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Metabolic dynamics of Colorado Plateau Biological Soil Crusts Trent Northen March 13th 2012, Moab UT Berkeley Lab ms.lbl.gov Effect of changing rainfall on the crust • Models predict changes in precipitation amounts and seasonality • Complement the descriptive studies with detailed mechanistic studies (systems approaches) on the dynamics of wetting to help predict crust responses to changing rainfall patterns Goal: Define the functional dynamics in natural communities in response to a single-wetting event. a b d 0 min Green Butte, Colorado Plateau 15min e 30 min after wetting Belnap J., 2006. Hydro. Process. a: USGS Canyonlands Research Station b: Belnap etal c: Zhang, Y. 2005. Chin. Sci. Bulletin. d: Garcia-Pichel AEM 2001 e: Gerritsen 2002 BIODIDAC Pulse-Activity Event and Biological Soil Crust • Life in the edge of habitability • Long periods without water • Suddenly: Green Butte sampling location Moab, Utah late June 2011 Microcoleus vaginatus Microcoleus vaginatus Belnap J., 2006, Hydrol. Process. Image. Ferran Garcia-Pic Microcoleus vaginatus PCC 9802 Genome Annotation • Hand-curated 385 genes out of 5264 predicted by IMG/ER. Pathway Overview • Validated the putative functional assignment of 201 genes including 168 enzymatic functions (via homology and active site checks). •Out of these 201 genes, 135 matched their IMG functional prediction, 9 had different functional assignments, and 15 had unique functional assignments. Generated with KEGG Atlas Microcoleus Vaginatus PCC 9802 Genome Annotation & Modeling • • • • • • • • • • • • Pathways Predicted Pathways NOT Predicted TCA Cycle Pentose Phosphate Cycle Glycolysis Photosynthesis-Light Reactions Photosynthesis- Antennae Proteins Peptidoglycan Biosynthesis Glycogen Biosynthesis 20 Amino Acids Biosynthesis Homolactic Fermentation Nitrate Assimilation Ammonium Assimilation Sulfate Reduction • • • • Mixed Acid Fermentation Heterolactic Fermentation Nitrogen Fixation Syctonemin Biosynthesis Experimental design We simulated rain and followed the behavior of the crust for 6 days (day and night) Wet-up Gas phase and pore water metabolites dynamics Microarray Expression Analysis DNAase treat whole transcriptome amplifica on RNA from crust amplified cDNA Label with Cy3 array represents 6130 genes from M.vaginatus PCC9802 and FGP-2 data analysis RMA normaliza on hybridize to Nimblegen expression array k-means clustering Correlation plot Early phase clusters – preliminary sorting Cluster 1 – peaks at 3 min – Includes only a handful of genes with known functions – signaling, TA loci?, methylases, DNA repair, ATPases Cluster 2 – increases after 3 min upto 9 h – Includes nitrate and sugar transport genes, cyanophycinase, RNA binding proteins, fatty acid metabolism, phycobilisomes, CCM genes, several proteases, cell division FtsZ, glycogen synthesis, aa synthesis Cluster 3 – increases after 15 min up to 9hr. Includes chlorophyll synth, ATP synthase, respiration, electron transport, nitrogen metabolism, pili, ribosomal genes, sulfate transport Cluster 4 – peaks at 15 min. chaperones and oxidative stress, DNA repair, signaling and regulation Cluster 5 – peaks at 0 and decreases –– oxidative stress (includes orange carotenoid, catalase, chaperones), phycobilisome degradation prtn, chlorophyll binding prtn, trehalose synthase, ppGPp synthetase, PHA synthase, cyanophycinase, signaling and regulation Diel genes – core clusters Cluster 1: peaks at dusk – no photosynthesis genes. Ribosomal genes, respiration (cytochrome oxidases) Cluster 2 - peaks at day – Most PSII genes, most phycobilisome genes, GroES/EL chaperones and Clp proteases, oxidative phosphorylation Cluster 3- peaks at night – PSI and some PSII, Carboxysome assembly, Rubisco genes 3 2 I Dry genes core clusters • Cluster 1 – decreases from start of dry; includes translation and cell division genes • Cluster 2 – increases from start of dry; includes oxidative stress, glycosyltransferases • Cluster 3 – decreases 1 hr after start of drying; includes oxidative phosphorylation, chlorophyll synthesis • Cluster 4 – increases 3 hrs after drying • Cluster 5 – peaks at 3 hrs from start of dry; includes signaling, sucrose synthase Dynamic responses to pulsed-activity event Relative activity Heterotroph 2 Heterotroph 1 Cyanobacteria Time T0: Meta-genomics Meta-proteomic analysis To define crust composition Reconstructing the microbial food web Photosynthetic cyanobacteria provide fixed C and N to an otherwise nutrient poor environment. A complex network of microbial interactions results in C sequestration or mineralization. Acknowledgem ents LBNL Trent Northen Benjamin Bowen Aimee Johnson Kriti Sondhi Nicholas Jose David Soendjojo Eoin Brodie Julian Fortney Nicholas Bouskill Rohit Salve JBEI Aindrila Mukhopadhyay Lara Rajeev Eric Luning Chris Petzold JGI Cheryl Kerfeld Seth Axen ASU/LBNL Ferran Garcia-Pichel Ruth Potrafka LBNL LDRD Support Thanks for your attention Dry-down