Cloning and Characterization of a Gene, pbpF , Encoding a New Penicillin-Binding Protein, PBP2B, in Staphylococcus aureus

A previously unrecognized penicillin binding protein (PBP) gene,pbpF , was identified inStaphylococcus aureus . This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that ofS. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and β-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed inEscherichia coli . The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected fromS. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B inS. aureus cell membrane that covalently binds penicillin. The internal region ofpbpF and PBP2B protein were found in all 12S. aureus strains tested by PCR and immunoblotting.