Correlation of NE 1545
Expression and Cell Size in
Nitrosomonas europaea
Exposed to Aromatic
Hydrocarbons
Caslin Gilroy
Dr. Lewis Semprini
Environmental Engineering
Wastewater Treatment Plant
N. europaea
The process of nitrification exhibited by N.
europaea is represented by the following
reactions:
2H+ + 2e- + NH3 + O2  NH2OH + H2O
This step is catalyzed by the enzyme AMO
H2O + NH2OH  NO2- + 5H+ + 4eThis 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
Control
B
40 mM Benzene
Membrane thickness decreased from 103 + 11 nm to 71 + 4 nm
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
100%
10%
90%
9%
80%
8%
70%
7%
60%
6%
50%
5%
40%
4%
30%
3%
20%
2%
10%
1%
0%
0%
0
10
20
30
40
[Toluene] uM
% Activity
50
% Size Decrease
60
70
% Size Decrease
% Activity
Toluene and Cell Size
100%
10%
90%
9%
80%
8%
70%
7%
60%
6%
50%
5%
40%
4%
30%
3%
20%
2%
10%
1%
0%
0%
0
5
10
15
[Phenol] uM
% Activity
% Size Decrease
20
25
% Size Decrease
% Activity
Phenol and Cell Size
100%
10%
90%
9%
80%
8%
70%
7%
60%
6%
50%
5%
40%
4%
30%
3%
20%
2%
10%
1%
0%
0%
0
50
100
150
[Aniline] uM
% Activity
% Size Decrease
200
250
% Size Decrease
% Activity
Aniline and Cell Size
100%
10%
90%
9%
80%
8%
70%
7%
60%
6%
50%
5%
40%
4%
30%
3%
20%
2%
10%
1%
0%
0%
0
20
40
60
80
[p-Cresol] uM
% Activity
100
% Size Decrease
120
140
% Size Decrease
% Activity
p-Cresol and Cell Size
Compound
Dipole
pKa
Moment
Water
Log Kow
EC50
Solubility
Max
Size
Conc.
Change
Max
NE1545
Change
Aniline
2.30D
4.87
387 mM
0.90
55 mM
-5%
55 mM
19-fold
Phenol
1.70D
9.95
882 mM
1.50
10 mM
-6%
22 mM
15-fold
p-Cresol
1.50D
10.26 176 mM
1.98
80 mM
-5%
60 mM
17-fold
m-Cresol
1.45D
10.99 222 mM
1.98
4 mM
-2%
8 mM
2-fold
o-Cresol
1.45D
10.26 231 mM
1.98
7 mM
-2%
8 mM
2-fold
Ethylbenzene
0.40D
1 mM
3.15
60 mM
-3%
20 mM
Toluene
0.36D
28.30
5 mM
2.70
20 mM
-1%
63 mM
p-Hydoquinone
0.00D
10.35 536 mM
0.60
175 mM
-2%
60 mM
2-fold
Compound
Dipole
pKa
Moment
Water
Log Kow
EC50
Solubility
Max
Size
Conc.
Change
Max
NE1545
Change
Aniline
2.30D
4.87
387 mM
0.90
55 mM
-5%
55 mM
19-fold
Phenol
1.70D
9.95
882 mM
1.50
10 mM
-6%
22 mM
15-fold
p-Cresol
1.50D
10.26 176 mM
1.98
80 mM
-5%
60 mM
17-fold
m-Cresol
1.45D
10.99 222 mM
1.98
4 mM
-2%
8 mM
2-fold
o-Cresol
1.45D
10.26 231 mM
1.98
7 mM
-2%
8 mM
2-fold
Ethylbenzene
0.40D
1 mM
3.15
60 mM
-3%
20 mM
Toluene
0.36D
28.30
5 mM
2.70
20 mM
-1%
63 mM
p-Hydoquinone
0.00D
10.35 536 mM
0.60
175 mM
-2%
60 mM
2-fold
Membrane Stability
1
2
3
4
5
6
7
8
Membrane Stability
1
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 Semprini
Dr. Tyler Radniecki
Dr. Kevin Ahern