Unlikely Remediator: Engineering D. radiodurans for toxic metal cleanup

Morgan Etheridge

merA

Bacterial mercuric reductase

• Reduces Hg(II) + NADPH NADP+ + H+ +Hg(0)22,23

MD735 Strain

MD736

MD737

Determining mer operon copy number and mercury resistance

• (1) ~600 ng genomic DNA from each strain was electrophoresed in agarose gel

Determining mer operon copy number and mercury resistance

• (1) ~600 ng genomic DNA from each strain was electrophoresed in agarose gel

• (2) gel was blotted and hybridized to a radiolabeled 1.5-kb EcoRI-BglII fragment of pMD726, containing part of merA and all of merD.

Determining mer operon copy number and mercury resistance

• (1) ~600 ng genomic DNA from each strain was electrophoresed in agarose gel

• (2) gel was blotted and hybridized to a radiolabeled 1.5-kb EcoRI-BglII fragment of pMD726, containing part of merA and all of merD.

• (3) The number of disintegration counts in each of the hybridizing bands was determined and used to calculate the mer copy number per cell.

# merA copies per cell

R1 0MD735 1MD767 10MD736 10MD737 150

• Growth curves for each of the strains were determined by inoculating 5 x 106 cells of ∼each into growth medium containing 50 μM Merbromin or 50 μM HgCl2.

Merbromin

Order of resistance

• BL308 > (MD737, MD736) > MD735 > MD767 > R1

There is a lag in cell growth…What does that tell us?

• As detoxification occurs…– Toxic effects on the outer cell wall?– Toxic effects on the membranes responsible for

cell transport (and growth)?

• Wild-type D. radiodurans did not grow in HgCl2…Why?

Hg(II) + NADPH NADP+ + H+ +Hg(0)22,23

Multiple Resistance

• MD764

An unlikely hard drive?

References

• Brim, H., McFarlan, S.C., Fredrickson, JK., Minton, K.W., Zhai, M., Wackett, L.P., Daly, M.J. 1999. Engineering Deinococcus radiodurans for metal remediation inradioactive mixed waste environments. Nature Biotechnology: 18. 85 – 90.

• Battista, J.R. 1997. Against all odds: The survival strategies of Deinococcus radiodurans. Annual Review of Microbiology: 51. 203 – 224.

• Wong, P.C., Wong, K., Foot, H. 2003. Organic data memory: Using the DNA approach. Communications of the ACM: 46. 95 – 98.