Biochemical characterization of the naturally occurring oxacillinase OXA-50 of Pseudomonas 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 beta-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 beta-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 beta-lactamase genes, one of which encodes an inducible cephalosporinase and the other of which encodes a constitutively expressed oxacillinase.

FIG. 1.

Comparison of the OXA-50 sequence (in boldface) with the sequences of other class D β-lactamases representative of the principal lineages of this group of β-lactamases. The previously proposed class D β-lactamase consensus sequence and numbering (DBL) (4) is indicated below the alignment (uppercase letters are the currently recognized invariant residues, and lowercase letters are the others). Boxed amino acid sequences are those conserved for oxacillinases. The vertical arrow indicates the cleavage site for the leader peptide of OXA-50. The enzymes included in the alignment are described in detail in Table 2.

FIG. 2.

SDS-PAGE analysis of purified enzymes from E. coli BL21(pET-1) (lane 1) and the culture extract (lane 2). A total of 25 μg of each sample was submitted to electrophoresis. Lane M, molecular mass markers, with sizes indicated on the left.