transcriptional responses of a hot spring microbial mat to nutrient additions space grant consortium...
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Transcriptional responses of a hot spring microbial
mat to nutrient additions Space Grant Consortium Research Symposium
Zureyma Martinez, ASU/NASA Space Grant
April 21, 2012
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Introduction
Microorganisms can regulate
metabolic processes by
changing expression of
genes.
In hot springs, microbial
mats fix N at different times
of the day based on
expression of the nifH gene
and nitrogenase protein
(Steunou et al. 2008).
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IntroductionBison Pool
Alkaline hot spring (pH
8) with a microbial mat
at 55oC
C, N, and metal storage
gene expression in
response to nitrogen (N),
phosphorus (P), and iron
(Fe) addition
Bison Pool
+ Nitrogen
N
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IntroductionMicrobial mats are dominated
by cyanobacteria that use the
Calvin Cycle to fix CO2
Key enzyme is the Ribulose
1,5-bisphosphate carboxylase/
oxygenase (RubisCO)
Large subunit of RubisCO is
encoded by rbcL
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Introduction
Reductive tricarboxylic
acid cycle
Alternative pathway for
CO2 fixation
Key enzyme is ATP
citrate lyase encoded
by aclB
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IntroductionNitrogen fixation
Nitrogenase requires iron (Fe) and molybdenum (Mo)
nifH encodes subunit of nitrogenase
Microbes typically use trace concentrations of metal
Mo storage protein encoded by mop
Other N assimilation genes, like the assimilatory nitrate reductase require Mo
N2 NH4+
Nitrogenase
Dixon and Kahn 2004 Nature Reviews Microbiology
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MethodsBison Pool samples incubated overnight in bottles
at in situ temperatures without nutrient addition
(C) and with nutrient addition (N, P, Fe, NP, NFe,
PFe, and NPFe)Extract DNA/RNA
PCR amplify w/ primers:
rbcLacbLnifHmop
Reverse
Transcribed RNA into
cDNA
DNase
Degrade DNA
cDNA
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Results
RubisCO gene was expressed in almost all treatments
Reductive TCA cycle genes expression appears to be more transient
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ResultsN
O 3- ( M
)
0
10
20
30
40
50
60
70
NH 4+
( M
)
0
1
2
3
4
5
6
7
P ( M
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Fe
( M)
0.000
0.005
0.010
0.015
0.020
0.025
NH4+ Addition: 62.5 mMNO3
- Addition: 62.5 mM
mop expression not detected in any samples
nifH expressed even in presence of nitrate and ammonia
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Summary & Future Work
Used gene expression as proxy for physiological
processes (CO2 fixation and N2 fixation)
RT-PCR on heterotrophic carbon assimilation pathways
Clone and sequence putative rbcL and aclB genes
qPCR on nifH to quantify expression between treatments
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Acknowledgements
Marcia Kyle
Jess Corman
Amisha Poret-Peterson
James Elser
Ariel Anbar
Alisa Glukhova
Christie Sabin