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Biochemical Pathways Underlying Multi-metal Resistance in Pseudomonas fluorescens
By
Jacob Fabio Costanzi
An Undergraduate Thesis submitted to the Department of Chemistry and Biochemistry at
Laurentian University
In partial fulfilment for an Honours Bachelor of Science Degree in Biochemistry.
Approved by:
Supervisor:
Date: _____________________
Second Reader:
Date: _____________________
Abstract
The goal of this research project was to bestow a multiple metal stress consisting
of five metals (Al, Fe, Zn, Ca, Ga) on a model biological system, in this case the
Pseudomonas fluorescens (ATCC 13525 strain) bacterial strain. There is of great interest
in attempting to observe how bacteria are able to thrive under stress conditions. By
making use of various techniques such as high pressure liquid chromatography (HPLC)
and blue-native polyacrylamide gel electrophoresis (BN-PAGE), a full metabolic and
enzymatic investigation were conducted. Using previous knowledge of how the microbe
can effectively eradicate single metal stressors, the goal was to uncover how the cells are
able to reconfigure their internal biochemical processes in order to effectively stave off
stress. Enzyme activity analysis revealed the up-regulation of several key enzymes most
importantly isocitrate lyase (ICL) and acylating glyoxyalate dehydrogenase (AGODH).
These enzymes were a part of a specific pathway within the TCA cycle which saw a large
production of oxalate in the stress spent fluid samples, thus hinting at the possibility that
oxalate serves as a means for scavenging metal stress. The electron transport chain (ETC)
was also shut down evidenced by a down-regulation of both complex I and complex IV
indicating the cells are applying new measures to produce ATP.
II
Acknowledgements
This research project was the culmination of four years of study in the
biochemistry program; however it was my first individual research opportunity. This was
a brand new endeavour and it compared to nothing in which I have experienced
throughout my four years. This project was entirely my own, and would take a lot of
persistence and determination while following the research plan and goals established by
Dr. Appanna. I learned a lot about responsibility, self- assurance and also time
management which are vital skills that can be implemented in every aspect of life.
Even though this project is an individual project, there are many resources within
the laboratory that I was able to utilize in order to ensure I was on the right track
throughout my research. Firstly, I would especially like to thank Dr. Vasu Appanna for
allowing me the honour of working within this rewarding research setting while
introducing me to his research team which have all been very helpful. I would like to
thank Christopher Auger as he was the main supervisor that oversaw my research.
Without his help this project would have been extremely difficult. He guided my research
but also challenged me to find motivation within to obtain the results that I sought after.
He was a reservoir of knowledge and had answers for all of my questions. I also owe a lot
of credit to Azhar Alhasawi, Sungwon Han, Sean Thomas and Varun Appanna who were
all very knowledgeable with the techniques used and could offer advice whenever
needed. I would also like to thank my fellow undergraduate researcher Mohammad
Barez. Going through this difficult research experience was much easier when doing it
with a partner as a lot of help could be shared. Lastly, I would like to thank my family
who have constantly provided support for my studies in hopes that it will pay off one day.
III
Table of Contents
Contents
Abstract ............................................................................................................................... II
Acknowledgements ........................................................................................................... III
Table of Contents .............................................................................................................. IV
List of Figures ................................................................................................................... VI
List of Schemes .............................................................................................................. VIII
List of Tables .................................................................................................................... IX
List of Abbreviations ......................................................................................................... X
1.0. Introduction .................................................................................................................. 1
2.0. Objectives and Hypotheses ........................................................................................ 17
3.0. Methodology .............................................................................................................. 18
3.1. Media ..................................................................................................................... 18
3.2. Agar Slant preparation .......................................................................................... 21
3.3. Growth Culture and Preculture Preparation ........................................................ 21
3.4. Inoculation of Control and Stress Media ............................................................... 23
3.5. Bacterial Cell Treatment........................................................................................ 23
3.6. Control Sample ...................................................................................................... 24
3.7. Stress Sample ......................................................................................................... 25
3.8. Bradford Assay41 .................................................................................................... 26
3.9. Preparation for blue-native polyacrylamide gel electrophoresis (BN-PAGE)...... 27
3.10. Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) ............................. 27
3.11. Enzyme Activity Assays ........................................................................................ 30
3.12. Complex I ............................................................................................................. 31
3.12. α Ketoglutarate dehydrogenase (αKDH) ............................................................. 32
3.13. Malate Dehydrogenase (MDH) ........................................................................... 32
3.14. Fumarase ............................................................................................................. 32
3.15. Isocitrate Dehydrogenase/NADP......................................................................... 32
3.16. Isocitrate Dehydrogenase/NAD ........................................................................... 33
3.17. Malic Enzyme ....................................................................................................... 33
3.18. Pyruvate Kinase ................................................................................................... 33
3.19. Isocitrate Lyase .................................................................................................... 33
IV
3.20. Complex IV........................................................................................................... 33
3.21. Acylating Glyoxylate Dehydrogenase (AGODH) ................................................ 34
3.22. Pyruvate Phosphate Dikinase (PPDK) ................................................................ 34
3.23. High Pressure Liquid Chromatography (HPLC) preparation ............................ 34
3.24. HPLC Process ...................................................................................................... 35
4. Results ........................................................................................................................... 37
4.1 HPLC Results .......................................................................................................... 38
4.2 Blue Native PAGE results ....................................................................................... 41
5.0 Discussion ................................................................................................................... 48
6.0 Conclusions and Future Research ............................................................................... 54
References ......................................................................................................................... 56
V
List of Figures
Figure 1
The different types of pollution and how they contaminate
various areas in the surrounding environments4.
2
The various sources of metal pollutants in the environment
and how they affect local soil and water systems12.
4
Demonstrates how metal toxicity can have local and systemic
effects when in contact with the body20.
7
Illustrates the process of soil washing. This process is beginning
to become more common as a remediation tool, but by the mere
complexity and cost its effectiveness may be limited30.
10
Demonstrates the growth curve used to anticipate the growth
times of the microbe. The curve with triangle markers represents
the control sample, the curve with the square markers represents
the multiple metal stress and the line with circle markers
represents reconditioned cells grown in multiple metals36.
22
Both media samples as the bacterial growth has reached the
stationary phase. The control (A) appears to be more of a
white colour, while the stress (B) is orange. Notice how both
samples appear to be turbid indicating confluency.
37
Represents an example of one of the standards, in this case
oxalate; used to identify the metabolites in the various samples.
38
Figure 8
Chromatogram for the control cytoplasm fraction.
39
Figure 9
Chromatogram for the stress cytoplasm fraction.
39
Figure 10
Chromatogram for the control spent fluid sample.
40
Figure 11
Chromatogram representing the stress spent fluid sample.
40
Figure 12
Representative chromatogram (n=3) data demonstrates the
relative level of metabolites present in each fraction. Each
metabolite is identified by the retention time.
41
Gel result obtained when doing an enzyme analysis assay of
complex IV.
42
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 13
Figure 14
Negative control to complex IV. The reaction is designed to
VI
Figure 15
Figure 16
Figure 17
Figure 18
Figure 19
Figure 20
Figure 21
Figure 22
Figure 23
Figure 24
Figure 25
Figure 26
Figure 27
have no activity in the control or the stress samples.
42
The purpose of this gel is to analyze the enzyme activity of
acylating glyoxylate dehydrogenase in both the control and
stress samples.
43
Negative control for acylating glyoxyalte dehydrogenase,
the reaction is designed not to show any activity in either
the control or stress samples.
43
In-gel enzyme analysis of isocitrate dehydrogenase/
nicotinamide adenine dinucleotide phosphate in both
the control and stress samples.
43
Negative control for isocitrate dehydrogenase/nicotinamide
adenine dinucleotide phosphate.
44
This gel represents the enzyme activity assay that compares
the activity of Isocitrate dehydrogenase/ nicotinamide adenine
dinucleotide.
44
Analysis of the enzyme activity of fumarase in both the control
and stress samples.
44
Relative enzyme activity of complex I in both the control and
stress samples.
45
Enzyme activity determination of malic enzyme in both the
control and stress samples.
45
In-gel analysis of malate dehydrgenase enzyme activity in both
the control and stress samples.
45
Enzyme activity analysis of alpha-ketoglutarate dehydrogenase
in both the control and stress samples.
46
The activity of isocitrate lyase is compared in both the control
and stress samples.
46
Enzyme activity analysis of pyruvate kinase in both the control
and stress samples.
46
This enzyme analysis was performed to identify the level
of pyruvate phosphate dikinase enzyme activity in both the
control and stress samples.
47
VII
List of Schemes
Scheme 1
Scheme 2
Scheme 3
Examples of how metals interact with the amino acids on
enzymes to lead to their inactivation. In schematic A, the
compound undergoes intramolecular binding. In Schematic
B, the compound undergoes intermolecular binding.
Abbreviations: P, protein; E, enzyme; SH, thiol group; M,
methionine (amino acid)18.
6
Previous studies on how P. fluorescens eradicates various
types of metals individually, but there is uncertainty as to how
multiple metals are effectively eradicated36.
14
Illustrates how aluminum stress can welcome a host of reregulation among the bacterial internal pathways both in
the TCA and the ETC38.
16
Scheme 4
This is the basis of any of the enzyme activity analysis reactions.
