Abstract
Mount St. Helens erupted on May 18th, 1980 and covered the ground with sulfur
dioxide-infused ash and magma.
Four samples were collected from each mountain
zone: scortched forest, clear cut forest, blow down forest, debris, and the negative control
taken where the land was not altered by the 1980 eruption.
separate pre-made nutrient agar plates.
Ten samples were grown on
Four bacterial colonies from each plate were
chosen randomly to test in Sulfur, Indole production and Motility (SIM) media tubes.
Three bacteria that tested positive for sulfur reduction were chosen to be used in Terminal
Restriction Fragment Length Polymorphism Analysis (t-RFLP) for a methods
comparison of t-RFLP to Applied Fragment Length Polymorphism (AFLP).
DNA was
extracted from ten frozen soil samples and PCR was completed on each sample. The
PCR products were purified and the DNA was digested using the restriction enzyme
Hhal.
The digested samples were analyzed using t-RFLP applications on an AFLP
setting on a model 4300 DNA analyzer.
My results compared the methods of a t-RFLP
on an AFLP application with the difference between the two being the AFLP starts with
digestion and a linkage ligation and the t-RFLP starts with PCR. My results
demonstrated that because my product was not tracked well throughout the experiment
and because of the use of a modified t-RFLP method on the AFLP setting the end DNA
gel did not produce the expected bands of product, but a single band of digest.
t-RFLP Method Comparison of Soil DNA analysis of Scorched Forest, Blown-down
Forest, Clear Cut Forest and Debris Zones of Mount St. Helens.
Jessica Plampin
Bio 401
4/30/08
Table of Contents
Section
Page Number
1. Abstract
2. Introduction
3
A. Importance of soil biology (Braatne, et al., 1999).
3
B. quinone profiling on Mount Pinatubo (Ohta et al., 2003).
3
D. rRNA analysis to determine soil microbial communities
on Mount St. Helens (Ibekwe et al., 2007).
4
E. Differences in debris zones on Mount St. Helens
(Moral et al., 1983).
5
F. Plant succession in the blown-down forest, scorched forest and
clear cut forests of Mount St. Helens. (Halpern et al. 1990).
6
G. RFLP vs. AFLP methodology (Olive et al. 1999).
7
H. My experimental hypothesis
8
4. Methods
A. Sites on Mount St. Helens (Gill et al., 2006).
9
B. Sample size.
10
1
Section
Page Number
C. Maps
11-13
D. DNA extraction of plated bacteria.
14
E. PCR
15
F. Purification and Digestion
16
G. t-RFLP
16
5. Results
17
6. Discussion
19
7. Acknowledgements
22
8. Literature cited.
23
2
Introduction
The first mold that was used to produce an antibiotic (penicillin) was cultured by
a microbiologist from a soil sample. There are many different types of soil on the earth
and any one of these may contain the bacterium to provide the world with the next
greatest antibiotic. Mount St. Helens erupted on May 18th, 1980 and covered the ground
with sulfur dioxide-infused ash and magma, which killed all of the vegetation that would
have deposited nitrogen into the soil.
Nitrogen is one of the fundamental elements
needed for plant survival and is obtained by plants from both the soil and the air.
Life
returned back to the mountain’s upper northern slope after just three years instead of the
predicted date of 1990 (Gill et al., 2006). This experimental study explored the
diversity of unknown bacteria and the diversity of sulfur reducing bacteria in the soil of
Mount St. Helens in the blown-down zone, scorched forest zone, debris zone, a clear cut
area in the scorched forest zone, and a southern mountain region unaffected by the blast
for a negative control.
The methods of studying microbial ecology/diversity using
molecular techniques are approached in this study. A terminal restriction fragment
length polymorphism (t-RFLP) technique to analyze the genetic signature of the
microbial community.
On Mount Pinatubo, in the Philippines, Ohta et al. (2003) measured the amounts
of nitrogen and carbon present in soil samples taken from seven different locations.
One location was a dead mudflow area, three samples were taken from a live growth mud
3
flow area, and three samples were taken from an area with sparse growth.
The samples
were taken to test for differences using standard microbiology nutrient agar plating
techniques in the bacterial populations based on depth. What they found was that both the
carbon and nitrogen content in all of the soil samples were extremely high and the types
of bacterial populations growing on each plate were all approximately the same.
Therefore, amount of plant life in each area did not affect the carbon and nitrogen content
in the soil.
