Membrane Phase Transition of Geobacter sulfurreducens

1
Membrane Phase Transition of Geobacter
sulfurreducens
79
Cally Scherber2, Vishard Ragoonanan1, Alptekin
Aksan1 1Biostabilization Laboratory, Department
of Mechanical Engineering, University of
Minnesota, Minneapolis, MN 2Department of Biology
Comparison of Dried vs. Hydrated Samples
Introduction
DRIED, 30ºC
Samples Geobacter Media OR Geobacter Media
6.75 Sucrose
Figure 4. Comparison of samples dried at 30ºC vs.
5ºC
FTIR Analysis
(1)G.sulfurreducens dried in 13.5 sucrose
solutions had the largest increase in the Tm
value. (2)G.sulfurreducens dried in media
solutions had larger Tm values than similar
samples that were undried and samples that were
dried-rehydrated. (3)G.sulfurreducens dried at
30ºC in media had slightly higher Tm values than
samples in 6.75 sucrose.
WHAT IS GEOBACTER SULFURREDUCENS?
FTIR Analysis
DRIED, 5ºC
G. sulfurreducens is an anerobic,
electricity-producing bacterium. Species of
Geobacter are of interest to researchers because
of their ability to harvest electricity from
organic waste material and their unique
functionality in microbial fuel cells. (fig 1)
DRIED, 30ºC
FTIR Analysis
Figure 5. Comparison of samples dried at 30ºC vs.
dried-re-hydrated vs. undried.
NOT DRIED
Samples Geobacter Media
FTIR Analysis
DRIED 30ºC, REHYDRATED
FTIR Analysis
FTIR SPECTRA ANALYSIS- Changes in
G.sulfurreducens membrane phase transition
temperature (Tm) correspond to physical changes
of the cellular membrane. These changes can be
observed through Fourier Transform Infrared
(FTIR) spectra analysis.
Figure 1. Cells of Geobacter Sulfurreducens
(http//www.geobacter.org/research/nanowires/)
WATERS ROLE IN THE CELLULAR MEMBRANE-
Changes in the water content resulting from
temperature and moisture levels can be
detrimental to a cell. When drying cells, damage
is mainly the result of physical changes of the
lipid bilayer within a cells membrane. As water
is removed from the lipid bilayer during drying,
the polar head groups of the membrane come closer
together, thereby allowing an increase of the
attractive forces between the lipids. This
attraction forces the transition from the liquid
crystalline to the gel phase. (fig 2)
HOW DOES FTIR WORK? As the cellular membrane
transitions from the liquid-crystalline to the
gel phase upon dessication, the frequency of the
CH2 stretch, corresponding to the lipid
membrane, decreases. This decrease in mobility is
observed through a downward shift in the wave
numbers of the CH2 peak position with respect to
temperature.3 (fig 6)
Figure 6. FTIR spectra for G.sulfurreducens
Geobacter Dried 30ºC
CH2 stretch2850cm-1
DRY
Undried
Membrane Phase Change
Dried-Rehydrated
The wave numbers corresponding to a specific
sample are plotted against temperature as shown
in figure 7. The dotted green line in the graph
demonstrates the decrease in vibrational
frequency that occurs when a sample is dried
versus a sample that is not dried. This
decrease in vibrational frequency corresponds to
an increase in the Tm value of the sample.
Gel Phase
Liquid Crystalline Phase
Liquid Crystalline
Figure 2. Molecular dynamics simulations of two
different membrane phases gel and liquid
crystalline. (www.physik.uni-bielefeld)
Geobacter Dried 5ºC
DRIED
CH2 Bond Vibration Frequency
UNDRIED
Upon re-hydration, the dry cellular membrane
transitions back into the liquid phase. As the
membrane transitions between these phases,
packing defects cause the lipid bilayer to
become leaky. This, in turn, can lead to an
osmotic gradient of solutes out of the cell,
which may result in cellular death 1. The
membrane phase transition temperature (Tm) is the
temperature at which the cell membrane
transitions from one phase to the other. This
temperature value is used to determine how
certain experimental and environmental conditions
affect the membrane of a cell.
Figure 8 Tm values of G.sulfurreducens under
varying experimental conditions.
Gel
Temperature C
Conclusions
Figure 7. Membrane Phase Changes corresponding to
cell dessication.
1 . G.sulfurreducens dried in 13.5 sucrose
solutions had a significantly higher Tm value
than G.sulfurreducens dried in media and 6.75
sucrose solutions. This shows that 13.5 sucrose
is too large of an amount and may be creating an
osmotic gradient that is forcing water out of the
cell. 2. G.sulfurreducens dried in media and
6.75 sucrose solutions at 5C had noticeably
higher Tm values than similar samples dried at
30ºC. This shows that it is more beneficial to
dry the cells at 30ºC rather than 5ºC. 3. In
every experiment, drying G.sulfurreducens
resulted in an increase in the Tm value of the
cell. It is apparent that whenever water is
removed from the cell, the Tm value of the cell
will increase. 4. It was not apparent whether or
not the addition of 6.75 sucrose prevented a
large increase in the Tm value of the
G.sulfurreducens samples. The samples dried in
media and the samples dried in 6.75 sucrose were
comparable in their Tm values. Due to this, it
would be unreasonable to say whether or not the
addition of sucrose aided in the prevention of
membrane changes upon dessication.
Results
HOW TO PREVENT MEMBRANE CHANGES
Since phase changes cause damage to cells, we are
interested in keeping our Geobacter samples in
the same phase throughout the drying and
re-hydrating process. 1. Drying in the presence
of sugar (sucrose or trehalose) has been shown to
play a role in reducing changes in the membrane
structure of a cell. As a result, the bacterial
cells will remain in the liquid-crystalline state
even when dried. In this sense, the sugar is
acting as a water substitute in the removal of
water. 2 2. Since cells are in the gel phase
at 5ºC and are in the liquid crystalline phase at
30ºC, drying in controlled temperature settings
of 5ºC or 30ºC prevents the cell from undergoing
membrane phase transition due to the
environmental temperature.
Comparison of Dried vs. Undried Samples

