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

2. 1.Introduction 2.Methods 3.Results 4.Summary 5.Future Work OUTLINE

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

5. 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

6. FePN Control NPPFeNPFeNFe 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 DESIGN Bison Pool Microbial Mat ~pH 8 T ~ 55 o C

7. 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

8. METHODS: QUANTITATIVE PCR (qPCR) ANALYSIS Extract DNA PCR amplify 16S rRNA genes Cycle Number (Ct) Copy Number 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 NH 4 + Addition: 62.5  MNO 3 - Addition: 62.5  M Fe Addition: 0.078  MP Addition: 7.8  M

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

11. ** * * *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.

12. 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

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