BIDIRECTIONAL PROMOTER

Operon mer

Over the ages, exposure to toxic elements enabled microorganisms to develop highly complex and efficient systems to overcome poisonous environmental conditions.. As an example of those systems, there is an operon (Mer operon), which function is to transform, by enzymatic reduction, mercury Hg2+ into Hg0, its volatile form. Operon Mer has been found in a wide range of bacteria that differ on number of genes involved. Those are usually located on plasmids and chromosomes and normally components of transposons and integrons. Mer operons can have different structure and are constituted by coding sequence of functional protein for regulation (MerR), transport (MerT, MerP and/or MerC, MerF) and reduction (MerA).

Transport: MerT and MerP transporter proteins

Transport of Hg in bacteria involves a complex system based in chemical interactions by cysteine residues. First, in periplasmic space Hg2+ is binded to a pair of cysteine residues on MerP protein. Then, it is transferred to another pair of cysteine residues on MerT, a cytoplasmic membrane protein. Finally, it is transferred to a cysteine pair at the active site of MerA (mercuric reductase), as represented below:

Regulation: MerR

MerR protein is responsible by regulation of mer proteins expressions. MerR binds to a region called MerO (operator) which is located upstream merT gene. MerO is between -10 and -35 region to RNA polymerase recognition site for the merPT promoter, which are structural genes. MerR itself has its own promoter (PmerR) that reads differently from merPT promoter.

Under Hg2+ influence, MerR binds to it, causing allosteric change in the protein, moving it from the operator region and allowing RNA polymerase access to transcriptional start site and start proteins transcription, regulating the bidirectional promoter depending on presence or absence of Hg, because MerR gene negatively regulates its own synthesis. It also has a really interesting characteristic to form a stable pre initiation complex with RNA polymerase, making it able to promptly answer to Hg presence in cytosol and starting transcription process.

Another remarkable characteristic of MerR is how it binds to Hg. The binding site is a homodimer and involves one cysteine in one monomer and two different cysteines on the other monomer. Unlikely other metalloenzymes that make strong bonds with their metal cofactors, MerR is capable of making strong but temporary binding to a metal ion.

This surprising mechanism of regulation and transport from Mer Operon inspired us to construct a treatment system (including biosensor, bioaccumulation and bioremediation) of mercury utilizing it. For this, we developed the key piece of all genetic constructions related to Hg! Check it out the Essential Biobrick!

Our construction: How it works?

To develop our biosensor, bioremediator and bioaccumulator in Escherichia coli DH5-alpha we designed a biobrick to be the key piece of many genetic construction related to mercury. The Essential Biobrick (BBa_K135001) is composed by the mer bidirectional promoter, having dual function: A) In reverse: transcription of the MerR regulator protein; and B) In forward: transcription of the MerP and MerT proteins, as represented below:

In mercury absence, MerR forms a MerR-promoter-operator complex, preventing RNA polymerase to recognize the promoter, consequently, messengers RNA MerPT will not be transcript. In presence of Hg2+, MerR protein binds to this element and dissociates from the promoter-operator complex, allowing MerPT expression.

Essential biobrick (BBa_K1355001) doesn’t have a transcription terminator, because it was made to be attached to others coding regions that permit a new function for bacteria. For this project, we used Escherichia coli DH5-alpha with Essential Biobrick attached to encoding region for Green Fluorescent Protein (BBa_E0840) to build a biosensor; as well as encoding region for Metal Binding Peptide (BBa_K346004) to build a bioaccumulator; and encoding region to mercury reductase (BBa_K1355000).

Composing the parts:

References

Barkay, T., Miller, S. M., & Summers, A. O. (2003). Bacterial mercury resistance from atoms to ecosystems. FEMS microbiology reviews, 27(2‐3), 355-384.

BIONDO, R. Engenharia Genética de Cupriavidus metallidurans CH34 para a Biorremediação de efluentes contendo Metais Pesados. 2008. São Paulo, Brasil.

Nascimento, A. M., & Chartone-Souza, E. (2003). Operon mer: bacterial resistance to mercury and potential for bioremediation of contaminated environments. Genetics and Molecular Research, 2(1), 92-101.

DASH, H. R.; DAS, S. Bioremediation of mercury and the importance of bacterial mer genes. 2012. International Biodeterioration & Biodegradation 75: 207 – 213.

HAMLETT, N. V.; et al. Roles of the Tn21 merT, merP and merC Gene Products in Mercury Resistance and Mercury Binding. 1992. Journal of Bacteriology 174: 6377 – 6385.

PINTO, M. N. Bases Moleculares da resistência ao Mercúrio em bactérias gram-negativas da Amazônia brasileira. 2004. Pará, Brasil.