Molecular Characterization of Microorganisms to Detoxify Mercury Pollutants and their Role in Enhancing Plant Growth

Abstract

Mercury pollution is a worldwide problem due to its toxicity to both human and animals. The level of mercury pollution in the environment is being increased day by day due to anthropogenic sources and activities like the discharge of industrial effluent from chlor-alkali industries, mining of metal and incineration of coal (Steenhuisen and Wilson, 2015). It is obvious that both forms of mercury (inorganic and organic) cause cytotoxic and neurotoxic effects to humans and animals. In plants, it poses adverse impacts at the cellular and subcellular level and their development. Therefore, there is an urgent need to realize the Hg-induced toxicity in humans and as well as in plants and its harmful effects by the consumption of contaminated nutrition. In this study, mercury resistant bacterial isolates were isolated from wastewater and heavy metal polluted soils collected close to tanneries of district Kasur, Itehad chemicals limited (pvt), Kala Shah Kaku, district Sheikhupura and Rohi-Nala, district Lahore, Pakistan. Eight out of 120 bacterial strains were screened out on the basis of nitrogen fixing ability, high level of Hg resistance (10-60μg/ml), H2S production and indole-3-acetic acid (IAA) production (4-40μg mL-1). Biochemical characterization, short sequence repeats (SSR) fingerprinting, 16S ribotyping and phylogenetic analysis characterized the selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478), Bacillus cereus AZ-3 (KT270479), Bacillus thuringiensis AZ-5 (KJ675627) Enterobacter sp. AZ-15 (KU558920), Salmonella enterica Z-A14 (KJ728670), Enterobacter cloacae Z-A15 (KJ728671), Pseudomonas putida Z-A22 (KJ728678) and Enterobacter asburiae Z-A20 (KJ728676). Phylogenetic relationship on the basis of merA and merB nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA and merB gene of already reported mercury resistant Gram positive bacteria. Restriction fragment length polymorphism (RFLP) analysis was applied to the amplification products of 16S rRNA, merA and merB genes (≈ 1.5, 1.3 and 0.5 kb) and specific restriction patterns were obtained after treatment with endonucleases EcoR1, Taq1, HinF1 and HaeIII. M A small scale reservoir containing Luria Bertani (LB) medium supplemented with 20 μg mL-1 and industrial effluent with same concentration of mercury (Hg+2), were designed to check the detoxification ability of selected isolates. It was found that 76% to 83% of mercury was detoxified by Bacillus spp. Scanning electron microscopic (SEM) analysis confirmed the deposition of HgS on the surface of bacterial cell membranes. Mercury resistant bacteria were immobilized in sodium alginate and checked for their capability to detoxify mercury from industrial effluent. Furthermore, Hg-resistant bacterial cells were grown in LB medium and genomic DNA was extracted by commercially available kit. The merF and merE genes (both are involved in Hg+2 transportation through bacterial cell membrane) were amplified by PCR from genomic DNA by using forward and reverse primers. In case of merE gene, the designed primers containined the designed restriction sites of restriction endonuclease enzymes, SacI and HindIII, respectively. The primers were designed to introduce a SacI site at the start of the mer genes and a HindIII site at the end. The PCR product was digested with SacI and HindIII and ligated into similarly digested pHLV vector. A thrombin cleavage site was inserted into pHLMerE before merE gene sequence due to the presence of methionine genetic codons in merE gene sequence. In case of merF gene, the designed primers containined the designed restriction endonuclease enzymes, Xhol and Sphl sites respectively. The recombinant plasmids HistagtrpΔLE- thrombin site-merE (pHLMerE vector) and KSI-MerFm-Histag (pET31b+ vector) where cysteine residues of MerFwt were mutated with serine in MerFm by site directed mutagenesis, were transformed into competent DH5α E. coli cells. Successful transformants were screened by colony PCR using same primers. The DNA sequence was confirmed by the dideoxy sequencing method. For both genes, the constructed both supercoiled plasmids were isolated from the DH5α strain and retransformed into C43(DE3) E. coli over expressed cells which grow well in the minimal medium used for isotopic labeling. The expression of merFwt (wild type) and merFm (mutated/modified) was obtained in LB, minimal media and isotopically labeled M9 medium. Expression of the fusion protein was induced by adding IPTG. The inclusion bodies containing the fusion protein were separated by centrifugation and then solubilized in the binding buffer. Native MerE and Summary xi MerF proteins were purified by FPLC. The polypeptide was monitored on each step by SDSPAGE. Samples (MerFm protein) for 1H-15N 2D HSQC (Heteronuclear Single-Quantum Correlation) experiment were prepared by resolublizing lypholized protein in NMR buffer. Hydrogen-deuterium fractionation experiment was done with different fractions of D2O. The number, placement and symmetry of peaks of HSQC NMR spectra confirmed the structural MerFm. Moreover, Hg-resistant bacteria were checked for their abilities of nitrogen fixation and phosphate and potassium solubilization qualitatively. IAA produced by selected bacteria was confirmed qualitatively and quantitatively by colorimetric method, TLC, HPLC and GC-MS analysis. Finally, mercury detoxification and IAA producing bacteria were inoculated in mercury amended soil which resulted significance increases in seed germination, shoot length, root length and shoot fresh weight, root fresh weight, number of pods per plant, number of seeds and weight of seeds respectively of chickpea (Cicer arietinum L.) in pot experiments. The bacterial consortium of Hg-resistant and IAA producing bacteria was also inoculated in non-mercury amended soil and the significant increase in yield of wheat (Triticum aestivum L.) and masoor (Lens culinaris L.) was observed in fields. In conclusion, selected bacteria used in this study as a consortium A7(Bacillus sp. AZ-1, Pseudomonas putida Z-A22 and Enterobacter cloacae Z-A15), may be used as an excellent biofertilizer for decreasing mercury pollution and enhancing plant growth in mercury contaminated soil to make a sustainable agricultural land.