IGEM:MIT/2007/Heavy Metals

Reviews

• Excellent Summary of Bacterial Metal Resistance (a must read)

1. Silver S and Phung le T. A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. J Ind Microbiol Biotechnol. 2005 Dec;32(11-12):587-605. DOI:10.1007/s10295-005-0019-6 |

• A Good Overview on Pollution (though slightly back-dated): Quantitative assessment of worldwide contamination of air, water and soil by trace metals [1]
• A summary of Pollutants from the EPA [2]
• Review Papers on Bioremediation

2. Paul D, Pandey G, Pandey J, and Jain RK. Accessing microbial diversity for bioremediation and environmental restoration. Trends Biotechnol. 2005 Mar;23(3):135-42. DOI:10.1016/j.tibtech.2005.01.001 |
3. Valls M and de Lorenzo V. Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS Microbiol Rev. 2002 Nov;26(4):327-38. DOI:10.1111/j.1574-6976.2002.tb00618.x |
4. Bruins MR, Kapil S, and Oehme FW. Microbial resistance to metals in the environment. Ecotoxicol Environ Saf. 2000 Mar;45(3):198-207. DOI:10.1006/eesa.1999.1860 |

• Paul - discusses advantages/disadvantages/problems of bioremediation
• Bruins, Valls - discusses various kinds of bioremediation systems

Heavy Metal Binding Proteins/Peptides

There are several classes of metal binding proteins to consider. (for review, see Cobbett02, below)

First, those that bind to many metal types (Cd, Hg, Pb, Cu, Zn, etc). These are nice in that they are multifunctional, but the disadvantage is that they will also bind non-toxic ions like Zn, thus decreasing their efficiency.

• • Metallothioneins (MT's) -- small animal proteins which contain many cysteine motifs (usually CxxC)
  • Phytochelatins (PC's) -- plant peptides of the form (EC)n-G where n=# repeats (usually 2-10, but can be as many as 20) (see the Bae papers)

Also, certain artificially selected sequences bind selectively to certain metals.

• • The MT-like motif CCAA was found to highly selective for Hg, while CAAC had broad specificity (see DeSilva02)
  • The motif (CGCCG)3 was found to bind both cadmium and mercury (see Pazirandeh98)

Seems that Mejare01 does a nice job of comparing the different Cd-binding methods which have been tried. According to them, the methods which gave the biggest increase (>60-fold) in binding capacity were those that expressed entire MT proteins (or domains of them). However, they did not consider the papers where PCs were used, which seem to have much higher binding capacities.

The Bae papers seem to indicate that the EC20 motif can bind both Cd and Hg (higher affinity for Hg), and the absolute accumulation is higher than for the MT-derived peptides. Also, EC20 binding still works in the presence of up to 200mM salt and even EDTA. Might be a good idea to ask them for their Lpp-OmpA-EC20 expression construct!

Binding Sequences To Try

• The full Pazirandeh98 Hg/Cd-specific binding sequence (CGCCG) is: CGCCGKGHCGCCGKGHCGCCG
• For good non-specific binding, best bet seems to be the PC-derived EC20 sequence: (EC)20-Gly

• From DeSilva02, the MerP-derived Hg-specific binding sequence (GMTCCAA) could be repeated 3x: GMTCCAAGMTCCAAGMTCCAA
• For Cd-specific binding, could later use the CadA-derived sequence (GFTCANC)