• G.sulfurreducens dried in 13.5 sucrose solutions
  had the largest increase in the Tm value.
• G.sulfurreducens in media, 6.75 sucrose, and
  13.5 sucrose solutions all had higher Tm values
  when dried.
• G.sulfurreducens dried in media had similar Tm
  values as G.sulfurreducens dried in 6.75
  sucrose.

References
Methods
Comparison of Samples at 5ºC vs. 30ºC
1. Samuel B. Leslie, Eitan Israeli, Bruse
Lighthart, John H. Crowe, and Lois M. Crowe.
1995. Trehalose and Sucrose Protect Both
Membranes and Proteins in Intact Bacteria during
Drying. Applied and Environmental Microbiology.
3592-3597 2. John F. Carpenter, John H. Crowe.
1989. An Infrared Spectroscopic Study of the
Interactions of Carbohydrates with Dried
Proteins. American Chemical Society. 3916-3922 3.
L. Beney, Y. Mille, P. Gervais. 2004. Death of
Escherichia Coli During Rapid and Severe
Dehydration is Related to Lipid Phase Transition.
Applied Microbial and Cell Physiology. 65
457-464.

• The experiments that were completed compared a
  variety of environmental and experimental
  conditions such as G. sulfurreducens samples in
  the presence and absence of sucrose, samples
  dried compared to samples undried, and samples
  dried at 5 ºC compared to 30ºC.
• Fourier Transform Infrared (FTIR) Analysis was
  then used to determine how these varying
  conditions affect the temperature at which the
  cellular membrane of the Geobacter transitions
  between the liquid crystalline and gel phases.

(1)G.sulfurreducens dried at 5ºC had a higher Tm
value than samples that were dried at 30ºC in
both 6.75 sucrose and media solutions.
(2)G.sulfurreducens dried at 5ºC in media had
slightly higher Tm values than similar samples
dried in 6.75 solutions of sucrose.

• Samples
• Geobacter Media OR
• Geobacter Media 6.75 Sucrose OR
• Geobacter Media 13.5 Sucrose

DRIED, 30ºC
Acknowledgements
FTIR Analysis
Figure 3. Comparison of dried vs. undried samples
This research was supported by University of
Minnesotas Undergraduate Research Opportunities
Program.
FTIR Analysis
UNDRIED