The dead area had less bacterial growth while the other areas had a greater
degree of bacterial growth. The most common type of bacteria found at all the locations
was from the group Actinobacteria. As Actinobacteria were the most abundant bacteria
found on Mount Pinatubo, they should also be most abundant type of bacteria on Mount
St. Helens. Standard microbiology nutrient agar plating techniques will be used to
receive the total growth of soil bacterial populations from Mount St. Helens’ zones.
In the experiment implemented by Ibekwe et al. (2007), they performed a DNA
analysis, an rRNA analysis, and a phospholipid fatty acid (PLFA) extraction of soil from
Mount St. Helens, 17 years after the volcano erupted. They performed these tests in
order to determine the microbial communities around living lupine matter, dead lupine
matter, bare soil and regular tree matter soil being used as a control. This experiment
was implemented to determine the effects of nitrogen and carbon limitation on microbial
communities in these areas. The presence of lupines, dead or alive, resulted in greater
amounts of carbon and nitrogen found in the soil samples because lupines do not intake
4
much of either element for survival. The phospholipid markers showed there to be
mostly fungi present rather than gram positive bacteria. There was also an increase in
the presence of actinomycete bacterial growth around the lupines. The denaturing
gradient gel electrophoresis (DGGE) profiles of the 16S rRNA analysis confirmed that
the bacterial communities of forest soil compared to the bare soil and live lupine soil
differed. The forest soil and the dead lupine soil communities were very similar.
Therefore, the live lupine soil and dead lupine soil, which contained more carbon and
nitrogen than the other soil samples, still differed in the types of bacteria present. A 16S
rRNA analysis also confirmed that there were different classes of microbes present and
that live lupines stimulated the greatest diversity of microbes.
Samples will be taken in
the different zones of Mount St. Helens and will be growing bacterial colonies as well as
performing a t-RFLP DNA analysis.
DGGE is an electrophoretic separation method
based on differences in melting behavior of double stranded DNA fragments (Fisher and
Lermann, 1979). The t-RFLP is also an electophoretic separation method but it is based
on fragment length.
In 1983 Roger Del Moral published an article regarding the regeneration of
vegetation in the debris zone of Mount St. Helens.
Moral sectioned off 42 plots on the
mountain in 1980 and in 1981 he resampled 37 of the plots and established 20 new plots.
The plots were located in Butte Camp, Pine Creek, and the south Fork of the Toutle
River. What he found was that Butte camp and Pine Creek both contained thick mud
5
deposits from the debris flow during the 1981 eruption. Therefore, both of these areas
plots contained little growth regeneration. The plots located at the south fork of the
Toutle River contained dead trees from the blast, but the lower herb layer was protected
by a layer of snow. Therefore, these plots regenerated growth quickly and contained the
most vegetation (Moral, 1983). According to this study the debris zone on the South
Fork of the Toutle River should be highly vegetated today. This would also mean that
the diversity of soil bacteria would also be high in numbers as plant diversity and
microbial diversity coincide as determined by Ibekwe et al. (2007).
Plant Succession in the scorched forest zone, blow down zone, blow down with
snow zone, and clear cut zone was analyzed by Halpern et al. (1990). Halpern
compared the plant succession of each zone relative to the distance to the crater, slope,
and air-fall tephra or “volcanic ejecta comprised of ash and pumice” (Halpern et al.,
1990). The blow-down forest is described as the area where the volcanic eruption had
enough force to break down or up root mature trees. The Blown-down forest with snow
is described as the same as the blow-down forest, but at a higher elevation so the
vegetation under the snow would have survived the blast. The scorched forest is
described as the narrow band of trees that is located on the edge of the blow-down forest
where the blast was less extreme but the intense heat from the blast caused the trees to
die. Clear cut forests are described as previously logged forests that were located in the
blow-down zones. Halpern found that plots from the blow-down zone and scorched
6
forest zone when compared for similarity in 1996 where 41% similar in foliage. The
blow-down zone and clear cut forest had a 37% similarity and the scorched forest and
clear cut forest had a 34% similarity. The heterogeneity of the species present in each
zone was negatively correlated to the tephra depth and positively correlated to the cover
of tree rootwads. This study should directly correlate to the diversity of soil microbes in
these zones as noted by Ibekwe et al. (2007).
The experiment by Olive et al. focused on comparing methods for bacterial DNA
analysis. When the AFLP was used it required digestion and ligating each digestion to
linkers as one of the first steps.
