Isolation And Characterization Of A Cadmium-Resistant Pseudomonas Putida And Cloning Of Key Genes Involved In Cadmium Resistance

A cadmium resistance bacteria was isolated from sewage sludge samples collectedin WuHan Steel Factory. The strain was identified as Pseudomonas putida bymorphological observation, plentiful physiological-biochemical experiments and 16SrDNA sequence analysis, named P. putida CD2. Maximal tolerant concentrations (MTCs)for the strain were determined. Strain CD2 exhibited high MTC values for a largespectrum of divalent metals.12000 mutants were obtained by using Tn5-B21 insertion mutagenesis, 12 mutantsshowed substantial decrease in resistance to cadmium by screening the library. The DNAsequences of the contiguous region from the Tn5 insertion sites were determined byinverse PCR. Six genes involved in cadmium resistance were identified: cadA2, czcC1,czcB1, czcA1, colR and colS. The last five genes belonged to two operons, czcCBA1 andcolRS, respectively.Disruption of czcCBA1 in Pseudomonas putida CD2 severely impaired theresistance to divalent heavy metal ions, especially to Cd²⁺, Zn²⁺ and Co²⁺. The threeproteins, CzcC1, CzcB1 and CzcA1 made up of a protein-complex spanning the entirecell wall. As a cation-proton antiporter, the protein-complex transfered excessive metalions to outside without energy providing. Disruption of cadA2 in P. putida CD2 onlyresulted in the sensitivities to Cd²⁺, Zn²⁺ and Pb²⁺. cadA2 encoded a P-type ATPase. Byhydrolyzing the ATP, the protein structure was changed and formed cation channel, thencarried the metal ions to Periplasmic space. The homologs of czcCBA1 and cadA2 hadbeen reported in other metal-resistant bacteria.coIRS was a typical Histidine kinase two-component signal transduction (TCST)system, and played a functional generalist, which was concerned with root-colonizingability, communication of bacteria and regulation of Tn4652 transposition. In this work,we presented a novel function of colRS, which regulated the metal resistance orhomeostasis. Disruption of colRS resulted in the substantial decrease of divalent metalresistance, especially for Mn²⁺, the novel fuction had been identified by geneticcomplemention.Utilizing the promoterless lacZ gene on Tn5-B21, the metal ions inducibleexpressions of the three genes could be assessed by mensurating theβ-Galactosidaseactivities. Zn²⁺ was the most effective inducer for induction of PczcC1, the induction wasabout 85 fold in the wild-type background, and Cuv was also effective for czcC1, cadA2was distinctly induced by Cd²⁺, Pb²⁺ and Zn²⁺ with the following order of effectiveness: Cd²⁺≥Pb²⁺≥Zn²⁺. The induction of PcolR was unobvious.The aforementioned results and conclusions might be scientific significance in theresearch of the metal resistant mechanisms in bacteria.