The microbiota inhabiting in metal polluted environment develops strong defense mechanisms to combat pollution and sustain life. Investigating the functional genes of the eukaryotic microbiota inhabiting in these environments by using metatranscriptomics approach was the focus of this study. Size fractionated eukaryotic cDNA libraries (library A, < 0.5 kb, library B, 0.5-1.0 kb, and library C, > 1.0 kb) were constructed from RNA isolated from the metal contaminated soil. The library C was screened for Cadmium (Cd) tolerant genes by using Cd sensitive yeast mutant ycf1Δ by functional complementation assay, which yielded various clones capable of growing in Cd amended media. One of the Cd tolerant clones, PLCg39 was selected because of its ability to grow at high concentrations of Cd. Sequence analysis of PLCg39 showed homology with DHHC palmitoyl transferases, which are responsible for addition of palmitoyl groups to proteins and usually possess metal coordination domains. The cDNA PLCg39 was able to confer tolerance to Cd-sensitive (ycf1Δ), Copper-sensitive (cup1Δ) and Zn-sensitive (zrc1Δ) yeast mutants when grown at different concentrations of Cd (40-100 μM), Cu (150-1000 μM) and Zn (10-13 mM), respectively. The DHHC mutant akr1Δ transformed with PLCg39 rescued from the metal sensitivity indicating the role of DHHC palmitoyl transferase in metal tolerance. This study demonstrated that PLCg39 acts as a potential metal tolerant gene which could be used in bioremediation, biosensing and other biotechnological fields.
Keywords: Cadmium; Metal tolerance; Metatranscriptomics; Palmitoyl transferase; Zinc finger, Yeast complementation.