Mercury resistance and accumulation in

Escherichia coli with cell surface expression of

fish metallothionein

Kristen Soroka

http://buyorganictilapia.com/

Kuo-Hsing et al. 2010

How does Mercury get into the environment?

http://www.nap.edu/openbook.php?isbn=0309071402&page=17

What are the health effects of Mercury?

Methyl Mercury

• Attacks the nervous system of humans and developing fetuses

• Vision, speech, hearing, or walking impairment

• Disturbance in sensations

• Muscle weakness

Elemental Mercury

• Headaches

• Tremors

• Emotional changes

• Neuromuscular changes

• Kidney effects

• Respiratory failure

• Death

http://www.epa.gov/hg/effects.htm

What is Metallothionein (MT)?

Capasso et al. 2003

Eight Plasmids!• A control plasmid for the cytosol expression and

outer membrane expression.

• Cytosol and outer membrane expression for:• Human MT• Mouse MT• Tilapia MT

pET-30aCytosol expressionHuman (mt1A) = pET-HmtMouse (mt1) = pET-MmtTilapia (mt) = pET-Tmt

Cell Surface ExpressionControl = pET-OmpCHuman = pET-OmHmtMouse = pET-OmMmtTilapia = pET-OmTmt

<http://www.synthesisgene.com/vector/pET-30a.pdf>.

Cytosol Plasmid Construct• Control : pET-30a

• pMAL-p2X-MT1A encodes human MT (Hmt) and pMBP-TEV-MT1 encodes mouse MT (Mmt) (Kao et al. 2006) as cited in Kuo-Hsing et al. 2010

• Tilapia liver (Tmt)

• Primers

• Cut-Sites : BglII and SacI

Outer Membrane Plasmid Construct

• Control : pET-30a pET-OmpC• OmpC gene from E. coli K19• Primers : OmpC-F and OmpC-R• Cut-Sites : NdeI and BamHI

• Cut-Site : PstI

• “These plasmids were designed to express outer membrane fusion protein in which the MT was integrated into the seventh outer membrane loop of the OmpC protein” (Kuo-Hsing et al. 2010).

Primers

MT Expression in Cytosol

• Kanamycin resistance

• IPTG

• SDS-14% PAGE gel

Kuo-Hsing et al. 2010, Fig 1a.

No MT Hmt Mmt Tmt

Cellular Location of Cytocolic tMT

Kuo-Hsing et al. 2010, Fig 2b.

Alexa Flour 594 donkey anti-rabbit IgG antibody

MT Expression in Outer Membrane

• Similar expression methods

• Isolating the nonpolar membrane protein.

• SDS-14% PAGE gel

Kuo-Hsing et al. 2010, Fig 1b.

OmTm

tOm

Hmt

OmM

mt

pET-30a

pET-Om

pC

Cellular Labeling of outer membrane targeted tMT

Kuo-Hsing et al. 2010, Fig 2a.

Alexa Flour 488 donkey anti-rabbit IgG antibody

Disk Assay and Resistance

Kuo-Hsing et al. 2010

Disk Assay

• If the bacteria is susceptible the to the metal then a zone of inhibition will surround the plate

• The smaller the zone of inhibition means it is more resistant to the metal

http://www.antimicrobialtestlaboratories.com/Zone_of_Inhibition_Test_for_Antimicrobial_Activity.htm

Kuo-Hsing et al. 2010

Cytosol expression

Outer membrane expression

Sequence, Structure, and Function

• CXCC (mammalian) CXXXCC (fish)• (Sucdiero et al. 2005) as cited in Kuo-Hsing et al

2010

• A change in primary structure influences the tertiary structure of proteins (Capasso et al. 2003)

• Structure dictates Function

(Capasso et al. 2003, Fig 2)

Discussion• “Our results showed that the limitation of

intracellular accumulation could be overcome by anchoring the metal-binding proteins directly to the cell membrane for improving adsorption (Fig 3) and reducing the toxicity (Table 2)” (Kuo-Hsing et al. 2010)

More about Mercury• http://fishadvisoryonline.epa

.gov/General.aspx

• Hocking River

• Mill Creek

• Clear Creek

• Gold Shop Mercury Capture System

http://www.epa.gov/international/toxics/mercury/asgm.html

References• Capasso, Clemente, Vincenzo Carginale, Orlando Crescenzi, Daniela Di Maro, Elio Parisi, Roberta Spadaccini,

and Piero Andrea Temussi. "Solution Structure of MT_nc, a Novel Metallothionein from the Antarctic Fish Notothenia coriiceps." Cell Press 11 (2003): 435-43. ScienceDirect. 1 Dec. 2013 <http://www.sciencedirect.com/science/article/pii/S0969212603000443>.

• Kuo-Hsing, Lin, Chien Mei-Gang, Hsieh Ju-Liang, and Huang Chieh-Chen. "Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein." Applied Microbiology and Biotechnology 87 (2010): 561-69. SpringerLink. 01 June 2010. Springer. <http://link.springer.com/article/10.1007/s00253-010-2466-x>.

• "Health Effects." EPA. 9 July 2013. Environmental Protection Agency. 01 Dec. 2013 <http://www.epa.gov/hg/effects.htm>.

• "PET-30a-c(+) Vectors." Map. Novagen. Dec. 1998. 1 Dec. 2013 <http://www.synthesisgene.com/vector/pET-30a.pdf>.

• "Reducing Mercury Pollution from Artisanal and Small-Scale Gold Mining." EPA. 29 Aug. 2013. Environmental Protection Agency. 03 Dec. 2013 <http://www.epa.gov/international/toxics/mercury/asgm.html>.

• "Zone of Inhibition Test for Antimicrobial Activity." Antimicrobial Test Laboratories. 02 Dec. 2013 <http://www.antimicrobialtestlaboratories.com/Zone_of_Inhibition_Test_for_Antimicrobial_Activity.htm>.