Characterization of the blaCARB-3 gene encoding the carbenicillinase-3 beta-lactamase of Pseudomonas aeruginosa

Abstract

We have isolated the blaCARB-3 structural gene encoding the CARB-3 carbenicillinase of Pseudomonas aeruginosa strain Cilote, tested the specificity of blaCARB-3 DNA probes and determined the nucleotide sequence of blaCARB-3. Three restriction fragment probes internal or delimiting the blaCARB-3 structural gene were hybridized with purified plasmid DNA coding for 18 other beta-lactamases (Blas). Under high-stringency conditions, only blaPSE-1, blaPSE-4, and blaCARB-4 sequences cross-hybridized with blaCARB-3. Sequencing of blaCARB-3 identified the structural gene which encodes a polypeptide product of 268 amino acids with a calculated estimated Mr of 29,246 for the mature form of the protein. Homology studies and computer analysis of primary structures confirmed that CARB-3 is a class-A Bla. The CARB-3 carbenicillinase differs from PSE-4 at two positions: Phe (PSE-4) instead of Leu188 (CARB-3), and Glu (PSE-4) instead of Ala266 (CARB-3), which changes the isoelectric value from (PSE-4) 5.4 to 5.75 (CARB-3). The possible effects of these two mutations were examined by comparisons on the 2 A crystal structure of the Staphylococcus aureus penicillinase, and they were shown to be silent substitutions causing no changes in the phenotype. The nucleic acid hybridization studies and sequence data confirmed that carbenicillinase-encoding bla genes are closely related and that blaCARB-3 is a variant of blaPSE-4.