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Molecular characterization of an operon, cueAR, encoding a putative P1-type ATPase and a MerR-type regulatory protein involved in copper homeostasis in Pseudomonas putida

Department of Biological Sciences, Science Drive 4, National University of Singapore, Singapore117543¹

Author for correspondence: Sanjay Swarup. Tel: +65 874 7933. Fax: +65 779 2486. e-mail: dbsss{at}nus.edu.sg

The authors have characterized a chromosomally localized two-gene operon, cueAR, which encodes a putative P1-type ATPase, CueA, and a MerR-type metalloregulatory protein, CueR, in Pseudomonas putida PNL-MK25. Disruption of cueAR by the insertion of mini-Tn5::gfp into the wild-type strain led to a mutant strain with a sixfold reduction in its tolerance to copper; however, the tolerance of this mutant strain to the other seven related transition metals tested was not affected. The sensitivity of the mutant strain was attributed to a higher level of accumulation of intracellular copper, suggesting the involvement of CueA in copper export. Insertion of the cloned cueAR operon into the copper-sensitive mutant strain fully restored its tolerance to copper. cueA::gfp expression studies confirmed that the cueAR operon was transcriptionally regulated by copper and CueR. Studies done on the mutant strain complemented with cueR and cueA revealed partial functional redundancy of cueA and cueR, respectively, in copper tolerance. Thus, the results of this study clearly suggest the involvement of cueAR in copper homeostasis in P. putida.

Keywords: copper tolerance, CPx-type ATPase, mini-Tn5::gfp mutagenesis

Abbreviations: GFP, green fluorescent protein; MIC, minimal inhibitory concentration

The GenBank accession number for the sequence reported in this paper is AF390440.

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