They all attempt to couple the activity of the enzyme to formazan
precipitation in gel43.
31
Scheme 5
Full schematic of how some of the cellular processes are
reconfigured to attempt to give a cell a chance to survive
the multi-metal stress.
VIII
52
List of Tables
Table 1
Various chemicals and their perceived level of toxicity15.
Table 2
Demonstrates the different types of plants and metals they
can extract from the soil and how they do it. Abbreviations:
PE, phytoextraction; PV, phytovolitalization; RF,
rhizofiltration; CA, chelate assisted phytoextraction; C,
continuous phytoextraction35.
Table 3
5
12
The ingredients required to prepare both the media, the stress
requires the addition of the metals.
19
The trace metals that are added to each media, these are
prepared in advance by lab staff.
19
Table 5
Metals that are used to stress the bacteria.
20
Table 6
Illustrates the reagents used to prepare the agar slants.
21
Table 7
Denotes the reagents used to create the cell storage buffer
(CSB).
24
Table 8
The preparations for the Bradford Assay.
26
Table 9
The Blue-Native preparation for the proteins for each fraction.
27
Table 10
Table illustrates the reagents used in the preparation of the
gradient gel. It should be noted that the ½ stack is used for
one gel and the full stack is for 2 gels.
28
Table 4
IX
List of Abbreviations
ADP
adenosine diphosphate
AGODH
acylating glyoxylate dehydrogenase
αKGDH
alpha ketoglutarate dehydrogenase
APS
ammonium persulfate
ATCC
american type culture collection
ATP
adenosine triphosphate
BN-PAGE
blue-native polyacrylamide gel electrophoresis
CoA
coenzyme A
CSB
cell storage buffer
˚C
degree Celsius
DCIP
2,6-dichlorophenolindophenol
dH2O
distilled water
ddH2O
deionized distilled water
ETC
electron transport chain
g
relative centrifugal force
HPLC
high pressure (performance) liquid chromatography
ICL
isocitrate lyase
ICDH/NAD
isocitrate dehydrogenase/nicotinamide adenine dinucleotide
ICDH/NADP
isocitrate dehydrogenase/nicotinamide adenine dinucleotide
phosphate
INT
iodonitrotetrazolium
L
liter
X
µg/mL
microgram per millilitre
µL
microliter
mA
milli amp
mg/mL
milligram per millilitre
mL
milliliter
mM
millimole per liter
M
mole per liter
nm
nanometer
NAD+
nicotinamide adenine dinucleotide (oxidized)
NADH
nicotinamide adenine dinucleotide (reduced form)
NADP
nicotinamide adenine dinucleotide phosphate (oxidized)
NADPH
nicotinamide adenine dinucleotide phosphate (reduced)
PPDK
pyruvate phosphate dikinase
PK
pyruvate kinase
PMS
phenazine methosulfate
TCA
tricarboxylic acid cycle
TEMED
N,N,N,N-tetramethylethylenediamine
rpm
revolutions per minute
XI
1.0. Introduction
The environment is very important to the health and safety of its inhabitants. This
does not deal strictly with humans but also any wildlife and ecological habitats. Since the
turn of the 19th and 20th centuries, we have become more reliant on industrialization1.
Many of the luxuries in which we enjoy today are as a result of industrialization. With the
great advancements we have made as a society in terms of industrialization,
modernization and even urbanization, there is also a cost that may in turn threaten the
stability of the environment and ultimately the planet. The downside to every major
advancement is certainly the pollution that is left behind by the various processes in
which we employ2. Pollution endangers many aspects of our environment and in turn can
harm the very fabric which supports life. To continue to prosper both economically and
existentially, we must consider ways to minimize our output of harmful products and find
more efficient ways in which various industrial processes can be carried out1. Also, for
the damage that has already been done, more effective and cheaper methods must be
implemented to clean up areas that have already been impacted.
Some of the most prominent areas that can acquire pollution are the soil and water
sources near and around properties where they are subjected to run off or contamination
by other means3. Soil is very absorbent in the sense that when pollutants come into
contact with it, they can percolate deeper into the soil with the aid of rain water or
through thawing of the snow in the spring, expanding the area of contamination. It may
not be apparent at first thought, but when glancing at figure 1, the effects of pollution can
influence large areas.
1
Figure 1: The different types of pollution and how they contaminate various areas in the surrounding
environments4.
The search for an adequate example of this problem is not an arduous task as we
experience it readily while living in a city like Sudbury. A major economic asset comes
from the mining sector; however, along with the large financial aid it provides for the
city, the industry as a whole (development and further exploration) may subject the
environment to many types of pollution, with ground pollution being a major source. In
Sudbury there are examples of contamination in the lakes and properties that have been
affected in large part by the mining sector5. So not only is this a problem that can occur
anywhere but it is pertinent to this city.
2
The term pollution is a general term used to describe a wide variety of sources
that contaminate an environment. There are many sources of pollution but the focus will
mainly be on metal pollutants in the environment. Metal pollutants occur as a result of
many processes in our society. There is evidence that many sectors that are responsible
for producing the vast majority of metal pollutants such as the agricultural sector, the
mining sector, the water treatment sector, the burning of fossil fuels (vehicle emissions)
and other industrial processes6. These materials are a result of the by-products and waste
products from these various industrial processes mentioned above. For example, in the
agricultural sector, metal pollution in the environment may occur as a result of the
pesticides or fertilizers that are sprayed on crops which contain these toxic metals or
within the fecal matter of the farm animals7,8. Metals remain chelated to the organic
matter of the feces and once decomposition occurs, the metals are released and
accumulate in the soil8. Metals make their way into the soil often carried by run-off to
adjacent bodies of water, land and properties where they begin to leach through the
ground. This is commonly seen on roadways where run off from highways carries many
metal contaminants from the road forcing them into the grounds adjacent to these
roadways9. This concept brings forth the idea that metal pollutants are not only imminent
at the site of contamination but as they leach and percolate through the ground, they have
the potential to contaminate vast areas. Also if pollution reaches a body of stagnant
(ponds) or moving water (rivers and streams), metals can be carried vast distances and
affect the wildlife within and all that use it for nutrition and habitat10. Not only is the
water at harm itself but there is the opportunity for the metals to be absorbed by the
underlying soil of these bodies of water which in turn can cause further complications11.
3
The figure below (figure2) gives a better indication of where metal pollution occurs in
our society and how the environment is affected.
Figure 2: The various sources of metal pollutants in the environment and how they affect local soil
and water systems12.
One of the major factors that aid the migration of metal pollutants through the soil
is acid rain. Along with other sources of pollution in the environment such as the burning
of fossil fuels and other sources; the result is an acidification of rain13. This acid rain falls
onto the ground which reduces the pH of the soil. This helps to solubilize any metals that
lie within the soil already; promoting their migration deer and deeper into the soil
ultimately contaminating larger areas14. In general, pollution is harmful to the
4
environment but when analyzing how one type of pollution affects another, the
magnitude of this problem is quickly seen. Table 1 below lists some common metals that
are frequently used in industry and categorizes them according to toxicity upon exposure.
Table 1: Various chemicals and their perceived level of toxicity 15.
Nontoxic
Aluminum
Bismuth
Calcium
Cesium
Iron
Lithium
Magnesium
Manganese
Molybdenum
Potassium
Strontium
Rubidium
Sodium
Low toxicity
Barium
Cerium
Dysprosium
Erbium
Europium
Gadolinium
Gallium
Germanium
Gold
Holmium
Neodymium
Beryllium
Promethium
Rhenium
Cadmium
Rhodium
Chromium
Samarium
Cobalt
Scandium
Copper
Terbium
Hafnium
Thulium
Tin
Ytterbium
Yttrium
Actinium
Antimony
Praseodymium
Boron
Moderate to high toxicity
Indium
Tantalum
iridium
Thallium
Lead
Thorium
Mercury Titanium
Nickel
Tungsten
Niobium Uranium
Osmium Vanadium
Palladium Zinc
Platinum Zirconium
Polonium
Radium
Ruthenium
Silver
Metal pollution does cause a substantial amount of harm to biological systems. It
affects humans in many ways causing a multitude of problems and there are many studies
being done, even within our lab to determine how a biological system (bacteria and
eukaryotic cell lines) reacts to metal pollutants. There has been a large amount of
documentation on the effects of metals and metalloids such as arsenic, lead, mercury and
cadmium as they are often found in the highest abundance in any contaminated area due
to their heavy traditional uses16.
There are however some metals that do provide
biological function and are necessary for the dietary and nutritional needs of the body.
Metals such as iron, zinc and calcium are essential for the body17,18. The difference
between a therapeutic function and a toxic function does also rely heavily on many
5
factors such as dose, exposure time and frequency of exposure15. There are a host of
conditions that result with exposure to metals most namely metal toxicity15, which is an
acute response to those metals that are poisoning an individual18. Noxious metals are able
to obstruct enzymatic functions very readily in the body. When metals are absorbed by
the body, they often form complexes with proteins especially those which contain iron
sulfur compounds such as cysteine and methionine. These complexes ultimately decrease
the function of a protein or enzyme as it is unable to bind any substrates17,18. Scheme 1
below illustrates how metals typically cause dysfunction in proteins or enzymes.
