Comparative study of merA genes in arabidopsis for phytoremediation

Mercury is a highly potent neurotoxin impacting the function and development of the central nervous system in people and wildlife. Exposure to it in the form of vapours or organic methylmercury leads to neurological and behavioural issues. Moreover, mercury exposure is toxic to the digestive system, organs and the immune system (World Health Organization, 2017). Mercury pollutants in the environment are present in elemental, inorganic and organic forms. Global heavy metal contamination are increasing in soil due to industrialisation. Therefore, phytoremediation processes using plants to eradicate heavy metals provide efficient and ecologically sound approaches to sequestration.  This article establishes several merA gene candidates for mercury phytoremediation purpose.

Mer operon and its application in remediation of mercury

A wide range of Gram-positive and Gram-negative bacteria have a unique way of resisting and subsequently transforming toxic forms of mercury to nontoxic forms. The mer operon is found in certain bacteria. It confers them resistance to mercury by reducing toxic mercuric compounds to volatile mercury. The operon includes several functional genes along with promoter, regulator and the operator gene. This is shown in image 1 below.

Typical mer operon found in microbes

Typical mer operon found in microbes

However, not all organisms possess all the genes of the operon. The mer operon can specify either narrow spectrum or broad spectrum resistance to that organism (Dash and Das, 2012). Narrow spectrum is capable of remediating inorganic mercury compounds, whereas broad spectrum remediates organomercuric and inorganic compounds. Moreover, merA and merB genes give this characteristic spectrum resistance since they are involved in functional reduction of mercuric compounds. The functional genes merA and merB are code for mercuric reductase and organomercurial lyase respectively (Heidenreich et al., 2008). The mercuric reductase enzyme is responsible for NADPH+ assisted reduction of Hg(II). Furthermore, the lyase is responsible for reducing organomercurial compounds with the help of the enzyme reductase. Some organomercurial compounds are methylmercury and phenyl mercuric acetate. Besides these main functional genes, other genes involved are, regulators (merR and merD) and transporter proteins (merP, merT and sometimes merC) (Tangahu et al., 2011).

This operon confers mercury resistance by reducing Hg ions to volatile mercury. This is then released into the atmosphere. Therefore, the mer operon poses as a highly advantageous candidate in mercury bioremediation studies.

merA genes in phytoremediation

As established, mer operon is found in bacteria and archaea groups. This gives them a wide range of mercury resistance. Scientists in the past have cloned merA genes into other bacteria along with certain plants to use them. This is because they act as remediation agents in cleaning up mercury contaminated environments. The table below lists different studies that have cloned bacteria-sourced merA genes into different plants.

No. Author/ Year Gene cloned Mercury Volatilization Plant Species used
1. Lyyra et al., (2007) merA and merB 95 ng mercury Eastern cottonwood.
2 Hussein et al., (2007) merA and merB 7 µg Tobacco.
3 Haque et al., (2010) merA 467 µg Tobacco.
4 Im Choi et al., (2007) merA 12 µg Populus sp.
3. Che et al., (2003) merApe9 (variant of merA) 900 µg Populus deltoides.

Systematic review of merA genes used in phytoremediation studies

The efficiency of merA function in transgenic plants was assessed on mercury volatilization capacity. As seen from the above table, the highest efficiency of mercury volatilization in a particular plant by merA gene alone was in Populus deltoids at 900 µg/gm of leaf per minute. Che et al., (2003) have used the same modified merA sequence (MerApe9), as used by Rugh et al., (1996). Also, they conducted one of the first merA related mercury phytoremediation studies.

Establishing merA gene candidates for phytoremediation

These candidates were established based on their homology with the primary gene variant, merA of E.coli which was modified into merApe9 gene by Rugh et al., (1996). Using BLAST tool, the original gene merA nucleotide sequence was searched against the NCBI database. Following that, the top 10 homologous sequences (output) were studied. These homologous sequences can also be explored further for their phytoremediation potential.

