Functional vs Organismal views of Ecology. One organism: Population genetics Many organisms: Ecology No organisms: Ecosystems

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1 Functional vs Organismal views of Ecology One organism: Population genetics Many organisms: Ecology No organisms: Ecosystems

2 The trade-off between precision and relevance Another trade-off exists: resolution and replication

3 Some basic questions about microbial communities 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

4 Work by Antonio Izzo

5 Species accumulation curves for four mycorrhizal communites Number of Species Horton et al Stendell et al Douglas-fir and Hemlock Horton and Bruns Number of Soil Samples

6 Biolog method (a culture approach) Uses microtiter plates with various carbon substrates Color development (tetrazolium) indicates substrate enabled growth Usually analyzed by Principle Components Analysis (PCA) or similar multivariant methods

7 Biolog method Principle components analysis Color development in different substrates

8 Advantages and disadvantages of Biolog method Fast & cheap It s a functional assay Limited to aerobic, heterotrophs that grow well in culture Reproducibility between labs Nothing is identified

9 Phospholipid Fatty Acid (PLFA)Analysis Extract PLFA from substrate (e.g. soil, other environmental samples) Analyze extract via gas chromotography Usually analyzed by Principle Components Analysis (PCA) or similar multivariant methods

10 PLFA (phospholipid fatty acid) Analysis

11 Advantges and disadvantages of PLFA Relatively Fast & cheap Nothing is identified

12 Sequence based approaches = rrna genes + PCR Why are rrna genes used so extensively? Universally present Universal primer sites High copy number Huge database enables identification at least at higher levels Carl Woese

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14 Advantages and disadvantages of protein coding sequences Coupling with 16S provides multigene approach Fewer alignment problems Resolution may be greater that 16S Potentially gets at function Primers must be degenerate Most of the universal protein targets do not involve unique ecosystem functions

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16 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

17 Assumptions of clone and sequence 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

18 Typical PCR reaction denature annealing of primers extension

19 Chimera formation via partial extensions

20 Assumptions of clone and No extraction bias sequence approaches 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

21 Giovannoni et al What can we say about unique sequences?

22 Achenbach and Coates 2000 photosynthetic Fe reducing, obligate anarobe Non-Fe reducing, facultative anarobe

23 From Achenbach and Coates 2000 * *

24 Problems with quantification Quantifying % of clone pool ignores the amplification and other biases just discussed (but is still commonly done!) Solutions Create probes from the sequences determined and probe unamplified environmental extracts (rrna). Quantitative PCR for particular targets

25 Advantages and Disadvantages of Sequence approach to community analysis Remains the best way to identify a pool of total unknowns Produces an imperfect quantitative picture. Cost and effort limit the number of replicate samples

26 DGGE - Denaturing gradient gel electrophoresis & TGGE - temperature gradient gel electrophoresis Amplify portion of rdna gene using a primer with a 5 GC clamp Load pool of amplicons onto denaturing gradient gel Slightly different products are separated by sequence differences that cause different levels of partial denaturation.

27 From Ward et al Mol. Biol. Rev

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

29 Heteroduplex formation: a feature of all PCR reaction with complex mixtures of similar products denature denature Reannealing of strands annealing of primers homoduplex heteroduplex extension extension

30 SSCP - single stranded conformational polymophisms Amplify DNA Denature templates and snap cool them Run productions on non-denaturing gel Migration is based on single-stranded confirmation of templates.

31 SSCP gel From Schmalenberger & Tebbe Mol. Ecol. 2002

32 T-RFLP (terminal restriction fragment length polymorphism) Amplify pool of sequences with one of the primers labeled Digest with a restriction enzyme A B C Each amplicon produces a single detected fragment B A C