It was good in identifying new strains of bacteria and
was extremely discriminative. Olive suggested this method be used for laboratories that
are interested in frequent epidemiological studies as the DNA sequencer is expensive and
can become cost efficient. The RFLP started with PCR then digestion with no linking
ligation step. This is the only difference between the AFLP and RFLP methods. The
RFLP method is also good for identifying new strains of bacteria and is also
discriminative, but cannot differentiate between two closely related strains of bacteria.
A RFLP is more cost efficient and will result in the same amount of output that the AFLP
would give.
Both methods use a big piece of DNA and cut it into smaller pieces to
provide a unique pattern of a gel that can be analyzed to determine diversity and changes
in diversity.
If the methods were modified there should be no differentiation in output
of end product.
7
For my experiment, I was planning to take soil samples from the blow down zone,
scorched forest zone, debris zone, a clear cut area in the scorched forest zone, and a
southern mountain region unaffected by the blast for a negative control.
For another
control the depth of soil sample taken would only contain the top two inches of soil, since
the diversity of bacteria are known to change with depth.
I would then be culturing the
sulfur reducing bacteria found in each of the soil samples.
I would also perform a
t-RFLP analysis on my samples of unknown bacteria in order to determine the types of
bacterium present and the most abundant bacterium present.
I would then perform
separate t-RFLP analyses on my sulfur reducing bacteria and compare my results with the
overall unknown bacteria present.
I hypothesized that the density of the sulfur reducing
bacteria band of my t-RFLP would be denser in the zones closest to the blast zone as
these zones received more ash and magma than the others (Halpern et al. 1990).
I also
believed that the diversity of microorganisms would differ from zone to zone with the
scorched forest and blow down forest being the most similar as each zone received
different amounts of ash, magma, and debris (Halpern et al. 1990).
As the hoped for
results did not accumulate the analysis of this experiment changed so that the diversity of
bacteria was no longer being analyzed, instead the methods used and the possible error in
the experiment were examined.
8
Methods
Collecting 20 soil samples from Mount St. Helens’ Zones
I collected all 20 of my soil samples on January 21, 2008, to try to avoid the
winter peak when road closures and snow are often problems. The scortched forest
zone (sample/Figure 1.1) and clear cut forest zone (sample/Figure 1.2) were taken at Iron
Creek Falls, which is eight miles down Highway 131. The blow down forest zone
(sample/figure 1.3) was taken at Elk Rock where a sign was posted that read, “Natural
Regeration Growth of Forest.” The debris zone (sample/Figure 1.4) was taken at the
North Fork of the Toutle River by the Toutle River Dam.
The negative control
(sample/Figure 1.5) was taken at the southern side of the volcano where the land was not
altered by the 1980 eruption.
I collected this sample at Merwin Park in Ariel, WA.
All
samples are shown in Figures 2 relative to the driving directions and in Figure 3 relative
to the blast zone and location within each zone.
The spade used to dig out the samples
was disinfected with a bleach water solution between samples. All samples (except
debris) were buried under at least a half foot of snow. At the debris sample site there
was no snow present.
samples (Zobell, 1934).
The snow will not effect any of the microbes present in the
I only used the top half inch of the soil for each of my samples
and used small soil sample jars to collect each sample in order to keep my samples
consistant. Ten samples were stored on ice and 10 samples were placed in a cold
storage container until they were all transferred to St.Martin’s University for proper
9
storage. Two samples from each site were frozen at -21 °C for 3 weeks before DNA
analysis. Freezing the samples likely killed the bacteria which prevented them from
replicating, but the samples could still be used to determine the bacterial diversity after
conducting DNA analysis. Two samples from each site were refrigerated at 4 °C for 3
weeks before bacterial growth on nutrient agar plates and sulfur reducing analysis.
Figure 1.1. Scorched Forest near Meta Lake 13
years after the eruption. Sample 1 was taken
in an area like this. (www.fs.fed.us 2005)
Figure 1.2. Clear cut forest 19 years after the
eruption. Sample 2 was taken in an area
like this (www.fs.fed.us 2005)
10
Figure 1.3. Blow down forest after six years of
regeneration. Sample 3 was taken in an area
like this. (www.fs.fed.us 2005)
Figure 1.4. North fork of the Toutle RiverDebris area. Sample 4 was taken in this
area. (www.fs.fed.us 2005)
Figure 1.5. Merwin State Park in Ariel, WA. Southern side of
Mt. St. Helens where control sample was collected.