Data analysis

BLAST results of the merA gene sequence revealed 100 closely related matches from the NCBI database. The closest one was sequence from a plasmid (Figure 2) that contained the entire mer operon expressed first in E.coli (Barrineau et al., 1984).

Accession number: K03089

Gene description:  Plasmid NR1 mercury resistance (mer)operon

Gene type: Protein coding

Organism: E.coli

The data was taken from GenBank.

Graphical output of BLAST analysis of merA sequence of interest

Graphical output of BLAST analysis of merA sequence of interest

The most similar sequence, belonging to NR1 plasmid of E.coli, is 93% identical to the query sequence, with a low E-value score (Image 2). The next 10 sequences that matched the query merA sequence belonged to 5 different species apart from E.coli. All of them matched to 92% identity. These include, Klebsiella, Citrobacter, Alcaligenes, Salmonella, and Providencia spp. They all showed same E-value as well as Identity score of 92%.

BLAST analysis of merA sequence of interest

BLAST analysis of merA sequence of interest

 

Inference

The matched sequences belonged to either plasmids or complete sequences, where none of them have been clearly annotated as merA genes. This implies that there is a need for further evaluation and thorough investigation of these sequences and their functions. In case of the first matched sequence, E.coli NR1 plasmid (Accession no. K03089), it is a mer operon plasmid consisting of merA, merR and merT coding regions. As shown in Table below, all the plasmids and complete genome have been annotated to recognise coding regions and genes. MerA genes are found in all the matches, except for KY749247 (S.enterica) which has not been annotated.

Accession No. Sequence Description mer genes present
K03089 (E.coli) Plasmid NR1 mercury resistance (mer) operon merA, merR, merT.
 CP018625 (E.coli) Plasmid pFORC44_1 (complete sequence) merA, merE.
KX832927 (P.rettgeri) Plasmid p16Pre36-NDM (complete sequence) merR, merT, merP, merC, merA, merD, merE.
CP022348 (K.michiganensis) Strain K516 chromosome (complete sequence) merR, merD, merA, merC, merP.
KY887594 (K.pneumoniae) Strain Kp55 plasmid pKp55 (complete sequence) merE, merD, merA, merC, merP, merT, merR.
KY887593 (C.freundii) Strain Cf53 plasmid pCf53 (complete sequence) merE, merD, merA, merC, merP, merT, merR.
KY887592 (C.freundii) Strain Cf52 plasmid pCf52 (complete sequence) merE, merD, merA, merC, merP, merT, merR.
KY749247 (S.enterica) Plasmid R1 (complete sequence) No genes annotated
CP019649 (S.enterica) Salmonella enterica subsp. enterica serovar Paratyphi B plasmid R1 merE, merD, merA, merC, merP, merT, merR.
CP021212 (E.coli) Strain B171, plasmid pB171_90 (complete sequence) merE, merD, merA, merC, merP, merT, merR.
KY623659 (A.faecalis) Strain GZAF1 plasmid pGZAF1_VIM merC, merA, mercuric transporter, transporter periplasmic component.

mer genes present in matched BLAST sequences

Further detailed analysis of individual sequence alignment revealed that a 12-bp long sequence present in query sequence was absent in the matched sequences. The sequence 5’-GCCGCATTCCGC-3’ was not found in any of the matched sequences. This could suggest that this section of the gene is not easily found and hence does not contribute to the enzyme’s functioning. The lack of this sequence section has contributed majorly towards the similarity scores of the matched sequences with the query sequence.

Further research on protein sequences

Consequently, the 11 sequences found to be highly similar to the original merA gene, suggests that this variant is commonly found  in microbes and is highly conserved. These sequences can be studied and modified further to derive other variants that show significantly higher mercury reduction capacity. More importantly, these sequences need be studied with respect to expressing efficiently in different plant systems, for commercial application of phytoremediation methods. In the next article, multiple sequence alignment studies of the protein sequence data of the selected merA gene of interest is studied. Through MSA, the conserved regions of the enzyme can be determined. It will shed light on the mode of action of the enzyme.

