16 Lec 16 Molecular methods - Molecular analysis of soil...

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Molecular analysis of soil microbial communities 1. Diversity, structure and quantity Eoin Brodie Lawrence Berkeley National Lab elbrodie@lbl.gov 510-486-6584
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Complexity of soil • Physical heterogeneity cm to m µ m to mm Particle size distribution And aggregate formation Results in complex 3D structure nutrients cm to km
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Complexity of soil • Chemical heterogeneity results from physical processes Tokunaga, LBNL
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Complexity of soil • Biological complexity results from O 2 diffusion C/N diffusion C/N diffusion O 2 diffusion O 2 NO 3 - Fe 3+ SO 4 2- CH 4 Aerobic respiration Denitrification Iron reduction Sulfate reduction Methanogenesis Microbial diversity
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The great plate count anomaly • Microscopy V Isolates – < 1 % of cells could be cultured 100 % 0.001 – 10 %
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Bacillus Pseudomonas Clostridium Escherichia Spirulina Staphylococcus Streptococcus Salmonella http://www.denniskunkel.com/ Classical Microbiology Methods: • Identify bacteria by morphology
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Why can’t we grow them? • Fastidious nature of soil microbiota – Due to physical/chemical heterogeneity • Some require a combination of chemical conditions only found in complex gradients – Due to microbe-microbe interactions • Some require metabolites/nutrients from other organisms – Due to microbe-plant interactions • Many require specific nutrients from plants
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Biomarkers molecules of biological origin, which can be used to indicate a biologically mediated-process or the presence of an organism. A biomarker must be unique to an organism and must come from the same organism each time no matter where we detect it, i.e. it should be independent of environmental influences Ideal biomarker found in all microbes taxonomic rank – to at least species, subspecies even better can be readily isolated from “living microbes” doesn’t persist “outside” microbes can be detected in very small quantities
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Cell macromolecules which may be useful as biomarkers Nucleic acids Proteins Lipids Polysaccharides Metabolites } } } Nucleic acids Proteins Lipids Polysaccharides Metabolites Found in all microbes Species level taxonomy Isolated from living cells Does not persist outside cell Low detection limit Independent of environment Suitable for community analysis
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Major advance with PCR (1983) Detection limits Nucleic acids have evolved with host organism Exception lateral gene transfer!! e.g. AB resistance How can we be sure biomarker is always from same organism? Structural or housekeeping genes are not/rarely transferred
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16 Lec 16 Molecular methods - Molecular analysis of soil...

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