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Interplay of Different Transporters in the Mediation of Divalent Heavy Metal Resistance in Pseudomonas putida KT2440

Anu Leedjärv,^1,2* Angela Ivask,^2, and Marko Virta¹

Department of Applied Chemistry and Microbiology, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland,¹ Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia²

Received 17 September 2007/ Accepted 27 November 2007

According to in silico analysis, the genome of Pseudomonas putida KT2440 encodes at least four Zn/Cd/Pb efflux transporters—two P-type ATPases (CadA1 and CadA2) and two czc chemiosmotic transporters (CzcCBA1 and CzcCBA2). In this study we showed that all these transporters are functional, but under laboratory conditions only two of them were involved in the mediation of heavy metal resistance in P. putida KT2440. CadA2 conferred Cd²⁺ and Pb²⁺ resistance, whereas CzcCBA1 was involved in export of Zn²⁺, Cd²⁺, and possibly Pb²⁺. CadA1, although nonfunctional in P. putida, improved Zn²⁺ resistance and slightly improved Cd²⁺ resistance when it was expressed in Escherichia coli. CzcCBA2 contributed to Zn resistance of a czcA1-defective P. putida strain or when the CzcA2 subunit was overexpressed in a transporter-deficient strain. It seemed that CzcA2 could complex with CzcC1 and CzcB1 subunits and therefore complement the loss of CzcA1. The CzcCBA2 transporter itself, however, did not function. Expression of cadA1, cadA2, and czcCBA1 was induced by heavy metals, and the expression levels were dependent on the growth medium and growth phase. Expression of cadA2 and czcCBA1 was nonspecific; both genes were induced by Zn²⁺, Cd²⁺, Pb²⁺, Ni²⁺, Co²⁺, and Hg²⁺. On the other hand, remarkably, expression of cadA1 was induced only by Zn²⁺. Possible roles of distinct but simultaneously functioning transporters are discussed.

Published ahead of print on 7 December 2007.

Requests for strains should be addressed to A. Ivask (E-mail: angela{at}kbfi.ee).