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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 GrantApril 21, 2012
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IntroductionMicroorganisms 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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IntroductionReductive 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
)0.00.20.40.60.81.01.21.41.61.8
Fe ( M
)
0.000
0.005
0.010
0.015
0.020
0.025
NH4+ Addition: 62.5 MNO3
- Addition: 62.5 M
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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AcknowledgementsMarcia KyleJess CormanAmisha Poret-PetersonJames Elser Ariel AnbarAlisa Glukhova Christie Sabin