Staphylococcus pseudintermedius MS5134 isolated from a pustule of a dog with impetigo was used for genome sequencing and cloning of an orf encoding a putative ET gene. To investigate the occurrence of this orf, 148 S. pseudintermedius isolates (99 from dogs diagnosed with superficial pyoderma and 49 from healthy dogs) collected from three veterinary institutes in Japan (Tokyo University of Agriculture and Technology, ASC Dermatology Service and Gifu University) were analyzed in this study. The S. pseudintermedius isolates were collected either from the skin lesions of dogs with superficial pyoderma or from the nasal

cavity of healthy dogs. Identification of S. pseudintermedius was carried out on the Y-27632 research buy basis of Gram-staining results, catalase, coagulase and β-galactosidase activities, and multiplex PCR to distinguish the thermonuclease (nuc) gene of S. pseudintermedius from that of buy I-BET-762 other staphylococcal species (Sasaki et al., 2010). Chromosomal DNA was isolated from S. pseudintermedius strains using the UltraClean Microbial DNA Isolation

Kit (MO BIO, Carlsbad, CA). The genome of strain MS5134 was sequenced using the whole-genome random-sequencing method as described previously at the Graduate School of Frontier Sciences, The University of Tokyo, Japan (Toh et al., 2010). From these DNA sequences, a putative ET gene homolog was identified based on sequence homology to ETD of S. aureus. The sequence data were analyzed using the blastp program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for amino acid homology searches, the clustalx Reverse transcriptase program (http://www.clustal.org) for multisequence alignments and phylogenic analysis and the signalp 3.0 program (http://www.cbs.dtu.dk/services/SignalP) for signal peptide

prediction. The theoretical pI and molecular weight were calculated using the expasy proteomics server (http://us.expasy.org/tools/pi_tool.html). The primers 5′-gggcatgcacatatgatgaagcc-3′ (forward primer) and 5′-ccagatctatcttctgattcagc-3′ (reverse primer) were used to amplify the novel orf by PCR. The PCR product was digested with SphI and BglII, subcloned into SphI–BglII-digested pQE70 vector (Qiagen, Valencia, CA) and transfected into Escherichia coli M15 cells (Qiagen). The final construct was confirmed by sequence analysis and was designated pQE-neworf-His. The recombinant protein encoded by the novel orf was harvested from the cytoplasmic soluble fraction of the transfected M15 cells using lysis buffer (BugBuster Protein Extraction Reagent; Novagen, Madison, WI), purified with TALON metal affinity resin (BD Biosciences Clontech, Mountain View, CA) and dialyzed against phosphate-buffered saline (PBS). The presence of the new ORF protein in the purified fraction was confirmed by immunoblotting with anti-His tag antisera (MBL, Nagoya, Japan) (data not shown).