Transcript G-16 Christie Sabin with Ariel

Nutrients and Microbial
Communities in Extreme
Environments
Christie Sabin
Mentors:
Amisha Poret-Peterson
Ariel Anbar
University of Arizona
April 21, 2012
OUTLINE
1. Introduction
2. Methods
3. Results
4. Summary
5. Future Work
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
INTRODUCTION
Bacterial community composition of lake changes in response to nutrients
All
Seasons
Control
Autumn
CNP
Spring
CNP
Summer
CNP
Newton and McMahon, 2011
INTRODUCTION
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)
quantitative PCR (qPCR) analysis of 16S rRNA genes
and
METHODS: EXPERIMENTAL DESIGN
Bison Pool
Control
N
P
Fe
x3
NP
NFe
PFe
High and Low Temperature Sites
Bison Pool
Bison Pool
Mound Spring
Mound Spring
Skippy’s Bathtub
Green Cheese
Hammer Spring
Hammer Spring
NPFe
Microbial Mat
~pH 8
T ~ 55oC
METHODS: TERMINAL RESTRICTION FRAGMENT
LENGTH POLYMORPHISM (T-RFLP) ANALYSIS
16S rRNA PCR Products
Extract DNA
PCR amplify
16S rRNA genes
FAM-labeled end
Restrict with
Different Enyzmes:
RsaI, MspI, HhaI
Fluorescence
Intensity
T-RF Size (bp)
T-RFLP generates a microbial community profile
METHODS: QUANTITATIVE PCR (qPCR) ANALYSIS
Extract DNA
PCR amplify
16S rRNA genes
(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
Copy Number
• Monitor PCR in real-time via fluorescent dye
Cycle Number (Ct)
RESULTS: WATER CHEMISTRY
NH4+ Addition: 62.5 mM
7
60
6
50
5
Fe Addition: 0.078 mM
N
P
PF
e
N
PF
e
tr
ol
C
on
N
P
PF
e
N
PF
e
N
C
on
Fe
0
Fe
0
P
1
N
10
Fe
2
N
20
3
Fe
30
4
P
40
N
NH4+ (M)
70
tr
ol
NO3- (M)
NO3- Addition: 62.5 mM
P Addition: 7.8 mM
0.025
1.8
1.6
P (M)
1.4
0.015
0.010
1.2
1.0
0.8
0.6
0.4
0.005
0.2
N
P
PF
e
N
PF
e
Fe
N
Fe
P
N
on
tr
ol
C
N
P
PF
e
N
PF
e
Fe
N
Fe
P
N
0.0
on
tr
ol
0.000
C
Fe (M)
0.020
RESULTS: T-RFLP ANALYSIS (Rep 1, RsaI)
DNA
cDNA
Fluorescence Intensity
• T-RFLP patterns differ
Control
between treatments:
DNA: C ~ P and N ~ Fe
N
cDNA: Control differs
• DNA and cDNA patterns
P
differ: Microbes
present, but express
rRNA genes differently
Fe
T-RF (bp)
RESULTS: qPCR ANALYSIS OF BACTERIAL 16S
rRNA GENES of DNA and cDNA
1014
1013
1015
DNA
Bacterial 16S rRNA
(copies g-1 microbial mat)
Bacterial 16S rRNA
-1
(copies g microbial mat)
1015
Rep 1
Rep 2
Rep 3
1012
1011
1010
*
109
*
108
107
1014
1013
cDNA
Rep 1
Rep 2
Rep 3
1012
1011
1010
*
109
*
108
107
C
N
P
Fe
NP
n.d.
NFe
PFe NPFe
n.d.
C
N
P
Fe
NP
n.d.
NFe PFe NPFe
n.d.
n.d.
n.d.
*Not normalized to wet weight of microbial mat. Error bars are SD on triplicate PCR reactions.
• 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
FUTURE WORK
• 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 TRFLPs 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
ACKNOWLEDGEMENTS
• Marcia Kyle
• Amisha Poret-Peterson
• Jessica Corman
• Zuri Martinez
• James Elser
• Ariel Anbar
• Alisa Glukhova