5. Samuelson P, Wernérus H, Svedberg M, and Ståhl S. Staphylococcal surface display of metal-binding polyhistidyl peptides. Appl Environ Microbiol. 2000 Mar;66(3):1243-8. DOI:10.1128/AEM.66.3.1243-1248.2000 |
6. Pazirandeh M, Wells BM, and Ryan RL. Development of bacterium-based heavy metal biosorbents: enhanced uptake of cadmium and mercury by Escherichia coli expressing a metal binding motif. Appl Environ Microbiol. 1998 Oct;64(10):4068-72. DOI:10.1128/AEM.64.10.4068-4072.1998 |
7. Kotrba P, Dolecková L, de Lorenzo V, and Ruml T. Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides. Appl Environ Microbiol. 1999 Mar;65(3):1092-8. DOI:10.1128/AEM.65.3.1092-1098.1999 |
8. Mejáre M and Bülow L. Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals. Trends Biotechnol. 2001 Feb;19(2):67-73. DOI:10.1016/s0167-7799(00)01534-1 |
9. Lu Y, Berry SM, and Pfister TD. Engineering novel metalloproteins: design of metal-binding sites into native protein scaffolds. Chem Rev. 2001 Oct;101(10):3047-80. DOI:10.1021/cr0000574 |
10. Bae W, Chen W, Mulchandani A, and Mehra RK. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol Bioeng. 2000 Dec 5;70(5):518-24. DOI:10.1002/1097-0290(20001205)70:5<518::aid-bit6>3.0.co;2-5 |
11. Bae W, Mehra RK, Mulchandani A, and Chen W. Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury. Appl Environ Microbiol. 2001 Nov;67(11):5335-8. DOI:10.1128/AEM.67.11.5335-5338.2001 |
12. Bae W, Mulchandani A, and Chen W. Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation. J Inorg Biochem. 2002 Jan 15;88(2):223-7. DOI:10.1016/s0162-0134(01)00392-0 |
13. Kotrba P, Pospisil P, de Lorenzo V, and Ruml T. Enhanced metallosorption of Escherichia coli cells due to surface display of beta- and alpha-domains of mammalian metallothionein as a fusion to LamB protein. J Recept Signal Transduct Res. 1999 Jan-Jul;19(1-4):703-15. DOI:10.3109/10799899909036681 |
14. DeSilva TM, Veglia G, Porcelli F, Prantner AM, and Opella SJ. Selectivity in heavy metal- binding to peptides and proteins. Biopolymers. 2002 Aug 5;64(4):189-97. DOI:10.1002/bip.10149 |
15. Opella SJ, DeSilva TM, and Veglia G. Structural biology of metal-binding sequences. Curr Opin Chem Biol. 2002 Apr;6(2):217-23. DOI:10.1016/s1367-5931(02)00314-9 |
16. Sousa C, Kotrba P, Ruml T, Cebolla A, and De Lorenzo V. Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB. J Bacteriol. 1998 May;180(9):2280-4. DOI:10.1128/JB.180.9.2280-2284.1998 |
17. Mauro JM and Pazirandeh M. Construction and expression of functional multi-domain polypeptides in Escherichia coli: expression of the Neurospora crassa metallothionein gene. Lett Appl Microbiol. 2000 Feb;30(2):161-6. DOI:10.1046/j.1472-765x.2000.00697.x |
18. Cobbett C and Goldsbrough P. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol. 2002;53:159-82. DOI:10.1146/annurev.arplant.53.100301.135154 |
19. Pazirandeh M, Wells BM, and Ryan RL. Development of bacterium-based heavy metal biosorbents: enhanced uptake of cadmium and mercury by Escherichia coli expressing a metal binding motif. Appl Environ Microbiol. 1998 Oct;64(10):4068-72. DOI:10.1128/AEM.64.10.4068-4072.1998 |
20. Chen S, Kim E, Shuler ML, and Wilson DB. Hg2+ removal by genetically engineered Escherichia coli in a hollow fiber bioreactor. Biotechnol Prog. 1998 Sep-Oct;14(5):667-71. DOI:10.1021/bp980072i |

Cadmium

Summary

• Cd causes damage to cells primarily by the generation of reactive oxygen species (ROS), which causes single-strand DNA damage and disrupts the synthesis of nucleic acids and proteins. Cd is also an inhibitor of the DNA mismatch repair system…Results of this study confirmed that Cd toxicity caused profound changes in gene expression in which several stress response systems were induced simultaneously.
• known carcinogen
• Most recent(June06) paper on e.coli bioaccumulation of Cd

