Nutrients and Microbial Communities in Extreme Environments

Christie Sabin

Mentors: Amisha Poret-Peterson

Ariel Anbar

University of Arizona

April 21, 2012

1. Introduction

2. Methods

3. Results

4. Summary

5. Future Work

OUTLINE

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

Bacterial community composition of lake changes in response to nutrients

INTRODUCTION

Newton and McMahon, 2011

All Seasons Control

Autumn CNP

Spring CNP

SummerCNP

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

FePNControl

NP PFe NPFeNFe

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 DESIGNBison Pool

Microbial Mat

~pH 8

T ~ 55oC

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

METHODS: QUANTITATIVE PCR (qPCR) ANALYSIS

Extract DNA

PCR amplify 16S rRNA genes

Cycle Number (Ct)

Co

py

Nu

mb

er

• 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

RESULTS: WATER CHEMISTRY

Control N P Fe

NFeNP

PFe

NPFe

NO

3- ( M

)

0

10

20

30

40

50

60

70

Control N P Fe

NFeNP

PFe

NPFe

NH

4+ ( M

)

0

1

2

3

4

5

6

7

Control N P Fe

NFeNP

PFe

NPFe

P ( M

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Control N P Fe

NFeNP

PFe

NPFe

Fe

(M

)

0.000

0.005

0.010

0.015

0.020

0.025

NH4+ Addition: 62.5 MNO3

- Addition: 62.5 M

Fe Addition: 0.078 M P Addition: 7.8 M

RESULTS: T-RFLP ANALYSIS (Rep 1, RsaI)

T-RF (bp)

Flu

ore

scen

ce I

nte

nsi

ty Control

N

P

Fe

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

C N P Fe NP NFe PFe NPFe

Bac

teri

al 1

6S r

RN

A(c

op

ies

g-1

mic

rob

ial

mat

)

107

108

109

1010

1011

1012

1013

1014

1015

Rep 1 Rep 2 Rep 3

DNA

C N P Fe NP NFe PFe NPFe

Ba

cte

rial

16

S r

RN

A(c

op

ies

g-1

mic

rob

ial m

at)

107

108

109

1010

1011

1012

1013

1014

1015

Rep 1 Rep 2 Rep 3

cDNA

* **

*

*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. n.d. n.d. n.d. n.d. n.d.

• 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

• Marcia Kyle

• Amisha Poret-Peterson

• Jessica Corman

• Zuri Martinez

• James Elser

• Ariel Anbar

• Alisa Glukhova

ACKNOWLEDGEMENTS