Scheme 1: Examples of how metals interact with the amino acids on enzymes to lead to their
inactivation. In schematic A, the compound undergoes intramolecular binding. In Schematic B, the
compound undergoes intermolecular binding. Abbreviations: P, protein; E, enzyme; SH, thiol group;
M, methionine (amino acid)18.
There are also other conditions such as skin rashes and infections (contact
dermatitis) which result when there is contact exposure to metal pollutants17,18,19. There
have also been indications that metal pollutants contribute to high cholesterol and
6
cardiovascular problems, problems within the GI tract, depletion of the immune system
and even endocrine disorders especially dealing with the thyroid gland17,19. There have
been many studies on how metals affect the neurological system and there may be links
between metal toxicity and neurological diseases such as multiple sclerosis (MS),
Alzheimer’s disease, Parkinson’s disease and many more18,19. Figure 3 illustrates the
various systems of the body that are impacted by metal toxicity.
Figure 3: Demonstrates how metal toxicity can have local and systemic effects when in contact with
the body20.
When analyzing all the harm that can be done to the human body, while dealing
with metal pollution, and seeing the amount of sources that emit metal pollution it is not
7
difficult to make the link that this is an imminent and concerning problem in our society.
If we are going to continually implement processes which result in the production of
metal pollution there may need to be modifications set in place that reduce the amount of
metal pollution released into the environment so it can be preserved. This is not just a
problem for humans but also wildlife with heavy concern for accessible water sources. In
cutting back on the pollution being produced, half of the battle is won but what can be
done with the environments that have already been contaminated and have accumulated
metal contaminants for many years? Systems must be put in place to rid these areas of
their contamination, but how? Are there even measures in place that are able to
effectively clean up contaminated soil?
The answer to this question is yes! However some of the methods that are in place
today can themselves be very complicated, costly and only temporary. The term used to
denote any “clean up” effort is remediation. Remediation by definition is quite simply the
direct reversal or halting of environmental damage21. There are many methods in place
that are used to effectively remediate contaminated areas but they may not be as effective
for remediating metal pollution. Remediation is fairly effective for organic pollutants but
for metals, there is a constant ongoing search to find more efficient methods22. Some
important techniques that are used for remediation today include: leaching ponds,
incineration, soil washing, and water treatment to name a few23,24,25. These methods are
decent methods but are all weighed down by cost, complication, inefficient remediation
and a detriment to the environment themselves; meaning that even though they may rid
an area of pollutants, there may be an introduction of pollutants or a transfer of pollution
to another area.
8
If we take incineration for example, while burning off pollutants in a
contaminated sample may rid that sample of pollutants, it also vaporizes other forms of
pollution which accumulate in the atmosphere and contribute to other problems such as
acid rain. These volatilized pollutants include metals, and if they bypass a filtering
process, they will be introduced into the atmosphere where there is a high possibility they
will cause secondary environmental contamination26. Leaching ponds as well, require
large areas for construction and as contaminated samples flow into the ponds, they are let
to sit open to the atmosphere. This method is used to extract desired substances from raw
materials and is commonly termed in situ leach (ISL). This method is often utilized in the
extraction of materials in mining27. This concept is also applied to remediate waste
products especially contaminated water which may be littered with dissolved metal
solutes and even radioactive substances. The contaminated water and any other chemicals
that are introduced into the pond in an attempt to cause separation or precipitation of any
dissolved metals or solutes are biologically available to cause sickness and disease as
wildlife may inadvertently make contact with these sources (ingesting, swimming)28.
Even though humans may know not to enter these areas, animals may come in contact
with the harmful chemicals inadvertently causing effects within wildlife29. The cost to
construct these ponds is immense as well.
Soil washing or extraction is a relatively arduous process that involves isolating a
contaminated area and excavating the soil and washing it by various leaching and
chemical techniques designed to precipitate contaminants in the soil30. This may be very
time consuming and difficult if the contamination covers a large area and also extends
deep into the ground. There are many different techniques of soil washing to isolate and
9
remediate contaminated soil specimens but they all focus on the same goal which is to
shed any metals or other pollutants from the sample. This method itself is very effective
for many types of pollution; however, metal contaminants are still very difficult to
eradicate31. This method must also account for dispersion of contaminants as a vast area
of soil may be affected. This process may also be burdened by high costs. In Figure 4
below, there is an example of how soil washing is carried out.
Figure 4: Illustrates the process of soil washing. This process is beginning to become more common
as a remediation tool, but by the mere complexity and cost its effectiveness may be limited 30.
Water treatment is one of the most advanced forms of remediation in our society,
and it is very important in ensuring that there is clean water for a city’s residents. As
water enters the treatment plant, there are many filtering processes involved; aimed at
removing contaminants from the water supply. However, as the degree of water pollutant
changes, so does the process of water purification32. Our water sources are subjected to
10
many forms of pollution ranging from sewage and other organic sedimentation to
resistant microorganisms and of course metal pollution. The process must account for
many types of pollutants and be able to eliminate them all. More steps may have to be
added to the process in order to ensure that the water being supplied to a city is free of all
harmful pollutants32. This will contribute to growing costs and also more complexity to
the process which may hinder this process in the future.
The physicochemical processes involved in pollution eradication today are very
complex, inadequate, and costly while being incapable of fully detoxifying metal
pollution. The threat of metal pollution never subsides so there must be a method in place
that can remediate toxic metals efficiently while minimizing the impact on the
environment.
Bioremediation is the process of remediation which utilizes a biological system.
This can be done with plants, bacteria, fungi and other types of fermentation cultures33.
Plants can be employed in a variety of facets in bioremediation; the use of plants for the
detoxification of polluted soil and water samples is known as phytoremediation34. There
are many different types of phytoremediation most notably, phytodegradation which
actually involves the breakdown of pollutants in a sample and phytoextraction which is
characterized by the bioaccumulation of pollutants within the tissues of the plant34.
Certain plants can be used for this process as they must be able to grow in metal
contaminated soils. This capability varies among plants (Table 2). Once the plant
successfully grows in contaminated conditions it must be able to uptake metals from the
soil, a capability that not all plants possess entirely. Processes can be undertaken to
enhance a plant’s accumulation capabilities, such as treating the soil with chelating
11
agents for example ethylenediaminetetraacetic acid (EDTA)34. The plants are able to
accumulate these harmful pollutants while still being able to grow and thrive are the most
sought after candidates. The only downfall is that the volume of plants required to
effectively neutralize an area may be quite high but this cost would not even near the cost
of some of the other methods34.
Table 2: Demonstrates the different types of plants and metals they can extract from the soil and how
they do it. Abbreviations: PE, phytoextraction; PV, phytovolitalization; RF, rhizofiltration; CA,
chelate assisted phytoextraction; C, continuous phytoextraction35.
Metal
Plant
Method
Comments
Pb
Brassica juncea
PE-CA
EDTA-enhanced uptake over
one cropping season resulted in
a 28% reduction in the Pb
contamination area
Cd
Zn
Thlaspi caerulescens
Silene vulgaris
PE-C
Phytoextraction of sludgeamended soils. Cd accumulation
was similar in all three species.
Zn accumulation in T.
caerulescens was 10-fold higher
than in other plants
Zn
Cd
Ni
Cu
Pb
Cr
Brassica oleracea
Raphanus sativus
Thlaspi caerulescens
Alyssum lesbiacum
Alyssum murale
Arabidopsis thaliana
PE-C
Sludge-amended soil
Se
B
Brassica juncea
Festuca arundinacea
Hibiscus cannibus
Lotus corniculatus
PE-C
PV
Water extractable B was
reduced between 24-52%
and total Se reduced between
13-48% by all species
U
Helianthus annus
RF
Removal of U from Ohio
ground water
12
This method can also be mimicked with microorganisms as well. Small single cell
fungi and bacteria are very adaptable and may be able to use metals as a form of nutrition
for energy production or simply they may be able to metabolize the metals so that they
are no longer in solution and can be easily removed from a sample.
A good method would be to utilize a highly adaptable microorganism which can
effectively live in conditions where metal pollution is high and metabolize these metals
into a form where they are no longer detrimental to the environment. This leads into the
research I will be performing. The use of microorganisms to eradicate metal pollutants
can be a very cheap and efficient alternative to the remediation techniques that are in
place today.
The microorganism that will be studied, for its efficiency in remediating metal
pollution is Pseudomonas fluorescens (P. fluorescens). P. fluorescens is a non-virulent
gram negative bacterium that is normally found in the soil. It is a very adaptable strain of
bacteria and can be grown in virtually any media. It gets its name (Pseudomonas
fluorescens) by its ability to fluoresce under ultra violet (UV) light36. This fluorescence is
not always emitted by this bacteria, it only fluoresces under low iron conditions when the
bacteria is deficient in iron (Fe3+)37. The specific bacterial strain that will be used is the
13525 American type culture collection (ATCC) strain.
There has been substantial research on this strain of bacteria especially by Dr.
Appanna and his research team. One thing that is known about P. fluorescens is that it is
very adaptable nature gives it the ability to grow and thrive in virtually any environment.
Also it is non virulent to humans and very cheap to maintain which makes it an enticing
vehicle of study. From previous work done by Dr. Appanna and his research team, there
13
is plenty of evidence that illustrates how P. fluorescens can effectively eradicate single
metals such as aluminum and zinc, but there is only minimal research on the eradication
of multiple metals38. Scheme 2 demonstrates some of the previous work done by Dr.