References

  • Barrineau, P., Gilbert, P., Jackson, W. J., Jones, C. S., Summers, A. O. and Wisdom, S. (1984) ‘The DNA sequence of the mercury resistance operon of the IncFII plasmid NR1’, Journal of Molecular and Applied Genetics, 2(6), pp. 601–619.
  • Che, D., Meagher, R. B., Heaton, A. C., Lima, A., Rugh, C. L. and Merkle, A. (2003) ‘Expression of mercuric ion reductase in Eastern cottonwood (Populus deltoides) confers mercuric ion reduction and resistance.’, Plant Biotechnology Journal, 1(4), pp. 311–319.
  • Dash, H. R. and Das, (2012) ‘Bioremediation of mercury and the importance of bacterial mer genes’, International Biodeterioration and Biodegradation. Elsevier Ltd, 75(November), pp. 207–213. doi: 10.1016/j.ibiod.2012.07.023.
  • Haque, S., Zeyaullah, M., Nabi, G., Srivastava, P. and Ali, A. (2010) ‘Transgenic tobacco plant expressing environmental E. coli merA gene for enhanced volatilization of ionic mercury’, Journal of Microbiology and Biotechnology, 20(5), pp. 917–924.
  • Heidenreich, B., Mayer, K., Sandermann, H. and Ernst, D. (2001) ‘Mercury-induced genes in Arabidopsis thaliana: Identification of induced genes upon long-term mercuric ion exposure’, Plant, Cell and Environment, 24(11), pp. 1227–1234. doi: 10.1046/j.0016-8025.2001.00775.x.
  • Hussein, H., Ruiz, O. N., Terry, N. and Daniell, H. (2007) ‘Phytoremediation of mercury and organomercurials in chloroplast transgenic plants: enhanced root uptake, translocation to shoots, and volatilization.’, Environmental Science & Technology, 41(24), pp. 8439–8446.
  • Im Choi, Y., Noh, E. W., Lee, H., Han, M, Lee, J. and Choi, K. (2007) ‘Mercury-tolerant transgenic poplars expressing two bacterial mercury-metabolizing genes’, Journal of Plant Biology, 50(6), pp. 658–662.
  • Lyyra, S., Meagher, R. B., Kim, T., Heaton, A., Montello, P., Balish, R. S. and Merkle, S. A. (2007) ‘Coupling two mercury resistance genes in Eastern cottonwood enhances the processing of organomercury’, Plant Biotechnology Journal, 5(5), pp. 254–262.
  • Rugh, C. L., Wilde, H. D., Stack, N. M., Thompson, D. M., Summers, A. O. and Meagher, R. B. (1996) ‘Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene’, Proceedings of the National Academy of Sciences, 93(8), pp. 3182–3187.
  • Tangahu, B. V, Abdullah, A. R. S., Basri, H., Idris, M., Anuar, N. and Mukhlisin, M. (2011) ‘A Review on Heavy Metals (As, Pb, and Hg) Uptake by Plants through Phytoremediation’, International Journal of Chemical Engineering, 2011, p. 31. doi: 10.1155/2011/939161.
  • World Health Organization (2017) Mercury and Health, World Health Organization. Available at: http://www.who.int/mediacentre/factsheets/fs361/en/.
Chandrika Kapagunta

Chandrika Kapagunta

Research Analyst at Project Guru
Chandrika is a nature enthusiast with special love for the marine world. Her Master’s degree in Marine Biotechnology and Scuba Diving experience has made her a strong advocate of environment and marine conservation, especially through bioremediation. She believes in finding solutions of everyday human problems in nature, be it medicines, technology or philosophy. Having worked as a volunteer at The Bombay Natural History Society and as a Senior Research Fellow at Central Institute of Fisheries Education, she has had exposure to the current state of the academic research, specifically in the field of environmental biotechnology.
Chandrika Kapagunta

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