Functional vs Organismal viewsof Ecology

• One organism:• Population genetics• Many organisms:• Ecology• No organisms:• Ecosystems

The trade-off between precision and relevanceAnother trade-off exists: resolution and replication

Some basic questions about microbialcommunities

• What is the structure: species present,abundance,"diversity" = richness + evenness

• Who is active, and what do they do?• Does this structure change with disturbance? if so how?• Does the structure effect the function?• What do individual species do in the community? - are

they "functionally redundant"• How can so many species co-exist?• Can we manipulate communities to improve a given

function (e.g. Bioremediation)• Can we identify useful gene products within these

communities

Work by Antonio Izzo

4030201000

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Horton et al. 1999Stendell et al. 1999Douglas-fir and HemlockHorton and Bruns 1998

Number of Soil Samples

Num

ber o

f Spe

cies

Species accumulation curves for four mycorrhizal communites

• Biolog method (a culture approach)

• Uses microtiter plates withvarious carbon substrates

• Color development (tetrazolium)indicates substrate enabledgrowth

• Usually analyzed by PrincipleComponents Analysis (PCA) orsimilar multivariant methods

Biolog method

Color development in different substrates

Principle components analysis

Advantages and disadvantages of Biologmethod

• Fast & cheap• It’s a “functional

assay”

• Limited to aerobic,heterotrophs that growwell in culture

• Reproducibilitybetween labs

• Nothing is identified

• Phospholipid Fatty Acid (PLFA)Analysis

• Extract PLFA from substrate (e.g.soil, other environmental samples)

• Analyze extract via gaschromotography

• Usually analyzed by PrincipleComponents Analysis (PCA) orsimilar multivariant methods

PLFA (phospholipid fatty acid) Analysis

Advantges and disadvantages of PLFA

• Relatively Fast &cheap

• Nothing is identified

• Why are rRNA genes used so extensively?

• Universally present• Universal primer sites

• Huge database enablesidentification at least at higherlevels

• Carl Woese

• High copy number

Sequence based approaches = rRNA genes + PCR

Advantages and disadvantages ofprotein coding sequences

• Coupling with 16Sprovides multigeneapproach

• Fewer alignmentproblems

• Resolution may begreater that 16S

• Potentially gets atfunction

• Primers must bedegenerate

• Most of the universalprotein targets do notinvolve uniqueecosystem functions

Sequence approach to community analysis

• Extract total DNA• Amplify genes• Clone amplicons• Sequence samples

from clone pool• Identify sequences via

phylogenetic analysis

• Extract Total RNA• Reverse Transcription

of rRNA into cDNA• Amplify rDNA• Clone amplicons• Sequence samples

from pool• Identify sequences via

phylogenetic analysis

Assumptions of clone andsequence approaches

• No extraction bias• No amplification bias• No cloning biases• Sequences retrieved are real and were from

living organisms• Phylogenetic placement is predictive of

functional attributes

annealing of primers

extension

denature

Typical PCR reaction

Chimera formation via partial extensions

Assumptions of clone andsequence approaches

• No extraction bias• No amplification bias• No cloning biases• Sequences retrieved were from living organisms• Cloning and PCR artifacts are unimportant• Phylogenetic placement is predictive of functional

attributes

What can we sayabout uniquesequences?

Giovannoni et al. 1996

Achenbach and Coates 2000

photosynthetic

Fe reducing, obligate anarobe

Non-Fe reducing, facultative anarobe

From Achenbach and Coates 2000

*

*

• Problems with quantification• Quantifying % of clone pool ignores the

amplification and other biases just discussed(but is still commonly done!)

• Create probes from the sequences determinedand probe unamplified environmentalextracts (rRNA).

• Quantitative PCR for particular targets

• Solutions

Advantages and Disadvantages ofSequence approach to community analysis

• Remains the best wayto identify a pool oftotal unknowns

• Produces an imperfectquantitative picture.

• Cost and effort limitthe number ofreplicate samples

• Amplify portion of rDNA gene using aprimer with a 5’ GC clamp

• Load pool of amplicons onto denaturinggradient gel

• Slightly different products are separated bysequence differences that cause differentlevels of partial denaturation.

DGGE - Denaturing gradient gel electrophoresis& TGGE - temperature gradient gel electrophoresis

From Ward et al 1998. Mol. Biol. Rev

DGGE gel From Ward et al 1998. Mol. Biol. Rev

denature

extension

annealing of primers

extension

denature

Reannealing of strands

heteroduplex

homoduplex

Heteroduplex formation: a feature of all PCRreaction with complex mixtures of similar products

SSCP - single strandedconformational polymophisms

• Amplify DNA• Denature templates and snap cool them• Run productions on non-denaturing gel• Migration is based on single-stranded

confirmation of templates.

SSCP gel From Schmalenberger & Tebbe Mol. Ecol. 2002

Amplify pool of sequences with one of the primers labeled

Digest with a restriction enzyme

A B C

BA

C

Each ampliconproduces a singledetected fragment

T-RFLP (terminal restriction fragment length polymorphism)