(www.fs.fed.us 2005)
11
Figure 2. Relative locations of samples: A-debris, B-blown-down, C-scortched forest, D-clear cut forest, E
-control (located furthur down 503). (www.fs.fed.us 2005)
12
Figure 3. Black blocks represent the sample locations in relation to the blast zone that they were taken
from. (www.fs.fed.us 2005)
13
DNA extraction of the plated bacteria
The samples remained refrigerated for two to four weeks before plating.
Refrigerating too long would have destroyed some of the original bacterial population
present in the samples as the bacterial DNA would eventually degrade. The ten
refrigerated samples were grown on separate pre-made nutrient agar plates. The nutrient
agar and SIM media were autoclaved using the Tuttnauer 2540E autoclave for 15 minutes
at 21psi and 121 °C to destroy any bacteria in the media.
In order to revive the bacteria
present in the samples the plates were incubated at 37 °C for 4 days.
Four bacterial
colonies were chosen randomly from the ten plates to test in Sulfur, Indole production
and Motility (SIM) media tubes in order to select for sulfur reducing bacteria present in
the soil. Bacteria that tested positive for sulfur reduction, which appeared black in SIM
media, were chosen to be used for Terminal Restriction Fragment Length Polymorphism
Analysis (t-RFLP) for diversity comparison.
DNA was extracted from the bacteria
using a sterile wire transfer loop to transfer the bacterial cells to a microcentrifuge tube
containing 500 µl of deionized water. The microcentrifugetubes were frozen and
thawed three times to lyse the bacterial cells, which released the cellular DNA. DNAase
is an active enzyme that catalyzes the bonds in DNA. The samples were vortexed using
the Thermolyne Maxi Mix PlusTM vortexer for 10 seconds and centrifuged using the
Beckman Microfuge® Lite Centrifuge at 10,000 x g for 30 seconds in order for the
cellular DNA and debris to become separated.
14
The supernatant was micropipetted into a
new microcentrifuge tube and stored at -24°C until ready to be used for Polymerase
Chain Reactions (PCR).
PCR of plated bacteria and soil samples
The three plated bacterial microcentrifuge tubes were then placed in a hot water
bath at 95 °C for 5 minutes to deactivate the DNAase in order to run PCR. The DNA
was extracted from the frozen soil using a MO BIO Laboratories, Inc. UltraCleanTM Soil
DNA Isolation Kit following the manufacturer’s instructions. The primers used were
the same used by Kirisits, M.J et al. and are shown in the table below. The 8f primer
was a specially labeled LiCor primer with an infrared dye for use on the DNA analyzer.
The 926r primer was a regular primer that came from Fisher Scientific.
Primer
8f 4000-31B IRDye 800-labled
oligo
5' AGAGTTTGATCCTGGCTCAG-3'
926r
5'-CCGTCAATTCCTTTRAGTTT-3'
Table 1. Primers suggested by Kirists, M.J et al.
To make the master mix for my DNA to replicate I used 32.5 µl of taq, 26 µl dNTS, 19.5
µl of each primer, 78 µl MgCl2, 130 µl of buffer, and 994.5 µl of nuclease free water.
One µl of each template was added to 49 µl of master mix. This was enough to make 26
PCR tubes of 50 µl each, which was the total number of samples I could run at one time.
I ran the mix on a Bio-Rad Gene Cycler for 35 cycles in order to make multiple replicates
of my soil DNA for the purification and digestion processes.
I tested the PCR products
by running a sample on a 1% argarose gel in 1x TAE buffer to make sure the PCR
15
process worked.
Purification and Digestion of the PCR products
Three PCR products per sample were combined and purified in order to separate
the DNA that was tagged from the 8f labled primer from the untagged DNA and PCR
by-products using the QIAquick kit (Qiagen, Valencia, CA) according to the
manufacturer’s instructions. According to M.J. Blears et al. (1998), the purified PCR
products were diluted with deionized water to concentrations between 15 and 40 ng/µl in
order for them to be properly digested by the restriction enzyme Hhal (40U) (New
England Biolabs, Inc., Beverly, MA) to produce restriction fragments. Approximately
100 ng of DNA were digested in 20µl of Hhal at 37°C for 6-12 hours. This was
followed by enzyme inactivation by warm water bath at 65°C for 20 minutes per M. J
Blears et al.
Running the t-RFLP on All the Purified and Digested Samples
The digested samples were analyzed using t-RFLP to determine diversity of
bacteria present in each sample.
To separate and analyze the DNA fragments were run
through a 5.5% polyacrylamide gel (KBPlusTM, LI-COR, Inc., Lincoln, NE) with a
thickness of 0.4 mm. The gel was prepared as the Li-Cor manual specified, except I
used 300 µl APS solution with 40 ml gel matrix and 30 µl Temed to make the gel matrix
fit the dimensions of the larger plates. Then as described by M. J. Blears et al. the gel
was allowed to polymerize over night.
