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Nutrients and Microbial Communities in Extreme Environments. Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012. OUTLINE. Introduction Methods Results Summary Future Work. INTRODUCTION. - PowerPoint PPT Presentation
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Nutrients and Microbial Communities in Extreme Environments
Christie Sabin
Mentors: Amisha Poret-Peterson
Ariel Anbar
University of Arizona
April 21, 2012
1. Introduction
2. Methods
3. Results
4. Summary
5. Future Work
OUTLINE
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
Bacterial community composition of lake changes in response to nutrients
INTRODUCTION
Newton and McMahon, 2011
All Seasons Control
Autumn CNP
Spring CNP
SummerCNP
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
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
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
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
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 mMNO3
- Addition: 62.5 mM
Fe Addition: 0.078 mM P Addition: 7.8 mM
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
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.
• 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
• Marcia Kyle
• Amisha Poret-Peterson
• Jessica Corman
• Zuri Martinez
• James Elser
• Ariel Anbar
• Alisa Glukhova
ACKNOWLEDGEMENTS