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

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 ?-lactamase gene, blaLUT-1, was cloned from the whole-cell DNA of the P. luteola clinical isolate LAM, which had a weak narrow-spectrum ?-lactam-resistant phenotype, and expressed in Escherichia coli. This gene encoded LUT-1, a 296-amino-acid Ambler class A ?-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 ?-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 ?-lactamases. No gene homologous to the regulatory ampR genes of class A ?-lactamases was found in the vicinity of the blaLUT-1 gene. The entire blaLUT-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 blaLUT-1 was found for each strain. These genes (named blaLUT-2 to blaLUT-6) had nucleotide sequences 98.1 to 99.5% identical to that of blaLUT-1 and differing from this gene by two to four nonsynonymous single nucleotide polymorphisms. The blaLUT 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.