21. Deng X, Yi XE, and Liu G. Cadmium removal from aqueous solution by gene-modified Escherichia coli JM109. J Hazard Mater. 2007 Jan 10;139(2):340-4. DOI:10.1016/j.jhazmat.2006.06.043 |

Contamination Levels/Limits

• MCL(Maximum Containment Level): 5 ppb (equal to about 45nM)
• MCLG(Maximum Containment Level Goal): 5 ppb

22. Staessen JA, Lauwerys RR, Ide G, Roels HA, Vyncke G, and Amery A. Renal function and historical environmental cadmium pollution from zinc smelters. Lancet. 1994 Jun 18;343(8912):1523-7. DOI:10.1016/s0140-6736(94)92936-x |
23. Satarug S, Baker JR, Urbenjapol S, Haswell-Elkins M, Reilly PE, Williams DJ, and Moore MR. A global perspective on cadmium pollution and toxicity in non-occupationally exposed population. Toxicol Lett. 2003 Jan 31;137(1-2):65-83. DOI:10.1016/s0378-4274(02)00381-8 |

Cadmium Contamination Sites

24. Ishihara T, Kobayashi E, Okubo Y, Suwazono Y, Kido T, Nishijyo M, Nakagawa H, and Nogawa K. Association between cadmium concentration in rice and mortality in the Jinzu River basin, Japan. Toxicology. 2001 May 28;163(1):23-8. DOI:10.1016/s0300-483x(01)00367-5 |

Plachimada, India: [3]

• • statistics on Pb, Cd, Cr levels in well water surrounding a Coca-Cola plant (2006)
  • pictures too :)

Promoters

Pubmed: Plasmid pI258 (from S.aureus) cadmium resistance (cadA) gene, complete cds [4]

25. Brocklehurst KR, Megit SJ, and Morby AP. Characterisation of CadR from Pseudomonas aeruginosa: a Cd(II)-responsive MerR homologue. Biochem Biophys Res Commun. 2003 Aug 22;308(2):234-9. DOI:10.1016/s0006-291x(03)01366-4 |

• Brocklehurst - contains sequence for ZntR/CadR and PzntA/PcadA

• • Semmie's article on CadC linked with GFP [5]

Mercury

Contamination Levels/Limits

• MCL: 2 ppb (equal to about 10 nM)
• MCLG: 2 ppb
• 20 ppb makes the fish mercury buildup significant

Polluted sites

• Onondaga lake [6] [7] [8]
• Salt Plains National Wildlife Refuge [9]

Detection (to assess our system)

• Gold Nanoparticles http://www.cah.ucf.edu/news/2005-Mercurynose.php

Promoter

26. Hamlett NV, Landale EC, Davis BH, and Summers AO. Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding. J Bacteriol. 1992 Oct;174(20):6377-85. DOI:10.1128/jb.174.20.6377-6385.1992 |
27. Park SJ, Wireman J, and Summers AO. Genetic analysis of the Tn21 mer operator-promoter. J Bacteriol. 1992 Apr;174(7):2160-71. DOI:10.1128/jb.174.7.2160-2171.1992 |
28. Condee CW and Summers AO. A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity. J Bacteriol. 1992 Dec;174(24):8094-101. DOI:10.1128/jb.174.24.8094-8101.1992 |
29. Gambill BD and Summers AO. Versatile mercury-resistant cloning and expression vectors. Gene. 1985;39(2-3):293-7. DOI:10.1016/0378-1119(85)90326-9 |
30. Hansen LH and Sørensen SJ. Versatile biosensor vectors for detection and quantification of mercury. FEMS Microbiol Lett. 2000 Dec 1;193(1):123-7. DOI:10.1111/j.1574-6968.2000.tb09413.x |

• Condee - Contains sequence for merO/P region
• Hansen - Plasmid with Pmer, MerR, and reporter (lux, lac, or gfp) tested in e.coli

Sponge

• Accumulation of Hg ions in cell membrane [10]

Current Industry Filtration Techniques

Mercury: approved by EPA - Coagulation/Filtration; Granular Activated Carbon; Lime softening; Reverse osmosis.