Pollution in the environment due to accumulation of potentially toxic metals results in deterioration of soil and water quality, thus impacting health of all living organisms including microbes. In the present investigation, a functional metagenomics approach was adopted to mine functional genes involved in metal tolerance from potentially toxic metal contaminated site. Eukaryotic cDNA library (1.0-4.0 kb) was screened for the genes providing tolerance to cadmium (Cd) toxicity through a functional complementation assay using Cd-sensitive Saccharomyces cerevisiae mutant ycf1 Δ . Out of the 98 clones able to recover growth on Cd-supplemented selective medium, one clone designated as PLCc43 showed more tolerance to Cd along with some other clones. Sequence analysis revealed that cDNA PLCc43 encodes a 284 amino acid protein harbouring four characteristic zinc finger motif repeats (CXXCXGXG) and showing partial homology with heat shock protein (Hsp40) of Acanthamoeba castellanii. qPCR analysis revealed the induction of PLCc43 in the presence of Cd, which was further supported by accumulation of Cd in ycf1 Δ /PLCc43 mutant. Cu-sensitive (cup1 Δ ), Zn-sensitive (zrc1 Δ ) and Co-sensitive (cot1 Δ ) yeast mutant strains were rescued from sensitivity when transformed with cDNA PLCc43 indicating its ability to confer tolerance to various potentially toxic metals. Oxidative stress tolerance potential of PLCc43 was also confirmed in the presence of H2O2. Present study results suggest that PLCc43 originating from a functional eukaryotic gene of soil community play an important role in detoxification of potentially toxic metals and may be used as biomarker in various contaminated sites.
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Heavy metal pollution poses a serious threat to soil, water, and the atmospheric environment. Many microbes sustain and respond to the metal stress, and the mechanisms involved in these processes remain unclear. The metagenomics and metatranscriptomics approaches were applied to study the structure and function of eukaryotic microbial communities in heavy metal-contaminated soils of two geographic locations situated in different climatic regions. To achieve this, amplicons of the hypervariable V4 region of 18S rDNA and cDNA synthesized from 18S rRNA extracted from these soils were generated and sequenced through paired-end sequencing chemistry on the Illumina-MiSeq platform. The NGS dataset processed by the Mothur pipeline and analyzed by Parallel-Meta 3 pipeline illustrated the presence of all the major eukaryotic phyla. Taxa diversity and community structure of micro-eukaryotes within and between the samples from two locations were compared. Clustering, heatmap, and PCA analysis supported the variation in taxonomic diversity and community structure in the datasets of these two sites. Analysis of taxa abundance in both sites identified marker organisms for the further characterization of such types of environments. A functional metatranscriptomics study revealed the identification of various expressed eukaryotic genes, which are involved in metal tolerance. These metal-tolerant gene families were phylogenetically related to the different eukaryotic lineages reported in the metagenomic analysis. Hence, this approach could be widely applied in microbial ecology to understand the role of active microbes in such specific environmental conditions.
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