Reverse transcriptase-real time PCR analysis of heavy metal stress response in a uranium resistant Pseudomonas aeruginosa strain isolated from Jaduguda uranium mine

A multimetal resistant Pseudomonas strain isolated from a uranium mine waste site of Jaduguda, India, was characterized for its potential application in bioremediation. Nearly complete 16Sr RNA gene sequence and fatty acid methyl ester analyses confirmed the identity of this bacterium as Pseudomonas aeruginosa. This bacterium exhibited high U-resistance i.e. up to an exposure of 6 h in 100 mg UL-1 solution (pH 4.0) and accumulation (maximum of 275 mg Ug-1 cell dry wt.) properties. Microcosm studies further proved the ability of the strain to remove soluble uranium (99%) from U-mine effluent and sequester it as U oxide and phosphate minerals while maintaining its viability. Considering the survival of this strain in U-mine site co-contaminated with other heavy metals, genetic basis of metal resistance was investigated. The bacterium was resistant to 3, 2 or 6 mM of Cu, Cd, or Zn, respectively. Polymerase chain reaction based detection followed by sequence identity and phylogenetic analysis revealed presence of specific metal resistance genes copA (copper resistance determinant) and czcA (RND type heavy metal efflux) in this isolate. Real-time PCR expression studies of these genes indicated significantly increased expression of both the genes in response to Cu, Cd, or Zn. Maximum up regulation of copA and czcA genes was observed following exposure (30 mm) to 25 μm of Cu or 10 μm Cd respectively. High levels of mRNA transcripts of copA and czcA genes in response to specific metals suggest that these resistance systems have important role in conferring metal resistance to the bacterium. Response of sodA an antioxidant Mn-cofactored superoxide dismutase gene to metal stress revealed that induction of this stress gene was not evident at lower concentration(s) of metals, the concentration(s) that cause maximum up- regulation of metal resistance genes. Higher test metal concentration or extended period of exposure, however, resulted in expression of sodA gene. The observed difference in transcriptional response could possibly be explained in light of role of heavy metals in their regulation. Metal resistance genes tend to provide cellular defense specifically at low concentration; while at higher metal levels strategies that are more general are adopted. In contrast, sodA gene is activated by superoxide free radical and not directly by metal cations, therefore shows increased expression only when the metal concentration cross certain threshold that generate enough reactive oxygen species by the interaction of metals and cellular components. Therefore it can be suggested that these metal responsive genes may serve as potential biomarkers to determine physiology and abundance of this bacterium when used for bioremediation activities in sites heavily contaminated with metallic pollutants. (author)