The gel was wrapped in wet paper towels and
16
cellophane in order to keep it from drying out.
The samples were electrophoresed on
the DNA sequencer for 20 minutes at 1500V, 35mA, 35W, 45°C in order for the DNA to
run smoothly.
I used a sharks tooth, 90 well comb and a LiCor 4300 DNA analyzer to
run my t-RFLP (AFLP Manual).
Analyzing the Diversity Analysis of the t-RFLP method of Soil and Bacterial DNA
My results compared the methods of a t-RFLP and an AFLP application.
My methods
were analyzed through the completion of the t-RFLP method modification on an AFLP
program.
Results
Three out of 48 Sulfur, Indole production and Motility (SIM) media tubes tested positive
for sulfur reduction and were used for DNA bacterial extraction. The extraction led to
PCR, which resulted in a 1% argarose gel to check for a product as shown in figure 4.
17
Figure 4. 1% argarose gel of first four PCR samples to check for product. The four bands from right to
left are of my sample. The upper bands of lines across represents the product at approximately 900bp the
lower bands of lines across Represents the primer at approximately 20bp.
After purification and digestion of the soil samples the product was analyzed on the
Model 4300 DNA analyzer which produced both a dark digest band on top and a
shadowy unknown band underneath as shown below in Figure 5.
The bands came out at
around 3 hours which is the known time for the digest to come out on the gel.
18
Figure 5. Model 4300 DNA analyzer’s gel of soil DNA and digestion. The top black band represented the
digestion that was used and the shadowy gray band underneath was an unknown band. The gel on the left
shows the entire 7 hour run. The gel on the right shows a close up of the bands produced.
Discussion
The PCR product as shown is Figure 4 seperated out in the 1% argarose gel, but the final
DNA bands in Figure 5 did not separate out as expected. This may have been due to the
increase of digest used in the digestion portion of the experiment as only the digest came
out in a clear dark band.
It also, would have been helpful if a ladder had been used on
19
the PCR to check the base pair location in order to keep better track of the product.
The
only way to know that the product received during PCR was the desired product would
be by using a ladder.
As the product was extremely close to the primers in the PCR gel
and their base pairs were far apart 900bp to 300bp, there was no evidence that the band
represented products.
The one dark band may suggest that there was little to no
digestion and since the analyzer only reads the florescent dye the band must be labeled
DNA. The digest may have not worked due to the modification of the digestion formula
where more restriction enzyme and DNA was added to the mixture and less water so that
the product would not get lost during this step. The negative consequence was that the
overload of restriction enzyme and DNA caused the digestion to fail in its purpose.
As
only 2 µl of digested product were inserted into gel this may not have been enough
product for the gel to read properly.
Also, when the samples were loaded into the gel
there was some sample contamination from some of the samples during the pipetting
process.
Therefore, if there was a problem in one of the samples the contamination
would make the problem occur in all of the contaminated samples as well.
band appeared after approximately five hours of running the gel.
The one dark
This time frame was
appropriate for product to be produced. The most likely cause of error in this
experiment would have been assuming that a t-RFLP procedure could be used in place of
an AFLP on the Model 4300 DNA analyzer.
An AFLP would have called for digestion
and a linker ligation process before the PCR process, whereas the t-RFLP starts with the
20
PCR and goes to digestion.
This reversal and removal of methods may have affected
my product when run on the Model 4300 DNA analyzer.
As the experiment did not
produce product as multi-bands the hypothesis could not be proven or disproven.
If this
experiment were to be conducted again it would be recommended that more samples be
taken from more locations as each zone covered a wide area of the mountain.
It would
also be wise to conduct this experiment in the summer so a sample from the crater zone
could be taken, as the crater is covered by snow during the other seasons.
The next step
for this experiment would be to test the digest on a 1% argarose gel with a ladder in order
to test it for desired product, and then repeat the digest and DNA gel using less digest so
that the DNA product would come out on the gel.
21
Acknowledgements
To Dr’s Coby, Olney, and Harman for all their support and encouragement during the
experimental procedure.
To Cheryl Guglielmo for providing guidance support in the lab.
To Saint Martin’s University for providing the funds and education needed for this
experiment to take place.
To Lynne Watanabe for being such an awesome help during the long lab hours.
To my Senior Seminar Classmates for all their input during the experimental process.
22
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23