In this context, we evaluated phage K, a known polyvalent phage with a broad host range that includes coagulase-positive and coagulase-negative staphylococci [20, 21]. We report here the identification of the phage tail-associated muralytic enzyme (TAME) of phage K (PCT publication no. WO2007/130655: publication date November 15, 2007) [22] and generation of a chimeric protein that combines the lethal

activity of TAME with the SH3b staphylococcal cell wall-binding domain of lysostaphin [23]. We demonstrated the efficacy of this chimeric protein in vivo using a rat nasal colonization URMC-099 cost model. Some of these findings were presented at the 2009 Madison Molecular Genetics of Bacteria and Bacteriophage meeting at the University of Wisconsin [24]. Methods Bacterial strains, bacteriophages, www.selleckchem.com/products/Temsirolimus.html plasmids, and growth conditions All bacterial strains used in this study are listed in two tables (additional file 1, Table S1, additional file 2, Table S2). Cell culture media were obtained from HiMedia labs (India). Phage K was obtained from the National Collection of Type Culture (NC07814-02) and propagated on S. aureus RN4220 [25]. The methicillin-resistant S. aureus (MRSA) strain B911 was used for bactericidal activity assays, and RN4220 was used for zymograms.Plasmid pET21a (Novagen, USA) was used for cloning and the constructs were expressed under the control of a T7 promoter. Plasmid pRG5

(ATCC) carrying full-length lysostaphin was used as a template for amplifying the SH3b domain. All cultures were grown in Luria Bertani (LB) broth at 37°C, 200 rpm. Ampicillin Terminal deoxynucleotidyl transferase (100 μg/ml) or isopropyl β-D-1-thiogalactopyranoside

(IPTG, 1 mM) were added to the cultures as needed. All reagents used in this study were purchased from Sigma (USA) unless otherwise stated. Sequence analysis and Identification of TAME The DNA sequence of phage K was obtained from the National Center for Biotechnology Information (NCBI) [GenBank: AY176327] [26]. Database searches were performed using BLASTN and BLASTP [27]http://​www.​ncbi.​nlm.​nih.​gov. Domain identification and protein family allocation was performed with the Pfam database [28]http://​pfam.​sanger.​ac.​uk/​ and the Conserved Domain Architecture Retrieval Tool [29]http://​www.​ncbi.​nlm.​nih.​gov/​Selleck LY294002 Structure/​lexington/​lexington.​cgi using the default parameters. Cloning of orf56 and its truncated forms Phage K DNA was prepared as previously described [26]. All DNA manipulations were performed according to the methods of Sambrook and Russell [30]. Briefly, the full-length orf56 gene was amplified from phage K DNA by polymerase chain reaction (PCR) using a forward primer containing a unique NdeI site: 5′-CCGGAATTCCATATGCGTAGAATAAGACCTAAG-3′ and a reverse primer incorporating an XhoI site: 5′-CCGCCGCTCGAGTTATTTCTTATCGTAAATGAATTGTGC-3′. Amplification was carried out using a Smart Cycler (BioRad, USA).