Biochemical Characterization of the Naturally Occurring Oxacillinase OXA-50 ofPseudomonas aeruginosa

Abstract

The bla_OXA-50 gene (formerly known as the PA5514 gene) is an oxacillinase gene identified in silico in the genome of Pseudomonas aeruginosa PAO1. By using a mutant strain of P. aeruginosa PAO1 that had an inactivated bla_AmpC cephalosporinase gene, the bla_OXA-50 gene was shown to be expressed constitutively in P. aeruginosa. This β-lactamase gene was cloned onto a multicopy plasmid and expressed in P. aeruginosa and Escherichia coli. It conferred decreased susceptibility to ampicillin and ticarcillin and, interestingly, to moxalactam and meropenem in P. aeruginosa but not in E. coli. Overexpression and purification enabled us to determine the molecular mass (25 kDa), the pI value (8.6), and the hydrolysis spectrum of the OXA-50 β-lactamase. It is a narrow-spectrum oxacillinase that uncommonly hydrolyzes imipenem, although at a low level. Very similar oxacillinase genes were identified in all P. aeruginosa isolates from various geographical origins tested. The weak variability of the nucleotide sequence of this gene (0 to 2%) corresponded to that found for the naturally occurring bla_AmpC cephalosporinase gene of P. aeruginosa. The study indicated that P. aeruginosa harbors two naturally encoded β-lactamase genes, one of which encodes an inducible cephalosporinase and the other of which encodes a constitutively expressed oxacillinase.