Appanna that has resolved the mysteries of how P. fluorescens is able to scavenge some
metals individually; this can help predict how the microbe will react to a multiple metal
stress.
Scheme 2: Previous studies on how P. fluorescens eradicates various types of metals individually, but
there is uncertainty as to how multiple metals are effectively eradicated36.
The research that has been done by Dr. Appanna and his research team on
multiple metal stress was exploratory work on how well they thrived under the stress
conditions as well as how the bacteria were able to expel the metals into the external
environment; the studies did not investigate the methods that bacteria employed to
enhance their survival under the given conditions. From the work that was previously
done with multiple metal stress, it can be seen that metals do impede the growth of this
14
bacteria as well36. Do to its highly adaptable nature, P. fluorescens is able to readjust
their internal metabolism which in turn helps them regain a high level of growth similar
but not quite equal to how they thrived prior to the introduction of the stress.
The bacterial internal metabolic pathways are the most important aspects of this
research; they can give us a very good idea at how the bacteria are effectively able to
reprogram their metabolism so they are able to thrive under a new condition. When in a
stressed environment, one of the most important pathways affected is the production of
ATP38. The latter is the most basic energy currency of the cell and is used to power all the
functions that go on within the cell. Once the production of ATP is diminished, the cell’s
growth and survival begins to slow. By rearranging the method used to fabricate ATP, the
microbe will be able thrive under new conditions.
Much has been uncovered of the metabolic re-regulation when the cell is faced
with single metal stressors but in nature, a cell may not have the convenience of being
stressed by one metal at a time. Depending on the environment that the cell is living in, it
may encounter multiple stressors at once. At this point the cell must employ an effective
method to ward of the stress. This may mean dealing with each stressor individually or
employing a broader method which will be used to safely discard all of the metal
stressors in one shot so to speak. Scheme 3 demonstrates how the internal metabolism of
the bacteria can be geared toward scavenging a single metal, in this case; this is work
done by Dr. Appanna’s research team where the bacteria were being stressed solely by
aluminum38.
15
Scheme 3: Illustrates how aluminum stress can welcome a host of re-regulation among the bacterial
internal pathways both in the TCA and the ETC38.
Information about how the cell survives in a multiple metal stress condition can
be obtained by analyzing processes within the cell. Bacterial cells are prokaryotic,
meaning that they lack the cellular organization when compared to a eukaryotic cell and
are essentially divided into a cytoplasmic and a membrane component. This means that
the metabolism can be easily analyzed by investigating what is occurring in each fraction.
Even though there is little intracellular organization for prokaryotic cells, energy
production and metabolic processes occur in specified areas of the cell. It can be seen that
glycolysis and the tricarboxylic acid cycle (TCA) takes place within the cytoplasm of the
cell and the ATP producing electron transport chain (ETC) occurs on the cellular
membrane fraction. When introducing a stressor to the cell, it may cause the reengineering of one or one or more of these processes ultimately allowing the cell to better
adapt to the conditions (which is seen in scheme 2).Understanding how the cell can find a
way to survive a multiple metal stress will be a useful tool to help maximize its potential.
16
Once a full understanding is obtained about the methods P. fluorescens uses to eradicate
a multiple metal stress, then this microbe may be potentially implemented into
bioremediation techniques39.
There are future research options that could be implemented as well, such as
designing a specific reaction vessel (bioreactor) for this microbe so that its metal
detoxification potential is maximized. Once this is implemented, bioremediation using P.
fluorescens may be a very popular technique used to clean up metal pollution in a variety
of settings; ultimately leading to the survival and benefit of the environment and its
inhabitants.
2.0. Objectives and Hypotheses
When an organism is challenged with a stress in its living environment, it has the
option to adapt to its surroundings or die. Luckily P. fluorescens is a highly adaptable
strain of bacteria and will quickly be able to adjust and thrive under a stress condition36.
The goal of this research project is to impose a multiple metal stress on P. fluorescens.
The stress, consisting of 5 metals (aluminum, iron (III), zinc, calcium and gallium) has
been previously studied by Dr. Appanna in the past however the aim is observe through
metabolic studies, how the cell is able to alter its internal biochemical pathways to
promote survival38. The main goal is to look at the regulation of the various metabolic
enzymes in both a controlled and stressed cell culture. The cells are grown in a citrate
media to provide a carbon source and the regulation of the stressed cells will be
compared to that of cells that have not been subjected to a multiple metal stress.
17
Analyzing enzyme regulation will provide information regarding how the cells are able to
combat the stress condition.
There has been previous research done on how this strain of bacteria is able to
combat individual metal stressors one at a time but there has not been extensive research
on how it deals with a multiple metal stress. When hypothesizing on how the cell will
react to the metal stress, there is strong evidence that the cell will implement a broad
method of scavenging the stress38. In terms of enzyme regulation and energy production,
in extrapolating from how the cell dealt with other forms of stress, it can be hypothesized
that there will be many changes in the TCA cycle which involve pathways specifically
designed to optimize ATP and essential metabolite production38,40.
3.0. Methodology
The methods used in this research project followed the protocol established by Dr.
Appanna’s research team in the Cellular response to a multiple-metal stress in
Pseudomonas fluorescens (1996)36. Some of the methods used in this study and the metal
concentrations were followed; however, the full protocol of this lab will be discussed in
detail below.
3.1. Media
The first step and one of the most important features of this research experiment
was the preparation of the media which was used to grow the bacteria. There were two
different media prepared, each of which was a normal phosphate medium where the
phosphate concentration was 0.64 mM. The normal phosphate medium was used in order
to provide an ample phosphate source to supply the bacteria to foster growth and to
18
ensure that the only influence on bacterial proliferation was attributed to the stress that
would be added to the medium (metals). The first medium that was prepared was the
“normal” medium and will serve as the control. The second medium was prepared in
exactly the same way as the control medium with the exception that the second medium
was impregnated with metals in order to provide a source of stress that will impede the
cell’s growth. This medium represented the stress. One important feature of preparing the
stress medium is that the stress metals must be allowed to chelate to the citrate carbon
source for 30 minutes. If this chelation does not occur, the metals will simply precipitate
out of the solution36. The chelation step is essential to ensure that the bacteria will take up
the metals. The five metals that were used to impose a stress on the bacteria were
aluminum (5mM), iron (5mM), zinc (3mM), calcium (2mM) and gallium (1mM)36. Also
added to both media was a sample of trace metals. The trace metals were added to
provide micronutrients needed by the bacteria to survive. The actual contents of both
media and the trace metals can be seen in table 3, 4 and 5 below.
Table 3: The ingredients required to prepare both the media, the stress requires the addition of the
metals.
Reagent
Normal Phosphate weight
(g)
0.06
0.03
0.8
0.2
4.0
Na2HPO4
KH2PO4
NH4Cl
MgSO4•7H2O
Citrate
Molecular Weight (g/mol)
142.0
136.1
53.5
246.5
210.1
Table 4: The trace metals that are added to each media, these are prepared in advance by lab staff.
Molecular
Weight (g/mol)
270.30
197.91
297.47
Trace Metal
Fe3+
Mn2+
Zn2+
Concentration
(µM)
2
1
0.5
19
Reagent
Grams/250 mL
FeCl3•6H2O
MnCl2•4H2O
Zn(NO3)2•6H2O
0.1350
0.0500
0.0002
110.99
281.10
170.48
241.98
Ca2+
Co2+
Cu2+
Mo2+
1
0.25
0.1
0.1
CaCl2
CoSO4•7H2O
CuCl2•2H2O
NaMoO4•2H2O
0.0275
0.0070
0.0043
0.0063
Table 5: Metals that are used to stress the bacteria.
Metal
Aluminum chloride
Iron (III) chloride
Zinc chloride
Calcium chloride
Gallium (III) nitrate
Molecular weight
(g/mol)
241.40
270.30
136.28
147.01
255.7
Concentration (mM)
Weight (g)
5
5
3
2
1
1.2070
1.3515
0.4088
0.2940
0.2557
For both media, the ingredients were dissolved in a small beaker containing
distilled deionized (ddH2O). As mentioned above for the stress media; the metals were
put through a chelation process for 30 minutes and once all the reagents were added, both
media (control and stress) were adjusted to a pH of 6.8. Once at the desired pH, both
media samples were adjusted to a volume of 1000 mL (1L). It was much safer to adjust
the pH to 6.8 prior to adjusting the volume as pH adjustment may overshoot the desired
volume. It is also much easier to adjust the pH of smaller volumes.
The prepared media was transferred into stoppered 500 mL Erlenmeyer flasks and
autoclaved for 45 minutes. This autoclave option was implemented to ensure that no
other microorganisms were living within the media and that the growth that was seen was
solely based on the survival of the desired microbe (P. fluorescens). Even after the media
has been autoclaved, the flasks that contain the media must be stoppered tightly with a
foam stopper and remain stoppered at all times to ensure no contamination will occur.
Any media that developed turbidity at room temperature were indicative of
contamination and were discarded.
