Team:Peking/Project/Bioabsorbent/FacilitationModule

From 2010.igem.org

Facilitation module

As previously shown in the ‘biosensor’ part, the PmerT promoter is a divergent promoter, and it regulates both the upstream merR’s expression level and the downstream Mer operon’s expression. In detail, the mer operon contains several genetically related gene, including merT, merP, merC, merB,merA, etc. also, there are new discovered gene related in mer operon such as merF and merG. Among them, merT, merP and merC have been revealed to play central part in the resistance to Hg (II).

Figure 1: The structure of common mer operon. It has a divergent promoter, which regulates upstream expression of MerR and downstream expression of MerT, MerP and MerC. Adapted from [1]

(1) merP

merP is a 72-amino acid periplasmic protein. It is processed from a 91-amino acid precursor by removal of a signal sequence by an energy-dependent process [1]. Structural studies have indicated that merP functions as a monomer and binds a single Hg (II) ion via its two cysteine residues at positions 14 and 17. Because of the quite ancient and wildly disseminated motif, merP is thought to play part in both membrane transiting and cell interior trafficking of metal cations.

Based on a comparison with other bacterial periplasmic binding, protein-dependent transport system, it has been proposed that Hg (II) diffused across the outer membrane. However, the inner membrane of bacteria is impenetrable for Hg (II). The Hg (II) in the periplasm then binds merP by the two cysteine residues. The Kd of fully reduced merP for Hg (II) is 3.7 uM, indicating that the binding affinity of MerP to Hg(II) is high enough. Factors including pH and oxidized level of the environment may influence the function of MerP.

（2）merT

As discussed above, merP binds Hg (II) via two cysteine residues. It it hypothesized based on the structure analysis that merP hands off the bound Hg(II) ion to the cysteine pair in the first transmembran e helix of MerT, so let’s see the structure and function of MerT.

merT is a 116-residue inner membrane protein[1]. By analysis of its structure, it is predicted that merT has three transmembrane motif, of which the first one can receive the Hg(II) from merP via a cysteine pair. This cysteine pair is accessible to the periplasmic side. The second pair of cysteines in MerT is predicted to lie on the cytoplasmic face of the inner membrane between the second and the third transmembrane helices. It is hypothesized that this cysteine pair is used to transfer Hg (II) to the cytoplasmic MerA, which is the main reduction agent.

(3) merC

For a long time, merC is considered to be of no use in the resistance to Hg (II) because mutations that affect the expression of MerC did not affect the resistance. The discovery in Thipobacillus isolated from mercury mines of operons that apparently had only merC and no merT genes forced reconsideration of the view that merC is vestigal. Cloning the Thiobacillus merC gene into E.coli revealed that its product was able to function in Hg (II) uptake, although not as effective as merT[1].

The 161-residue membrane-bound protein merC is predicted to have four transmembrane helices and is the largest protein in the mer operon. Studies concluded that merC is evolving differently than genes immediately adjacent to it in the operons where it occurs and maybe also evolving differently in different hosts. It has been proposed that merC may be needed under conditions of very high Hg (II) exposure, perhaps because merC system can uptake Hg (II) without the need of merP.

Figure 2: The summary of the function of MerT, MerP and MerC in the absorption of Hg (II) and their location within the cell. Note that the MerP binds Hg (II) in the periplasm and passes it to MerT, and then transfers it to MerA for reduction. Adapted from [1].

Since our bioabsorbent should capture Hg (II) as much as possible, aiming at this, artificial operon consisting of merP, merT, merC under the regulation of T7 promoter was constructed. T7 promoter was driven by constitutively expressed T7 polymerase. We hope that this design will endow our bioabsorbent with greater decontamination power.

Figure 3: The overall structure of the facilitation module. The constitutive expression of MerT, MerP and MerC are driven by T7 promoter. This facilitation module was expected to enable the bacteria to absorb more Hg (II) into the cytosol by facilitating the transmembrane process of Hg (II).

Reference:

1: Tamar Barkay, Susan M(2003). FEMS Microbiology Reviews. 27.355-384

2: Jon L.H, John Wilkie(2005). BioMetals. 18:429-436.

3: Andrea M.A.(2003). Genetics and Molecular Research. 2:92-101.

4: Peter A.Lund, Nigel L.Brown(1986). Gene. 52,207-214.