-5 and Q8r1-96 were amplified by using primer sets lysupPf5 (5′ CGC CAT TCT AGA TTA CTG AAC AA 3′) – lyslowPf5 (5′ TTT TGG ATC CGC AGG ACC TTC AGA C 3′) and lysQ8-up (5′ CGG ACA TCT AGA ATC ATG CAC TTG 3′) – tail13 (5′ GCC GCT TGG GTG ATT TGA TT

3′), respectively. The cycling program included a 2-min initial denaturation at 94°C followed by 35 cycles of 94°C for 15 sec, 59°C this website for 30 sec, selleck inhibitor and 68°C for 1 min, and a final extension at 68°C for 3 min. PCR products were gel-purified and cloned into the SmaI site of the plasmid vector pCR-Blunt (Invitrogen) under the control of the T7 promoter. The resultant plasmids were single-pass sequenced to confirm the integrity of cloned genes and electroporated into E. coli Rosetta/pLysS (Novagen) with a Gene Pulser II system (Bio-Rad Laboratories, Hercules, Calif.). Plasmid-bearing E. coli clones were selected overnight on LB agar supplemented with ampicillin and chloramphenicol, and suspended in 2xYT broth supplemented with antibiotics to give an OD600 of 0.1. After incubation with shaking for one hour at room temperature, gene expression was induced in the broth cultures with 3 mM IPTG. The induced cultures were incubated with shaking for another 5 hours and the cell density was monitored by measuring OD600 every 30 min. To disrupt cell membranes in endolysin-expressing cultures, a drop of chloroform was added after four hours of induction. Two independent repetitions were performed with each strain. Acknowledgements The authors are grateful to Dr. Olga FRAX597 mw Mavrodi for help with the screening of Q8r1-96 gene library for ssh6-positive cosmid clones.