20
3.2. Agar Slant preparation
The agar slant serves the same purpose of the growth media, so it must contain
similar reagents in order to foster the growth of the bacteria. The preparation was
essentially the same as for the control media above with the exception that Bacto agar is
added to cause solidification. The total volume of agar to be prepared varies but a 200
mL sample is enough to prepare approximately 20 slants. The agar slants were prepared
according to the ingredients in table 6 below.
Table 6: Illustrates the reagents used to prepare the agar slants.
Reagent
Na2HPO4
KH2PO4
NH4Cl
MgSO4•7H2O
Citrate
Bacto Agar
Weight (g)
1.2
0.6
0.2
0.04
0.8
3.3
Everything is dissolved in ddH2O (dH2O can be substituted), once dissolved the
solution is adjusted to a pH of 6.8 and the volume is adjusted to 200 mL. The agar media
again has to be autoclaved for 45 minutes, however agar solidifies so the slants must be
prepared quickly. The heated agar media is poured into test tubes and the test tubes are
laid on an elevated surface to create the slanted appearance.
3.3. Growth Culture and Preculture Preparation
Prior to the preculture being prepared, if there is no knowledge of how the
microbe grows, a growth curve must be created to determine the length of time the
bacteria require to reach confluency. Luckily for this research experiment the growth
21
curve was already done in a previous study by Dr. Appanna’s research team 37 and can be
seen below (figure 5).
Figure 5: Demonstrates the growth curve used to anticipate the growth times of the microbe. The
curve with triangle markers represents the control sample, the curve with the square markers
represents the multiple metal stress and the line with circle markers represents reconditioned cells
grown in multiple metals36.
The preculture is the initial growth of the bacteria that is used to inoculate the
media. This process is a sterile process and involves the use of a flame so proper
precautions and safety equipment must be used. The preculture is prepared by inoculating
100 mL of the control medium with the Pseudomonas fluorescens strain 13525 (ATCC).
In sterile conditions, an old agar slant containing the bacteria was used to inoculate a
brand new slant for the next person to use. Then, using the old slant, the preculture was
inoculated and allowed to grow for 24 hours in a gyratory water bath.
22
3.4. Inoculation of Control and Stress Media
After the preculture was allowed to grow for 24 hour, it was time to inoculate the
media. When looking at the growth curve in figure 5, the control and stress have varying
growth times so this must be taken into account when inoculating the media. The growth
curve is comprised of 3 basic phases, the lag phase is where the bacteria begin to multiply
and adapt to their surroundings, the exponential growth phase is where the bacteria
proliferate very quickly, and the final stage is the stationary phase where the bacteria
reach confluency36. The stationary phase is the ideal phase to terminate growth in order to
obtain bacterial cells that have thrived through the stress. Anything past this phase may
cause the cells to implement other methods in order to metabolize their own waste
products giving misleading metabolic results36.
Under sterile conditions, 200 mL of each the control and stress media were
inoculated with 1 mL of the preculture. The control sample was left to grow for 23 hours
and the stress sample for 27 hours. Once the bacteria have reached the stationary phase,
the media will become opaque, which is exemplified in Figure 6 below.
3.5. Bacterial Cell Treatment
After the bacterial cells have reached the stationary phase, growth was terminated.
At this point sterility is not an issue any longer so aseptic technique can be omitted. Each
sample; both the control and stress were treated differently in the centrifugation process
in order to isolate the bacterial cells.
23
3.6. Control Sample
For the control sample, there were 3 spins; two spins at 11 000 rpm (18 000 g)
and one at 50 000 rpm (215 000 g) both at 4˚C. Once growth was terminated at 23 hours,
the sample was dumped into a centrifuge tube, balanced with a water sample and spun for
15 minutes at 11 000 rpm (18 000 g) for 15 minutes. After the spin was complete the
solution separated into a supernatant and a pellet. The pellet was the clumping of the
bacteria and the supernatant was strictly the extracellular media or spent fluid. The spent
fluid could reveal a lot about how the bacteria thrived in their environment as it contained
waste products and other metabolites released by the cell. A 10 mL sample of the spent
fluid was taken and frozen for HPLC analysis.
The remaining spent fluid was discarded in the sink while the pellet was
resuspended using 0.85% NaCl. Once homogenized, the sample was balanced with a
water sample and respun using the exact same parameters as the first spin. This second
spin served to wash the pellet. After the spin was complete the supernatant which was the
NaCl washing was discarded along with any debris it removed from the pellet. The
remaining pellet was suspended in 1 mL of cell storage buffer (CSB), which serves to
preserve the cells and the reagents that comprise the buffer can be seen in table 7 below.
The cells were stored at 4˚C overnight.
Table 7: Denotes the reagents used to create the cell storage buffer (CSB).
Reagent
Weight (g)
Tris-HCl
0.3029 (50 mM)
PMSF
0.0090 (1 mM)
DTT or Tricarboxylic for sulfhydryl
0.0077g
protection/aconitase stability respectively
Adjusted to pH 7.6 and created 50 mL solution
24
3.7. Stress Sample
The stress sample also underwent 3 spins, one at 1500 rpm (340 g), one at 11000
rpm (18 000 g) for 15 minutes each, and one at 50000 rpm (215 000 g) for 3 hours. All
spins were at 4˚C. Once the growth was terminated at 27 hours, the sample was
transferred to a centrifuge tube, balanced with a water sample and spun for the first spin.
This was a low speed spin to attempt to rid the sample of the metals. Once the spin was
complete a sample of 10 mL of the supernatant was harvested for the spent fluid and
frozen immediately, the remaining supernatant was discarded. The pellet was washed
with 0.85% NaCl , balanced with a water sample and respun at 11 000 rpm (18 000 g).
After this spin the washings were discarded and the pellet was suspended in CSB and
stored at 4˚C overnight.
The following day both the control and the stress samples were transferred into
test tubes for sonication. Sonication uses sound to lyse the cells and separate them
according to cytoplasmic or membrane fractions. The process was always carried out on
ice to avoid denaturing the proteins. The cells were subject to sonication for 15 second
intervals with 5 minute breaks in between. These breaks were a necessary precautionary
measure to avoid denaturing proteins. Once the control and stress fractions have been put
through the sonication process, both samples were put into centrifuge tubes, balanced and
spun for the long high speed spin (50 000 rpm or 215 000 g for 3 hours). This spin
separated each sample into cytoplasmic and membrane fractions.
After the spin was complete, the fractions were separated into 4 different
fractions. The 4 fractions included: control cytoplasm, control membrane fractions, as
well as stress cytoplasm, stress membrane fractions. The cytoplasmic fractions were the
25
supernatants in both the control and stress sample. The pellet represented the membrane
fraction for both the control and stress samples and had to be resuspended in 500 µL of
CSB. These tubes were stored overnight at 4˚C.
3.8. Bradford Assay41
The Bradford assay was implemented to quantify the protein that was present in
each fraction41. This was a fairly simple process. It involved creating a blank, a standard
and each of the fractions were done in triplicate (12 samples). In order to properly
quantify protein, both the cytoplasmic and membrane fractions were diluted. The
cytoplasmic fractions, both control and stress were diluted by adding 4 µL of sample to
96 µL of ddH20. The membrane fractions, both control and stress were diluted by mixing
2 µL of sample with 98 µL of NaOH and then boiling each sample for 5 minutes to
attempt to loosen any proteins that may still remain embedded in the membrane. Once all
4 fractions were prepared, the Bradford mixtures were prepared according to table 8
below. The absorbance was taken at 595 nm in order to quantify protein.
Table 8: The preparations for the Bradford Assay.
Fraction
ddH2O (µL)
Blank
Standard
Control Cytoplasm
Control Membrane
Stress Cytoplasm
Stress Membrane
800
750
770
770
770
770
Bradford Reagent
(µL)
200
200
200
200
200
200
26
Protein (µL)
50
30
30
30
30
3.9. Preparation for blue-native polyacrylamide gel electrophoresis
(BN-PAGE)
Once the protein concentrations were known, it was time to prepare the samples
for BN-PAGE. It was hopeful that each fraction could be prepared at 4 mg/mL however
sometimes low protein concentration limited our ability to do so. An example of
preparation can be seen below in table 9. The only difference between the cytoplasmic
and membrane fractions was the addition of 50 µL of 10% (w/v) malthoside which
served to ensure that there were no membrane bound proteins that remained lodged in
fragments of membrane.
Table 9: The Blue-Native preparation for the proteins for each fraction.
Fraction
Protein (µL)
Control
cytoplasm
Stress
Cytoplasm
Control
Membrane
Stress
Membrane
200
3X Blue-Native 10% malthoside
Buffer (µL)
(µL)
166.7
-
H2O (µL)
133.3
309
166.7
-
24.3
100
166.7
50
183.3
156
166.7
50
127.3
Proteins that are prepared for Blue-Native PAGE at the desired concentration can
be analyzed using the PAGE process.
3.10. Blue-Native polyacrylamide gel electrophoresis (BN-PAGE)
The process of BN-PAGE is very interesting. This form of electrophoresis
separates proteins according to their molecular weight but does it in a way that preserves
the native form of the protein. This allows the opportunity for enzyme studies to be done
on the separated enzymes.
