Metatranscriptomic analysis of metal contaminated soils for isolation of metal tolerant genes

Abstract

Environmental pollution due to accumulation of potentially toxic metals is an imminent universal problem that uncompromisingly endangers human health, biodiversity and ecosystems. Accumulation of heavy metals into the soil environment results into deterioration of soil and water quality, thus affecting the soil environment and its biotic as well as abiotic factors. Soil is also known as a hot spot for flourishing microbial communities and for countless phenomenon and mechanisms actively combating the heavy metal toxicity. Many of members of these microbial communities cannot be easily isolated or cultured in vitro under traditional microbiological methods, hence to appreciate their true functional diversity and their activities expressed in situ in response to various adverse environments, a new omic approach i.e., metatranscriptomics is developed which involves the use of pool of mRNA directly extracted from environmental samples. Here in this work, the potential of metal polluted agro-forestry soil from Pierrelaye (PL), France, was exploited through metatranscriptomic approach to mine the novel genes responsible for metal tolerance/resistance and to know the diversity of polluted soil at the time of sampling. To achieve this, a pilot scale sequence-based and function-based metatranscriptomic analysis on soil samples from polluted site PL was done. In the present investigation, three size fractionated cDNA libraries from total RNA of metal contaminated soil was synthesized and screened for Cd tolerant genes by yeast complementation system using Cd sensitive ycf1Δ mutant. Here, a total of 35 cDNAs identified as unique good quality sequences are reported out of which four cDNAs with full length ORFs are further characterised for bioinformatic analysis, expression studies and multi-metal tolerance profiling. Each of the four cDNAs screened for Cd tolerance were further characterised as ubiquitin fusion protein (UFP)/ cDNA PLBe1, von Willebrand factor type D domain (VWD)/ cDNA PLCe10, heat shock protein 40 Type I like protein (Hsp40 Type I like protein)/ cDNA PLCc43 and phytochelatin synthetase (PC synthase)/ cDNA PLCd43 as through bioinformatic analysis. Furthermore, as the focus of the study is eukaryotic community present in the site thus the amplicons of hypervariable V4 regions of total 18S rDNA and cDNA derived from total 18S rRNA were also sequenced through Illumina-MiSeq (2 x 250 bp). Here, multiple sets of degenerate primers were used to gain a complete and more understanding of eukaryotic community structure and members of the sampling site. The NGS datasets retrieved were further analysed using Mothur pipeline after quality check via FastQC, Analysis was performed against SILVA reference database which primarily target V3-V4 region of 18S rRNA gene. It was reported that soil samples were dominated by the Metazoa and Fungi followed by other eukaryotic phylum such as Cercozoa, Conosa, Ciliophora etc. Hence, this study reported that amplicon sequences retrieved from soil were of eukaryotic origin and inferred that soil sample had a substantial impact on the soil eukaryotic diversity. Hence, the outcomes of this study i.e., eukaryotic origin metal tolerant cDNAs as well as amplicons sequencing depicts the efficiency of work strategy where both sequence based as well as function based metatranscriptomic approaches were exploited to focus on and exploit only eukaryotic microflora for various active genes and phenomenon active in the soil at the time of sampling.