Interplay of different transporters in the mediation of divalent heavy metal resistance in Pseudomonas putida KT2440

Abstract

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 Cd(2+) and Pb(2+) resistance, whereas CzcCBA1 was involved in export of Zn(2+), Cd(2+), and possibly Pb(2+). CadA1, although nonfunctional in P. putida, improved Zn(2+) resistance and slightly improved Cd(2+) 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 Zn(2+), Cd(2+), Pb(2+), Ni(2+), Co(2+), and Hg(2+). On the other hand, remarkably, expression of cadA1 was induced only by Zn(2+). Possible roles of distinct but simultaneously functioning transporters are discussed.

FIG. 1.

Effects of cadmium (A) and lead (B) on the growth of P. putida KT2440 transporter-deficient strains. Dose-response curves for P. putida wild-type strain KT2440 (▪), czcA1 mutant strains KT2440.1, KT2440.21, KT2440.13, and KT2440.231 (▴), cadA2 mutant strains KT2440.4, KT2440.24, KT2440.34, and KT2440.243 (▾), and czcA1 cadA2 double-mutant strains KT2440.14, KT2440.241, KT2440.143, and KT2440.2431 (⧫) are shown. The relative growth indicates the growth efficiency in the presence of a metal compared to the growth in medium without the metal. The data are the means ± standard deviations of at least three independent experiments.

FIG. 2.

Effects of cadmium (A) and zinc (B) on the growth of the P. putida KT2440.2431 transporter-deficient strain complemented with transporter genes from a temperature-inducible plasmid. Dose-response curves for control strain P. putida KT2440.2431(pTIHis) (▴), a strain complemented with the cadA2 gene [P. putida KT2440.2431(pTIcadA2His)] (▪), and a strain complemented with the czcA2 gene [P. putida KT2440.2431(pTIczcA2His)] (•) grown at 28°C (solid lines) and 37°C (dashed lines) are shown. The relative growth indicates the growth efficiency in the presence of a metal compared to the growth in medium without the metal. The data are the means ± standard deviations of at least three independent experiments.

FIG. 3.

Effects of zinc (A) and cadmium (B) on the growth of the E. coli TH201 transporter-deficient strain overexpressing P. putida KT2440 transporter genes from a temperature-inducible plasmid. Dose-response curves for control strain E. coli TH201(pTIHis) (▴), a strain overexpressing the cadA2 gene [E. coli TH201(pTIcadA2His)] (•), and a strain overexpressing the cadA1 gene [E. coli TH201(pTIcadA1His)] (▪) grown at 37°C are shown. The relative growth indicates the growth efficiency in the presence of a metal compared to the growth in medium without the metal. The data are the means ± standard deviations of at least three independent experiments.