27
Firstly the process uses a gradient gel. The gel is poured in a way such that there
is a 4-16% (w/v) gel gradient in the resolving gel and the reagents can be seen in table 10
below. This was done by the use of a gradient former and pump. The gradient former
functions by adding the 16% gel then slowly mixing the 16% and 4 % in the proper
proportion as to produce the proper gradient until only the 4% gel remained. Therefore
from the bottom of the gel to the top, gel gradient ran from 16% to 4 %. Prior to pouring
the gel, it is very important that there are no leaks in the casting apparatus, as the gradient
may be affected. It was necessary to make sure that the glass plates were pressed tightly
together so the gel did not leak out. A check is usually done with water.
Once the resolving gel has been poured, it was overlaid with isopropanol until the
gel polymerized to eliminate any bubbles and help shape the top of the gel into a straight
edge. The gel took approximately 30-45 minutes to polymerize. Once polymerized the
isopropanol was removed with the aid of filter paper. The stacking gel was then poured
onto the resolving gel. The gel was poured right to the top of the glass plates then a wellforming comb was inserted. The gel was allowed to polymerize for about 30 minutes and
then let sit at 4˚C overnight.
Table 10: Table illustrates the reagents used in the preparation of the gradient gel. It should be noted
that the ½ stack is used for one gel and the full stack is for 2 gels.
Reagent
16% Separating gel
(µL)
4% Separating gel
(µL)
½
Stack(µL)
49.5% Acrylamide
3X BN Buffer
dd H2O
75% Glycerol
TEME
10% APS
937
967
223
773
0.8
7.6
234
967
1699
137
568
1000
Full
Stack
(µL)
273
1136
2000
1.0
9.7
2.5
15
5
30
28
After the gel had totally polymerized it was prepared for loading. Ideally it would
be desired to add 60 µg of protein to each well; however, depending on the concentration
of the BN prepared protein that may not be feasible. The wells of the gel were usually
loaded from left to right from the second well as the first well was reserved for the ladder.
The wells were loaded in the order of control cytoplasm, stress cytoplasm, control
membrane and stress membrane. If 60 µg of protein was added, it would translate to 15
µL if the protein fractions were prepared at 4 mg/mL. For the ladder, 10 µL of ferritin
and 10 µL of BSA were added to give an idea of protein migration42.
Once the plate was loaded it was transferred to the electrophoresis unit. This unit
held the plate in place throughout the process. The plate was overlaid with blue cathode
buffer, which was poured into the middle of the electrophoresis chamber. The anode
buffer was poured on the outside of the chamber up to pre-marked levels specific to the
number of gels. The actual electrophoresis was run at 4˚C to ensure that the proteins did
not denature.
The process of electrophoresis was fairly simple. At first the electrophoresis
apparatus was hooked up to the power source. Electrophoresis was run in 3 stages. The
first stage involved running the gel at 15 mA at 80 V for 30 minutes. This ensured that all
the proteins migrated through the stacking gel to the edge of the resolving gel. At this
point the second phase of electrophoresis commences and the power was changed. The
gel was raised to run at 25 mA at 150V until the mobile front migrated halfway down the
resolving gel. This was visualized by a blue line across the gel. At this point the blue
cathode buffered was replaced with a clear cathode buffer. The gel was then run at 25
29
mA at 300 V until the protein had finished their migration throughout the gel. Once
electrophoresis was complete, the gel was now prepared to be subject to enzyme probing.
3.11. Enzyme Activity Assays
The main basis of this project was to study enzyme regulation in both the control
and the stress fractions and compare how they differ in each scenario. The main vehicle
of research used was in-gel enzyme analysis. One of the benefits of using BN-PAGE in
general was that the proteins remained in their native state. With that being said, their
functionality should have been preserved. So by giving an enzyme its substrate it should
produce a product which can be measured. That was the basis of this technique; the
reactions done in gel were coupled to other reactions which produced a precipitate to
denote the presence and function of that enzyme. This is a qualitative measure as the
level of enzyme activity would produce a precipitate which appeared as a band in the gel
with an intensity that was proportional to its activity or regulation. A general example of
this enzyme reaction can be seen in Scheme 4 below. The enzyme is provided with its
substrate and as it converts the substrate into product, that reaction is coupled to another
reaction which leads to the reduction of NAD(P)+ to NAD(P)H and most importantly the
reduction of INT which is marked by a red formazan precipitate appearing as a red band
in the gel44. This process is the basis of these enzyme analysis reactions and each reaction
mixture contains all of the reagents to achieve this red band through the reduction of INT.
30
Scheme 4: This is the basis of any of the enzyme activity analysis reactions. They all attempt to
couple the activity of the enzyme to formazan precipitation in gel43.
These reactions were done with many enzymes, in an attempt to piece together
and differentiate the metabolic pathways of the control and stress samples. Each of the
reaction mixtures were 3 mL as each gel lane required 1.5 mL of reaction mixture. Once
the reaction mixtures were added, the gels were placed in a dark area until the reaction
was completed and a red band appeared in the gel. The reaction was then terminated with
a destaining solution and left covered overnight. The only exception was seen in complex
IV where a brown band signified enzyme activity however the gel was treated the same
(destained and left over night).
3.12. Complex I
The reaction mixture includes 150 µL (5mM) of NADH, 300 µL INT and 2550
µL of reaction buffer +KCN to top the volume up to 3 mL. The mixture was added to two
lanes of the gel that contain a control and stress membrane fraction.
31
3.12. α Ketoglutarate dehydrogenase (αKDH)
The reaction mixture involves 150 µL (5 mM) αKGDH, 3 µL (0.5 mM) NAD+, 3
µL (0.1 mM) CoA, 300 µL INT, 150 µL PMS, and 2394 µL of reaction buffer. The
reaction mixture was added to a sample containing membrane fraction.
3.13. Malate Dehydrogenase (MDH)
The reaction mixture includes adding 150 µL (5mM) malate, 15 µL (0.5 mM)
NAD+, 150 µL PMS, and 300 µL INT and 2385 µL of reaction buffer. The mixture was
added to the gel containing membrane fractions.
3.14. Fumarase
The reaction mixture consists of 150 µL (5 mM) of fumarate, 10 units (2 µL) of
malate dehydrogenase, 15 µL of NAD+, 300 µL INT, 150 µL PMS and 2383 µL of
reaction buffer. The reaction mixture was added to a gel sample that contained membrane
fractions.
3.15. Isocitrate Dehydrogenase/NADP
The reaction mixture is prepared by adding 300 µL (10 mM) Isocitrate , 30 µL
(1mM) NADP+, 300 µL INT, 150 µL PMS, and 2220 µL of reaction buffer. This mixture
was used to probe the cytoplasmic fractions. A negative control of this enzyme assay was
also performed to provide a comparison. The reaction was prepared without NADP+
substrate and more reaction buffer was added in its place (2250 µL).
32
3.16. Isocitrate Dehydrogenase/NAD
The reaction mixture is prepared by adding 300 µL (10 mM) Isocitrate , 30 µL
(1mM) NAD+, 300 µL INT, 150 µL PMS, and 2220 µL of reaction buffer. This mixture
was used to probe the cytoplasmic fractions.
3.17. Malic Enzyme
The reaction mixture is prepared with; 150 µL (5 mM) malate, 15 µL (0.5 mM)
NADP+, 300 µL INT, 150 µL PMS and 2385 µL reaction buffer. The reaction mixture
was added to a gel sample containing cytoplasmic fractions.
3.18. Pyruvate Kinase
The reaction mixture is 150 µL (5 mM) phosphoenolpyruate, 15 µL (0.5 mM)
ADP, 15 µL (0.5 mM) NADH, 10 units (2 µL) lactate dehydrogenase, 150 µL DCIP, 300
µL INT and 2368 µL reaction buffer. The reaction mixture was added to a gel sample
containing cytoplasmic fractions.
3.19. Isocitrate Lyase
The reaction mixture comprises of 60 µL (2mM) of isocitrate, 15 µL (0.5 mM)
NAD+, 10 units LDH (2 µL), 300 µL INT, 150 µL PMS and 2473 µL reaction buffer.
The reaction mixture was added to a gel sample containing cytoplasmic fractions.
3.20. Complex IV
The reaction mixture is prepared by adding 1.8 mL (5 mg/mL) of
diaminobenzidine, 600 µL (562.5 mg/mL) sucrose, 225 µL (10 mg/mL) cytochrome C
and 375
reaction buffer. This reaction mixture was added to gel containing membrane
33
fraction. A negative control for this enzyme was also done which involved eliminating
cytochrome c and adding more reaction buffer (1400 µL).
3.21. Acylating Glyoxylate Dehydrogenase (AGODH)
The reaction mixture is prepared by adding 225 µL (5 mM) glyoxylate, 29.7 µL
(0.66 mM) CoA, 37.35 µL (0.83 mM) NADP+, 450 µL INT, 225 µL PMS and 2032.95
µL reaction buffer. This reaction mixture was added to a gel sample containing the
cytoplasmic fraction. A negative control for this enzyme was also prepared which
involved removing glyoxylate and adding reaction buffer in its place (2160.6 µL).
3.22. Pyruvate Phosphate Dikinase (PPDK)
The reaction mixture is prepared by adding 150 µL (5 mM) phosphoenolpyruate,
150 µL adenosine monophosphate (0.5 mM), 15 µL (0.5 mM) pyrophosphate, 10 units (2
µL) lactate dehydrogenase, 15 µL (0.5 mM) NADH, 300 µL INT, 150 µL DCIP. The
reaction is added to a gel containing the cytoplasmic fraction.
