Determination Of Zn²⁺-resistant Function Of P-type ATPase Gene CadA1 In Pseudomonas Putida CD2

The cadA1 gene encodes a P-type ATPase in P. putida. The P-type ATPases are a family of transporters that mediate the uptake and extrusion of heavy metal ions. But little is known about the function of cadA1 in heavy metal resistance. The aim of the present work is to determine if the cadA1 gene is involved in tolerance to heavy metal ions in P. putida CD2.In this study, a mutant strain of P. putida CD2 was constructed by disrupting cadA1. The function of cadA1 was studied by determining the resistance of the cadA1 disrupted strain to different heavy metal ions. But no difference in the MIC was observed between P. putida CD2-1 and CD2. It was shown that the cadA1 disrupted strain did not change the ability in resistance to Cd²⁺,Zn²⁺,Co²⁺,Pb²⁺,Cu²⁺ and Mn²⁺ compared with the wild type.For determining the effect of CadA1 on heavy metal resistance, the cadA1 gene was A1so overexpressed in heavy metal-sensitive mutants of P. putida CD2. The overexpression of cadA1 increased resistance to Zn²⁺ in the czcCBA1 disrupted mutant of P. putida CD2. It indicates that the cadA1 gene confers Zn²⁺ resistance which has not been proven by disrupting cadA1, suggesting that the cadA1 gene is functional, but may be regulated by some unknow factors in P. putida CD2 that it cannot exert its function in heavy metal resistance.Further more, the regulation of cadA1 expression was studied by GFP fluorescence assay. A reporter plasmid pEGP-PCAD was constructed and used to monitor the expression of PcadA1 in response to heavy metal ions in P. putida CD2,CD2-2 and CD2-3. It showed that PcadA1 was specific, responding at a significant level to Zn²⁺ in P. putida CD2-2. But there was A1most no expression of PcadA1 in P. putida CD2 or CD2-3 when induced by heavy metal ions. It reveals that expression of cadA1 is induced by Zn²⁺ and this induction is inhibited by the presence of CzcCBA1 in P. putida CD2.In combination of the results obtained, it was demonstrated that cadA1 can confer resistance to Zn²⁺, but the level of CadA1 in P. putida CD2 is so low because of the presence of CzcCBAl that it contributes little to Zn²⁺ resistance. However, expression of cadA1 would increase to serve as a salvage pathway for Zn²⁺ resistance when czcCBA1 disrupted. To the best of our knowledge, this work has provided the first evidence that the cadA1 gene confers Zn²⁺ resistance in P. putida CD2.