CHARACTERIZATION OF THE PENA AND PENR GENES OF BURKHOLDERIA-CEPACIA-249 WHICH ENCODE THE CHROMOSOMAL CLASS-A PENICILLINASE AND ITS LYSR-TYPE TRANSCRIPTIONAL REGULATOR

Burkholderia cepacia is recognized as an important pathogen in the lun g infections of patients with cystic fibrosis. An inducible beta-lacta mase activity has been associated with increased resistance to beta-la ctam antibiotics in clinical isolates of B. cepacia. In this study, we report the revised sequence of the penA gene, which encodes the induc ible penicillinase of B. cepacia, and show that it belongs to the mole cular class A beta-lactamases and exhibits a high degree of similarity to the chromosomal beta-lactamase of Klebsiella oxytoca. Analysis of the nucleotide sequence of the DNA region directly upstream of the pen A coding sequence revealed an open reading frame (penR), the transcrip tion of which was oriented opposite to that of penA and whose initiati on was 130 bp away from that of penA. Two potential ribosome-binding s ites and two overlapping -10 and -35 promoter sequences were identifie d in the intercistronic region. The predicted translation product of p enR was a polypeptide of 301 amino acids with an estimated molecular s ize of 33.2 kDa. The deduced polypeptide of penR showed a high degree of similarity with AmpR-like transcriptional activators of class A and C beta-lactamases, with identities of 59 and 58.7% with Pseudomonas a eruginosa PAO1 AmpR and Proteus vulgaris B317 CumR, respectively. The N-terminal portion of B. cepacia PenR was predicted to include a helix -turn-helix motif, which may bind the LysR motif identified in the int ercistronic region. Induction of PenA by imipenem was shown to be depe ndent upon the presence of PenR. Expression of the cloned B. cepacia p enA and penR genes in Escherichia coli SNO302 (ampD) resulted in a hig h basal and hyperinducible PenA activity. These results suggest that t he regulation of the PenA penicillinase of B. cepacia 249 is similar t o that observed in other class A and class C beta-lactamases that are under the control of a divergently transcribed AmpR-like regulator.