3.23. High Pressure Liquid Chromatography (HPLC) preparation
HPLC is a technique used to separate metabolites by their retention time in a
column. Different columns have a better affinity to separate different metabolites
however the column used in the lab is a C18 column with a hydrophobic packing. It has
an amide cap which possesses a positive density charge so it can bind to negatively
charged metabolites such as organic acids. The actual HPLC machine contains an
Alliance separation module which separates compounds in a solution by their retention
time to the column. The process uses a 20 mM KH2PO4 (pH 2.9) mobile phase. The
HPLC machine is also equipped with a Waters UV-vis spectrometer that has a dual
34
absorbance detector that can take the absorbance of a solution at 210 nm which analyzes
carbonyl groups and 254 nm which analyzes CN bonds.
The solutions that were used for HPLC actually required very little preparation.
Only the cytoplasmic fractions and the spent fluid were analyzed using HPLC. The spent
fluid was a sample of the supernatant from the first 11 000 rpm spin for the control
sample and the 1500 spin for the stress sample so it did not require any preparation for
HPLC. For the cytoplasmic fractions, both control and stress, 200 µL of each were
pipetted into a separate eppendorf tube and boiled for 10 minutes to denature any proteins
in the fraction. Protein is forbidden to run through the machine as it can obstruct the
tubing.
3.24. HPLC Process
The flow rate was set to 0.7 mL/min and the instrument was allowed to pass
mobile phase for 30 minutes to ensure there was not any residual matter bound to the
column.
While this occurs, it was an excellent opportunity to prepare the samples to be
run. The only samples from the bacteria that were run were the spent fluid samples from
both the control and the stress and the cytoplasmic fractions from both the control and the
stress. The cytoplasmic fractions are spun at 10 000 rpm for 10 minutes to separate the
protein form the samples. The bacterial samples were run along with standards in order to
compare the retention times which will appear as peaks on the spectra. For example if
oxalate appears at 3.725 minutes, and the samples show a prominent peak near this
elution time, this may indicate that there is an abundance of oxalate in the sample. This
can be further confirmed by using a technique known as spiking where a fraction of
35
oxalate (or whatever metabolite being investigated) is added to the sample and run
through the HPLC again. If the peak that was suspected to represent oxalate; for example,
becomes larger and more prominent then this is a confirmation that oxalate is present in
the sample.
To ensure no protein runs through the HPLC machine, the vials are inserted into a
carousel and Pasteur pipettes are inserted into each vial. Each pipette was fitted with a
cotton plug that was stuffed deep into the pipette; this cotton plug acted as a filter and
only allowed metabolites to pass into the vials.
All standards were diluted to a concentration of 1mM (10 µL of standard + 990
µL of water). The bacterial samples were loaded similarly according to their fractions.
The control and stress cytoplasmic fractions were loaded in similar concentration and the
control and stress spent fluid samples were also matched. Each sample ran for 30 minutes
and the results were compared to the standards run. If there was any doubt, the spiking
technique could be utilized to clear any discrepancies.
36
4. Results
After the cell cultures were grown for their respective growth times in order to
reach the stationary phase, they were treated as described in the methods section. Figure 6
below demonstrates the appearance of the cultures once they have reached the stationary
phase. At this point the cells were ready to be harvested and treated for further study. The
results for this project are presented in two forms; there are gel data and HPLC data.
(A)
(B)
Figure 6: Both media samples as the bacterial growth has reached the stationary phase. The control
(A) appears to be more of a white colour, while the stress (B) is orange. Notice how both samples
appear to be turbid indicating confluency.
37
4.1 HPLC Results
The HPLC data are presented in chromatograms which can be seen below. Only
the data obtained from the first channel was analyzed for the both cytoplasm (figure 8
and figure 9) and the spent fluid fractions (figure 10 and figure 11). Below are examples
of the chromatograms that were obtained from the HPLC process along with an example
of one of the standards used for comparison to the chromatograms. The standard seen
below is oxalate (figure 7). The standards had a very sharp and distinct appearance, and
were marked with usually one major peak. The levels of the metabolites in each fraction
are compared in figure 12. There is an uncertainty of the identity of the metabolite
obtained in the spent fluid so only the retention times of both the control and the stress
are reported on the graph.
Figure 7: Represents an example of one of the standards, in this case oxalate; used to identify the
metabolites in the various samples.
38
Figure 8: Chromatogram for the control cytoplasm fraction.
Figure 9: Chromatogram for the stress cytoplasm fraction.
39
Figure 10: Chromatogram for the control spent fluid sample.
Figure 11: Chromatogram representing the stress spent fluid sample.
40
Figure 12: Representative chromatogram (n=3) data demonstrates the relative level of metabolites
present in each fraction. Each metabolite is identified by the retention time.
4.2 Blue Native PAGE results
The electrophoresis process was implemented to separate proteins so that enzyme
activity can be analyzed. Enzyme analysis was performed according to the procedure
described and the results can be seen below.
When enzyme activity is identified, it will appear as a red precipitate in the gel
(except for complex IV a brown precipitate is visible). An example of this can be seen in
figure 13 below. There were a total of 12 enzymes probed and their results can be seen
below. Only complex IV was shown as a whole gel. The remaining gels were cropped to
visualize only the bands where the enzyme activity occurred.
41
Figure 13: Gel result obtained when doing an enzyme analysis assay of complex IV.
Figure 14: Negative control to complex IV. The reaction is designed to have no activity in the control
or the stress samples.
42
Figure 15: The purpose of this gel is to analyze the enzyme activity of acylating glyoxylate
dehydrogenase in both the control and stress samples.
Figure 16: Negative control for acylating glyoxyalte dehydrogenase, the reaction is designed not to
show any activity in either the control or stress samples.
Figure 17: In-gel enzyme analysis of isocitrate dehydrogenase/ nicotinamide adenine dinucleotide
phosphate in both the control and stress samples.
43
Figure 18: Negative control for isocitrate dehydrogenase/ nicotinamide adenine dinucleotide
phosphate.
Figure 19: This gel represents the enzyme activity assay that compares the activity of isocitrate
dehydrogenase/ nicotinamide adenine dinucleotide.
Figure 20: Analysis of the enzyme activity of fumarase in both the control and stress samples.
44
Figure 21: Relative enzyme activity of complex I in both the control and stress samples.
Figure 22: Enzyme activity determination of malic enzyme in both the control and stress samples.
Figure 23: In-gel analysis of malate dehydrgenase enzyme activity in both the control and stress
samples.
45
Figure 24: Enzyme activity analysis of alpha-ketoglutarate dehydrogenase in both the control and
stress samples.
Figure 25: The activity of isocitrate lyase is compared in both the control and stress samples.
Figure 26: Enzyme activity analysis of pyruvate kinase in both the control and stress samples.
46
Figure 27: This enzyme analysis was performed to identify the level of pyruvate phosphate dikinase
enzyme activity in both the control and stress samples.
47
5.0 Discussion
The methods used throughout this project were very important in categorizing the
various enzymatic processes undertaken by the cell in order to thrive in the stress
conditions. When looking at the HPLC chromatograms, the control and stress cytoplasm
seem to have significant amounts of citrate and succinate, (determined by the comparison
to known standards) which can be seen in figures 8 and 9. When looking at the
prevalence for both metabolites, both are less prominent in the stress samples. The
relative level of these metabolites can be seen in figure 12, they are measured by the area
under the curve in µV*sec. The result of this observation can be attributed to a downregulation of a process or enzyme.
The spent fluid can also reveal a lot about the internal physiology of the cell. The
spent fluid is the extracellular media which usually contains the waste products of the cell
and may reveal how the microbe externalizes the metals36. In the control spent fluid
(figure 10), there is a large peak at 3.903 minutes and in stress spent fluid there is a
similar peak at 3.900 minutes (figure 11). When attempting to identify this peak against a
set of standards it is apparent that the metabolite may be oxalic acid. The standard for
oxalic acid (figure 7) sees a retention time of 3.725 minutes which is very close to the
retention times of the major peaks in both the control and stress spent fluids. As a result,
it is speculated that this peak in the spent fluid is representative of oxalic acid which may
be a method used for scavenging38,39. Also, by comparing the relative area under the
curve between the control and stress samples, it can be seen that in the stress, there is a
significantly higher incidence of this metabolite. This information can be utilized to infer
48
that under a multiple metal stress condition, the bacteria utilize oxalate to bind the metals
and remove them from the cells38,39.
At this point this is only an obscure indication and the use of the BN-PAGE results
and enzyme activity analyses, will further piece together the story of the metabolic
regulation of these bacteria. In total there were 12 enzymes probed that contributed to
various metabolic processes. These enzymes were analyzed to determine their relative
level of activity which will differentiate their role in a control versus a stress condition.
The first enzyme that was probed was complex IV. Complex IV is the final enzyme
subunit in the ETC prior to the synthesis of ATP. The results of the enzyme analysis in
figure 13 indicate that there is a down regulation for this enzyme in the stress sample as
the band which is brown, appears in the control and not the stress. This also hints at the
fact that the ETC may not be implemented by the bacteria under stress conditions. Metals
particularly have a profound effect on enzymes that contain iron sulfur clusters. Metals
bind to these iron sulfur clusters and cause the deactivation of the enzyme38. We also ran
a negative control (figure 14) to ascertain enzyme activity.
