Nutrients and Microbial Communities in Extreme Environments

1. Nutrients and Microbial Communities in Extreme Environments Christie Sabin Mentors: Amisha Poret-Peterson Ariel Anbar University of Arizona April 21, 2012

2. OUTLINE • Introduction • Methods • Results • Summary • Future Work

3. 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

4. INTRODUCTION All Seasons Control Bacterial community composition of lake changes in response to nutrients Autumn CNP Spring CNP SummerCNP Newton and McMahon, 2011

5. 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) and quantitative PCR (qPCR) analysis of 16S rRNA genes

6. METHODS: EXPERIMENTAL DESIGN Fe Control NP PFe NPFe NFe P N Bison Pool x 3 High and Low Temperature Sites Microbial Mat ~pH 8 T ~ 55oC Bison Pool Mound Spring Skippy’s Bathtub Hammer Spring Bison Pool Mound Spring Green Cheese Hammer Spring

7. METHODS: TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM (T-RFLP) ANALYSIS T-RFLP generates a microbial community profile 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)

8. METHODS: QUANTITATIVE PCR (qPCR) ANALYSIS Extract DNA PCR amplify 16S rRNA genes Copy Number Cycle Number (Ct) • 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

9. RESULTS: WATER CHEMISTRY NO3- Addition: 62.5 mM NH4+ Addition: 62.5 mM Fe Addition: 0.078 mM P Addition: 7.8 mM

10. RESULTS: T-RFLP ANALYSIS (Rep 1, RsaI) 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 Control Fluorescence Intensity N P Fe T-RF (bp)

11. RESULTS: qPCR ANALYSIS OF BACTERIAL 16S rRNA GENES of DNA and cDNA * * * * *Not normalized to wet weight of microbial mat. Error bars are SD on triplicate PCR reactions. n.d. n.d. n.d. n.d. n.d. n.d. • 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

12. 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 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

13. Marcia Kyle Amisha Poret-Peterson Jessica Corman Zuri Martinez James Elser Ariel Anbar Alisa Glukhova ACKNOWLEDGEMENTS