Functional vs Organismal views of Ecology · Functional vs Organismal views of Ecology •One...
Transcript of Functional vs Organismal views of Ecology · Functional vs Organismal views of Ecology •One...
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
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Horton et al. 1999Stendell et al. 1999Douglas-fir and HemlockHorton and Bruns 1998
Number of Soil Samples
Num
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f Spe
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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
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• 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)