Correlation of NE 1545 Expression and Cell Size in

Nitrosomonas europaea Exposed to Aromatic

Hydrocarbons

Caslin Gilroy

Dr. Lewis Semprini

Environmental Engineering

N. europaeaWastewater Treatment Plant

The process of nitrification exhibited by N. europaea is represented by the following reactions:

2H+ + 2e- + NH3 + O2 NH2OH + H2OThis step is catalyzed by the enzyme AMO

H2O + NH2OH NO2- + 5H+ + 4e-

This step is catalyzed by the enzyme HAO

Notice that there is a net gain of two electrons,which are used by the bacteria for energy

Why is this bacterium important? N. europaea prevents eutrophication in wastewater treatment plants.

Causes of Inhibition in Ammonia Oxidation

N. europaea is very sensitive to common inhibitors (CN, heavy metals, pH shifts, and organics).

Currently can monitor chemical inhibition, but cannot determine cause.

Therefore cannot neutralize inhibitors and wastewater treatment plant failure can occur.

Solution

Want to be able to detect nitrification inhibition AND determine cause of inhibition.

Do this by identifying genes expressed in response to specific inhibitors.

Create a biosensor to detect N. europaea’s gene expression and link expression to specific chemical contamination.

Shift in Cell Membrane Structure

A BA B

Membrane thickness decreased from 103 + 11 nm to 71 + 4 nm

Control 40 M Benzene

Possible Biosensor Gene Identified

NE 1545 linked to cell membrane size and organic compound exposure.

Hypotheses: Increased expression of NE 1545 is correlated

with exposure to certain aromatic hydrocarbons.

Exposure to these hydrocarbons is correlated with a decrease in membrane thickness.

Batch Experiments

Nitrite Assay

60 M-2%175 M0.60536 mM10.350.00Dp-Hydoquinone

2-fold63 M-1%20 M2.705 mM28.300.36DToluene

20 M-3%60 M3.151 mM0.40DEthylbenzene

2-fold8 M-2%7 M1.98231 mM10.261.45Do-Cresol

2-fold8 M-2%4 M1.98222 mM10.991.45Dm-Cresol

17-fold60 M-5%80 M1.98176 mM10.261.50Dp-Cresol

15-fold22 M-6%10 M1.50882 mM9.951.70DPhenol

19-fold55 M-5%55 M0.90387 mM4.872.30DAniline

ChangeChangeSolubilityMoment

Max NE1545Conc.

Max Size EC50Log KowWater pKaDipole Compound

60 M-2%175 M0.60536 mM10.350.00Dp-Hydoquinone

2-fold63 M-1%20 M2.705 mM28.300.36DToluene

20 M-3%60 M3.151 mM0.40DEthylbenzene

2-fold8 M-2%7 M1.98231 mM10.261.45Do-Cresol

2-fold8 M-2%4 M1.98222 mM10.991.45Dm-Cresol

17-fold60 M-5%80 M1.98176 mM10.261.50Dp-Cresol

15-fold22 M-6%10 M1.50882 mM9.951.70DPhenol

19-fold55 M-5%55 M0.90387 mM4.872.30DAniline

ChangeChangeSolubilityMoment

Max NE1545Conc.

Max Size EC50Log KowWater pKaDipole Compound

60 M-2%175 M0.60536 mM10.350.00Dp-Hydoquinone

2-fold63 M-1%20 M2.705 mM28.300.36DToluene

20 M-3%60 M3.151 mM0.40DEthylbenzene

2-fold8 M-2%7 M1.98231 mM10.261.45Do-Cresol

2-fold8 M-2%4 M1.98222 mM10.991.45Dm-Cresol

17-fold60 M-5%80 M1.98176 mM10.261.50Dp-Cresol

15-fold22 M-6%10 M1.50882 mM9.951.70DPhenol

19-fold55 M-5%55 M0.90387 mM4.872.30DAniline

ChangeChangeSolubilityMoment

Max NE1545Conc.

Max Size EC50Log KowWater pKaDipole Compound

60 M-2%175 M0.60536 mM10.350.00Dp-Hydoquinone

2-fold63 M-1%20 M2.705 mM28.300.36DToluene

20 M-3%60 M3.151 mM0.40DEthylbenzene

2-fold8 M-2%7 M1.98231 mM10.261.45Do-Cresol

2-fold8 M-2%4 M1.98222 mM10.991.45Dm-Cresol

17-fold60 M-5%80 M1.98176 mM10.261.50Dp-Cresol

15-fold22 M-6%10 M1.50882 mM9.951.70DPhenol

19-fold55 M-5%55 M0.90387 mM4.872.30DAniline

ChangeChangeSolubilityMoment

Max NE1545Conc.

Max Size EC50Log KowWater pKaDipole Compound

Membrane Stability1 2 3 4 5 6 7 8

Membrane Stability1 2 3 4 5 6 7 8 9 10

Future Study

Continued experimentation with NE 1545 to determine if it is an effective sentinel gene for our biosensor.

Acknowledgements

Howard Hughes Medical Institute Dr. Lewis SempriniDr. Tyler RadnieckiDr. Kevin Ahern