Molecular and Biochemical Characterization of the Natural Chromosome-Encoded Class A β-Lactamase from Pseudomonas luteola

Abstract

ABSTRACT Pseudomonas luteola (formerly classified as CDC group Ve-1 and named Chryseomonas luteola ) is an unusual pathogen implicated in rare but serious infections in humans. A novel β-lactamase gene, bla LUT-1 , was cloned from the whole-cell DNA of the P. luteola clinical isolate LAM, which had a weak narrow-spectrum β-lactam-resistant phenotype, and expressed in Escherichia coli . This gene encoded LUT-1, a 296-amino-acid Ambler class A β-lactamase with a pI of 6 and a theoretical molecular mass of 28.9 kDa. The catalytic efficiency of this enzyme was higher for cephalothin, cefuroxime, and cefotaxime than for penicillins. It was found to be 49% to 59% identical to other Ambler class A β-lactamases from Burkholderia sp. (PenA to PenL), Ralstonia eutropha (REUT), Citrobacter sedlakii (SED-1), Serratia fonticola (FONA and SFC-1), Klebsiella sp. (KPC and OXY), and CTX-M extended-spectrum β-lactamases. No gene homologous to the regulatory ampR genes of class A β-lactamases was found in the vicinity of the bla LUT-1 gene. The entire bla LUT-1 coding region was amplified by PCR and sequenced in five other genetically unrelated P. luteola strains (including the P. luteola type strain). A new variant of bla LUT-1 was found for each strain. These genes (named bla LUT-2 to bla LUT-6 ) had nucleotide sequences 98.1 to 99.5% identical to that of bla LUT-1 and differing from this gene by two to four nonsynonymous single nucleotide polymorphisms. The bla LUT gene was located on a 700- to 800-kb chromosomal I-CeuI fragment, the precise size of this fragment depending on the P. luteola strain.