Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated beta-lactamase with two molecular variants

Abstract

Klebsiella pneumoniae BA32, a clinical isolate from Buenos Aires, Argentina, was found to produce a plasmid-encoded beta-lactamase (FOX-1) which conferred resistance to broad-spectrum cephalosporins and cephamycins. Resistance could be transferred by conjugation or transformation into Escherichia coli K-12 via a 48.5-kb plasmid (pGLK1) that produced two FOX-1 molecular variants with isoelectric points of 6.8 and 7.2 and apparent molecular sizes of 37 and 35 kDa, respectively. The kinetic study revealed that the two variants had very similar substrate and inhibition profiles. These values resemble those of chromosomally mediated class C (group 1) cephalosporinases. The structural gene of FOX-1 (blaFOX-1) was cloned into a 2,270-bp PstI-PstI fragment and was expressed in E. coli TG1. The deduced 382-amino-acid sequence of FOX-1 exhibited a high degree of similarity with chromosomally encoded AmpC beta-lactamases of Pseudomonas aeruginosa, Serratia marcescens, Enterobacter cloacae, E. coli, and Citrobacter freundii. These findings suggest that FOX-1 is a plasmid-mediated AmpC-type beta-lactamase that is encoded by a single gene and that has two molecular variants.