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Topological analysis of a manganese exporter protein, MntP, in
Escherichia coli and its response to Reactive Oxygen Species
Eli Martinez, McNair Scholar and Lauren Waters, mentor
Department of Chemistry – University of Wisconsin Oshkosh
Abstract
Results
Manganese is an important trace nutrient involved in the detoxification of
reactive oxygen species (ROS). Manganese also acts as an enzyme cofactor for
superoxide dismutase and other enzymes. In bacteria, manganese and Mn-SOD
allow bacteria to withstand ROS created from internal and external metabolic
processes. Pathogenic bacteria require manganese to remain virulent in
environments with reactive oxygen species, such as the host organism. Yet,
manganese is toxic at high concentrations, so it is kept at low concentrations by
manganese homeostasis. In the model organism E. coli, manganese homeostasis
is maintained through the use of proteins that import and export manganese,
MntH and MntP, from within the cell. MntP is a transmembrane exporter protein
that we predict to traverse the membrane six times with the N- and C- terminals
located in the periplasm (Figure 1). Alkaline phosphatase (PhoA) is only active in
the periplasm. The orientation of MntP within the membrane was studied using
PhoA-MntP fusions. N- and C- terminal fusions of PhoA to MntP were created
using Polymerase Incomplete Primer Extension cloning. Using an alkaline
phosphatase assay, activity was seen with the C-terminal fusion, which was
confirmed to be oriented in the periplasm. Problematically, the ability of the
fusion proteins to export manganese was reduced, most notably with the Nterminal fusion, which was less than 50% active.
Orientation of MntP
N- and C- terminal fusions between alkaline phosphatase
and MntP were successful created through Polymerase
Incomplete Primer Extension Cloning. Sequencing of
plasmids containing the fusions yielded positive results of
a successful fusion between proteins (data not shown). A
mutation was noted within the alkaline phosphatase
coding region. A higher activity was seen with the Cterminal fusion to MntP than to the basal rate of activity
(Figure 4). The N-terminal fusion to MntP did not yield
activity greater than the basal rate of activity. The data
indicates that the C-terminal is located in the periplasm,
while the orientation of the N-terminal is unknown.
Figure 5: Manganese Sensitivity Assay
Figure 1: Amino Acid Alignment of MntP
Manganese Sensitivity
A manganese sensitivity assay was used to determine if
the created fusions were still able to function. Analysis
shows that the fusions to MntP were unable to
completely rescue a bacterial strain absent of mntP
(Figure 6). The fusion proteins were determined to have
less than 50% functionality.
The deletion (Δ) of MntP is not able to grow on plates
containing Mn.
Bioinformatics of the exporter protein, MntP, predicting six
transmembrane domains.
Additionally, the relationship of MntP to reactive oxygen species has yet to be
defined. The over expression of MntP, in comparison to the absence of the
protein, displayed a sensitivity to hydrogen peroxide. This phenotype of MntP
links the protein to reactive oxygen species resistance for the first time. In the
future, additional loop fusions for MntP are ongoing. By better understanding
MntP and the other cell machinery involved in manganese homeostasis, the
virulence of pathogenic bacteria has the potential to be reduced.
Physiological Assay
The over expression of MntP caused hydrogen peroxide
sensitivity in a sensitized strain of E. coli. ΔmntP grew
steadily over time, while the growth of E. coli with the
over expression of MntP stayed stagnant (Figure 7).
Methods
Conclusion
Orientation of MntP
The orientation of MntP was studied through the creation of N- and C- terminal
fusions to alkaline phosphatase. The fusion proteins were synthesized by
Polymerase Incomplete Primer Extension cloning (PIPE cloning). Unlike other
cloning methods, PIPE cloning is based off the premise of creating singlestranded DNA overhangs through Polymerase Chain Reactions (PCR), a
technique that amplifies DNA. The vector and insert DNA, mntP and phoA, will
have regions of homology at the single-stranded DNA overhangs. The bacterial
cell machinery is then utilized to recombine the vector and insert DNA.
Depicts the proposed model of six transmembrane regions, in addition to the
location of desired fusions between a reporter protein and MntP.
Fusions to MntP were able to partially grow on plates containing Mn. Cell
cultures plated on the media were diluted 10 fold to a final dilution of 10-6
from top to bottom.
Relationship between MntP and Reactive Oxygen Species
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The data obtained from the PhoA-MntP fusions will be
compared to GFP-MntP fusions. Additional fusion proteins
are to be created using an alternative method. Mutations
in MntP and other proteins will be created and tested
using the Manganese Sensitivity Assay and the Oxidative
Stress Assay.
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ΔmntP + pBAD24
80
log(OD600)
Alkaline Phosphatase activity
Physiological Assay
Additionally, we studied the effect of reactive oxygen species on bacterial cell
growth with and without MntP. A sensitized strain of E. coli was used in a growth
curve experiment, which had its ability to combat reactive oxygen species
reduced.
Figure 6: Functionality of Fusion Proteins
Orientation of N- and C- terminal fusions
An enzyme, alkaline phosphatase, assay was used to determine the activity of
the N- and C- terminal fusions. The alkaline phosphatase assay involves the
addition of a colorless substrate to cells containing the fusions (Figure 3). If
alkaline phosphatase is localized in the periplasm, a yellow pigment is produced.
Manganese Sensitivity
The N- and C- terminal MntP fusions’ functionality was assessed with a
manganese sensitivity assay based off previous research (Figure 5).
Figure 2: Proposed fusions to MntP
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Acknowledgements
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0
Δ PhoA + pBAD24 Δ PhoA + pMntP Δ PhoA + pHA-4 Δ PhoA + pYbhT Δ PhoA + pMntP- Δ PhoA + pPhoAPhoA
MntP
ΔmntP + pMntP
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Bacterial plasmid
0.1
0
PNPP (colorless)
PNP (yellow)
Figure 4: Alkaline Phosphatase Activity of Fusion Proteins
Data is representative of triplicate experiment.
Figure 3: Alkaline Phosphatase Reaction
P-nitrophenylphosphate (PNPP) is cleaved by alkaline phosphatase.
Absorbance is measured at 420 nm using a spectrophotometer.
The C-terminal of MntP was confirmed to be located in
the periplasm of E. coli. No conclusions can be drawn on
the N- terminal fusion, since MntP was not functional.
The functionality of the tested fusions to MntP was
determined to be reduced after they conveyed sensitivity
to manganese. Furthermore, the over expression of MntP
demonstrated sensitivity to hydrogen peroxide. This links
MntP to reactive oxygen species resistance, further
strengthening the relevance to pathogenic and symbiotic
bacteria.
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Time (hours)
Figure 7: Oxidative Stress Assay
A sensitized strain of E. coli was used that had its ability to combat reactive
oxygen species reduced.
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Thank you to Mary Seaman for her continued support. In
addition, we would like to thank Dr. Chris Bianchetti as
well. I would also like to thank Rilee Zeinert and other
students from Dr. Waters’ lab for their contributions to
the project. The UW Oshkosh McNair Scholars Program is
100% funded through a TRIO grant from the United States
Department
of
Education
PR/Award
Number
P217A120210. For 2014/2015, the UW Oshkosh McNair
Scholars Program will receive $216,000.00 each year in
federal funds.