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![Page 1: Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e885503460f94b8be74/html5/thumbnails/1.jpg)
Nutrients and Microbial Communities in Extreme Environments
Christie Sabin
Mentors: Amisha Poret-Peterson
Ariel Anbar
University of Arizona
April 21, 2012
![Page 2: Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e885503460f94b8be74/html5/thumbnails/2.jpg)
1. Introduction
2. Methods
3. Results
4. Summary
5. Future Work
OUTLINE
![Page 3: Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e885503460f94b8be74/html5/thumbnails/3.jpg)
INTRODUCTION
• Growth of microorganisms can be limited by nutrients like nitrogen, phosphorus, iron
• Nutrient limitation study of phytoplankton from Eastern Tropical North Atlantic
• N limited because CO2 fixation and chlorophyll concentrations increase with N addition
• N2 fixation is co-limited by P and Fe
Mills et al. 2004
![Page 4: Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e885503460f94b8be74/html5/thumbnails/4.jpg)
Bacterial community composition of lake changes in response to nutrients
INTRODUCTION
Newton and McMahon, 2011
All Seasons Control
Autumn CNP
Spring CNP
SummerCNP
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Objective of this project is to profile hot spring microbial
communities before and after addition of nitrogen,
phosphorus, and iron using T-RFLP analysis (Terminal
Restriction Fragment Length Polymorphism) and
quantitative PCR (qPCR) analysis of 16S rRNA genes
INTRODUCTION
![Page 6: Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e885503460f94b8be74/html5/thumbnails/6.jpg)
FePNControl
NP PFe NPFeNFe
x 3
High and Low Temperature Sites
Bison Pool
Mound Spring
Skippy’s Bathtub
Hammer Spring
Bison Pool
Mound Spring
Green Cheese
Hammer Spring
METHODS: EXPERIMENTAL DESIGNBison Pool
Microbial Mat
~pH 8
T ~ 55oC
![Page 7: Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e885503460f94b8be74/html5/thumbnails/7.jpg)
Extract DNA
PCR amplify 16S rRNA genes
T-RFLP generates a microbial community profile
16S rRNA PCR Products
Restrict with Different Enyzmes:
RsaI, MspI, HhaI
FAM-labeled end
T-RF Size (bp)
Fluorescence Intensity
METHODS: TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM (T-RFLP) ANALYSIS
![Page 8: Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e885503460f94b8be74/html5/thumbnails/8.jpg)
METHODS: QUANTITATIVE PCR (qPCR) ANALYSIS
Extract DNA
PCR amplify 16S rRNA genes
Cycle Number (Ct)
Co
py
Nu
mb
er
• Monitor PCR in real-time via fluorescent
dye (SYBR Green) that binds double
stranded DNA
• Include samples of known concentration
(copy number) to construct standard curve
• Inverse relationship between copy number
and Ct value
![Page 9: Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e885503460f94b8be74/html5/thumbnails/9.jpg)
RESULTS: WATER CHEMISTRY
Control N P Fe
NFeNP
PFe
NPFe
NO
3- ( M
)
0
10
20
30
40
50
60
70
Control N P Fe
NFeNP
PFe
NPFe
NH
4+ ( M
)
0
1
2
3
4
5
6
7
Control N P Fe
NFeNP
PFe
NPFe
P ( M
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Control N P Fe
NFeNP
PFe
NPFe
Fe
(M
)
0.000
0.005
0.010
0.015
0.020
0.025
NH4+ Addition: 62.5 MNO3
- Addition: 62.5 M
Fe Addition: 0.078 M P Addition: 7.8 M
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RESULTS: T-RFLP ANALYSIS (Rep 1, RsaI)
T-RF (bp)
Flu
ore
scen
ce I
nte
nsi
ty Control
N
P
Fe
DNA cDNA
• T-RFLP patterns differ
between treatments:
DNA: C ~ P and N ~ Fe
cDNA: Control differs
• DNA and cDNA
patterns differ:
Microbes present, but
express rRNA genes
differently
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C N P Fe NP NFe PFe NPFe
Bac
teri
al 1
6S r
RN
A(c
op
ies
g-1
mic
rob
ial
mat
)
107
108
109
1010
1011
1012
1013
1014
1015
Rep 1 Rep 2 Rep 3
DNA
C N P Fe NP NFe PFe NPFe
Ba
cte
rial
16
S r
RN
A(c
op
ies
g-1
mic
rob
ial m
at)
107
108
109
1010
1011
1012
1013
1014
1015
Rep 1 Rep 2 Rep 3
cDNA
* **
*
*Not normalized to wet weight of microbial mat. Error bars are SD on triplicate PCR reactions.
RESULTS: qPCR ANALYSIS OF BACTERIAL 16S rRNA GENES of DNA and cDNA
• With the exception of NPFe2, bacterial 16S rRNA copies in DNA appear to
be similar between treatments
• Bacterial 16S rRNA copies in cDNA may differ, but need to obtain numbers
for missing data and perform statistical analyses
• Normalization of samples to DNA/RNA concentration may reveal pattern
that is not evident from wet weight normalization
n.d. n.d. n.d. n.d. n.d. n.d.
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• Obtain missing data (DNA/RNA extraction, cDNA
synthesis, PCRs, T-RFLP and qPCR analyses)
• Repeat steps using archaeal primers
• Analyze all DNA and cDNA bacterial 16S rRNA T-
RFLPs and qPCR data
• In depth analysis of T-RFLPs, 16S rRNA gene copy
number, and water chemistry to assess extent of
microbial community composition changes in
response to nutrient addition
FUTURE WORK
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• Marcia Kyle
• Amisha Poret-Peterson
• Jessica Corman
• Zuri Martinez
• James Elser
• Ariel Anbar
• Alisa Glukhova
ACKNOWLEDGEMENTS