Acylating glyoxylate dehydrogenase (AGODH) is responsible for converting
glyoxylate and CoA into oxalyl-CoA. This is a modified metabolic pathway which occurs
as a result of the reengineered TCA cycle38. Looking at figure 15, there is evidence of an
upregulation of this enzyme in the stress sample. A negative control was also used for
comparison (figure 16), to help better visualize how the bands appear.
Isocitrate dehydrogenase/NADP (ICDH/NADP) is another enzyme that was analyzed.
It is responsible for the conversion of isocitrate into α-ketoglutarate. The activity of this
enzyme has increased in the stress sample (figure 17) and was even compared to a
49
negative control (figure 18) to confirm regulation. Alternatively the enzyme isocitrate
dehydrogenase/NAD experienced the opposite result. It sees a down-regulation in the
stress sample (figure 19) as this enzyme provides the substrate necessary for the ETC in
NADH38. As the ETC is being shut down there is no need for NADH.
Fumarase is the enzyme involved in the conversion of fumarate into malate in the
TCA cycle. This enzyme can be seen in figure 20 and seems to have a higher intensity in
the stress. There has been evidence that this enzyme’s function is down-regulated in the
stress however the evidence also eludes to the fact that there is an isoform of the enzyme
which may be activated under certain stress conditions. There are two isoforms of
fumarase: fumarase A and fumarase C. Fumarase A contains iron sulfur clusters which
are affected by metal stress but fumarase C does not contain these iron sulfur clusters and
as a result are not affected by the stress38. The gel is unclear but there is a band in the
control lane and a band in the stress lane but at lower migration. Due to the iron sulfur
clusters, fumarase A does not migrate as far a fumarase C in the gel38. This indicates that
this point of the TCA has been affected slightly in order to maintain the TCA cycle.
Complex I is another enzyme that functions in the ETC, it is the first enzyme in the
ETC and is responsible for abstracting an electron from NADH and passing them along
the ETC. By looking at the results obtained in figure 21, it can be seen that this enzyme is
in fact downregulated in the stress. Similar to complex IV, complex I contains iron sulfur
clusters that are affected by metal binding which inactivates the enzyme38. As a result,
this must mean that the ETC is being shut down altogether as complex IV is also downregulated. As a result of the multiple-metal stress, the cell shuts down the ETC. They
50
must also be implementing another means in order to produce ATP as the organism is
still proliferating.
Malic enzyme is an enzyme designed to convert malate into pyruvate, this is used to
generate NADPH for antioxidant defense38,40,42 The activity for this enzyme is upregulated in the stress sample (figure 22).Malate dehydrogenase, which is responsible for
the conversion of malate to oxaloacetate is also up-regulated in the stress (figure 23). The
gel shows three bands for this enzyme. These are all isoforms of the same enzyme. Under
stress conditions, the bacteria may modify the structure of the enzymes to better suit their
needs. This indicates that the TCA may be up-regulated as a whole in order to find a way
to provide the cell with a means of producing ATP as the ETC is being supressed.
α-Ketoglutarate dehydrogenase is another enzyme that was down-regulated under
stress conditions (figure 24). This enzyme is responsible for the conversion of alphaketoglutarate to succinyl-CoA, liberating NADH in the process that commences the ETC,
however the ETC is suppressed as we have seen by the down-regulation or complex I and
IV38. The suppression of the ETC can be attributed to the down-regulation of both
complex I and IV and also by the lack of NADH being produced to initiate the ETC.
Isocitrate lyase is an enzyme specifically utilized by the bacteria under certain stress
conditions38. It is responsible for converting isocitrate into glyoxylate and succinate and
forms the first step of the glyoxylate shunt. Under stress conditions, this enzyme is
upregulated (figure 25),
The remaining two enzymes that were probed were pyruvate kinase and pyruvate,
phosphate dikinase which both function near the end of glycolysis. Pyruvate kinase is
responsible for converting phosphoenolpyruvate into ATP and pyruvate in normal
51
conditions. In stress conditions, this enzymes is down-regulated (figure 26) which
indicates that ATP is not being produced by this way. Pyruvate phosphate dikinase is
responsible for the same reaction as pyruvate kinase, but utilizes AMP and PPi rather than
ADP. According to the gel results in figure 27, this enzyme does not function in this
process.
Having examined a wide variety of enzymes, it makes it easier to piece together the
networks that are involved in the functioning of the cell under multi-metal stress. The
bacteria, thanks in part to their highly adaptable nature are very capable of switching
gears so to speak and alter their metabolic processes in order to effectively thrive under
certain harsh conditions. We have seen this first hand in this experiment that as the
bacteria are introduced to an environment that contains a multiple-metal stress, they are
able to effectively grow and proliferate, not at the same rate as the control albeit but
nonetheless they quickly learn to overcome the impeding stress. Below is a summary
(scheme 5) of all the findings that have been observed so far.
Scheme 5: Full schematic of how some of the cellular processes are reconfigured to attempt to give a
cell a chance to survive the multi-metal stress.
52
To tie the whole story together and make sense of the results that were obtained it is
essential to try and find a link between what was obtained from the HPLC analysis and
the enzyme results. In the cytoplasmic fractions, the two metabolites that were the most
prevalent were citrate and succinate, and both were more prevalent in the control than the
stress which is seen in table 12. Citrate was utilized as the carbon source, the main food
for the cell, so it makes sense that within the cytoplasm, there would be a large incidence
of citrate. The explanation of the decrease in citrate from the control to the stress
cytoplasmic fraction may be attributed to the increased usage of citrate in the stressed
conditions38. Succinate, however may not be as easy to explain; but there are plenty of
leads to its explanation. Isocitrate lyase is the enzyme responsible for converting
isocitrate into glyoxylate and succinate. This enzyme is more active in the stress samples
so therefore succinate should by much more prominent in the metal-stressed cell which is
not what is occurring. Succinate, instead of being pooled by the cell must be quickly
converted into other metabolites in order to maintain a modified TCA cycle which may
occur in the stressed cells38.
In aluminum stress, succinate has been known to be implemented in special
pathway in the glyoxylate shunt where it is utilized to produce oxalate and further lead to
the production of ATP using oxalyl CoA transferase and succinyl CoA transferase40,44. If
this method is being utilized in a multiple metal stress, this would further explain the
lower levels of succinate seen in the stress cytoplasmic fractions.
The spent fluid can also be used to draw a link between the enzymatic results as
well. In the stress sample there was a large peak near where oxalate usually elutes (figure
53
7). In comparing the relative area under the curve between the control and stress samples
for this peak (figures 10 and 11) the stress has a significantly higher prevalence of
oxalate. As the activity of isocitrate lyase is already elevated producing higher levels of
succinate and glyoxylate in the metal-stressed cells, the enzymatic activity of acylating
glyoxylate dehydrogenase is also elevated producing oxalate at higher levels which finds
its way into the spent fluid. The cell, for some reason is pumping oxalate out of the cell at
a significantly higher level in the stress spent fluid (figure 12). The sheer level of this
metabolite in the extracellular media may indicate that in fact oxalate is used as a method
of scavenging metals. Oxalate may be used as a chelator in order to bind the metals, thus
eliminating them from within the cell40,44.
There is some evidence that suggests that phospholipids are utilized in the process of
metal detoxification, mainly phosphatidylethanolamine (PE), however we did not
investigate this phenomenon36.
6.0 Conclusions and Future Research
In conclusion, imposing a multiple metal stress on a P. fluorescens strain of bacteria
does impede cellular growth; however, the cells are able to overcome the source of stress
and thrive in the harsh conditions. This survival is attributed to the cell’s highly adaptive
nature. These bacteria are able to change their internal metabolism in a way that favours
their survival by ejecting the metals from within the cells.
Some previous work has been done by Dr. Appanna in order to solve the mystery of
how these cells are able to circumvent the impending threat in order to survive however
the metabolic and enzymatic pathways involved in this process are still a mystery36,38,39.
54
By using such techniques as HPLC and BN-PAGE, much has been uncovered of this
strain of bacteria’s ability to re-arrange their metabolism to favour their survival in a
metal-polluted environment.
In observing the up-regulation of several key enzymes, namely ICL and AGODH,
there is indication that the cell alters many processes especially the TCA cycle. With that
being said, the metabolic analysis of the cytoplasmic and spent fluid samples has
illuminated the fact that, in the stress there are major differences in certain metabolites. It
is noticeable that oxalate has a much higher incidence in the stress spent fluid, which may
indicate its role as a scavenger for a metals40,44.
There is still some evidence that can be obtained to further confirm this observation,
namely using native proteins. By analyzing the cell’s ability to produce ATP in both the
control and stress conditions, it may further indicate how the cell is producing the
necessary energy to thrive when stressed versus the control conditions.
Knowing that this microbe is effective at neutralizing metals both individually and in
combination, it may be worthwhile to stress it with different metal combinations and even
different metal concentrations in order to fully understand how efficient and potent their
metal eradicating capabilities are. By understanding how these cells are able to combat
noxious metals, their power can be harnessed to help remediate polluted samples on a
larger scale.
55
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