Gene resources from metal polluted soils using metatranscriptomic approach

Abstract

A metatranscriptomic approach was employed to study the functional genes responsible for providing tolerance to toxic metals and also to isolate and clone them into appropriate hosts for their expression. In the first step, total RNA was extracted from metal polluted soils from India and France and cDNA was synthesized selectively from polyadenylated mRNA of eukaryotes utilizing the SMART (Switching Mechanism At 5' end of RNA) cDNA synthesis kit. cDNAs were fractionated into different sizes (A < 0.5 kb, B 0.5–1.0 kb, and C 1.0–4.0 kb) by 2-dimensional agarose gel electrophoresis. The size fractionated cDNAs were digested with SfiI restriction enzyme before ligation into yeast shuttle plasmid vector pFL61. The cDNA libraries A, B and C were initially screened for genes involved in providing tolerance to copper by functional complementation using the copper sensitive mutant cup1Δ which is sensitive to copper at 150 μM on SD-Ura medium. More than 5x106 clones were screened from library C and 155 clones were rescued from metal sensitivity. Some of the clones showing high tolerance to Cu were further sequenced and analyzed for their homology and characterized further. Apart from these, the clones rescued from contaminated soil from France were further characterized for its potential role in providing tolerance to Cu as well as other metals such as Cd, Zn and Co. Some of the genes characterized in this study are aldehyde dehydrogenase, serine protease inhibitor, DHHC palmitoyl transferase, maltose fermentation regulatory protein and chitin synthase. Functional complementation, growth response and intracellular metal accumulation studies were performed and it was found that the genes were able to confer tolerance to yeast mutants against toxic concentrations of wide range of metals like copper (150-1000 μM), cadmium (40-100 μM), zinc (10-13 mM) and cobalt (2-50 mM). The results suggested that the expression of these genes is a part of the response of the soil eukaryotic microorganisms in relation to toxic metal stress. Characterization reveals that they are directly or indirectly involved in countering toxic metals and mitigating their harmful effects. Also, to establish a link between the organisms possessing these genes and the functional eukaryotic population in the polluted soil, molecular diversity study by Next-Generation Sequencing (NGS) was performed. Diversity of eukaryotic communities of the soil sampling sites was studied by sequencing 18S rDNA amplicons by high throughput sequencing on Illumina HiSeq platforms using suitable eukaryotic universal primers. Data analysis and taxonomic assignments revealed that phylum Amoebozoa and Opisthokonta were the major players of the metal polluted soil. This study demonstrated that metatranscriptomic approach has been particularly useful in understanding metal polluted soil ecology and can be massively fruitful to access metal tolerant species and genes whose products are important in areas of bioremediation, biosensing and other important biotechnological fields.