Biologic Ground Truth Life in the Atacama 2004 Science & Technology Workshop Ned Minkley Center for Biotechnology and Environmental Processes Carnegie Mellon University

Life in the Atacama 2004 Workshop2Carnegie Mellon Detecting Life in Extreme Environments Photographs from the November, 2003 trip to the Atacama

Life in the Atacama 2004 Workshop3Carnegie Mellon Life Detection Methods for Microorganisms Methods  Macroscopic vs. Microscopic  Microscopes  Light  TEM, SEM  Cultivation  Semi-solid surfaces (petri plates)  Liquid media (tubes, flasks)  Detection without Cultivation  PCR amplification of 16S rDNA  Molecular Phylogeny  Endolithic Biota  Atacama Desert Rocks  Atacama Desert Soils Limitations  Preparation artifacts  Extraction (e.g. biofilm)  Choice of Media  Extraction; inhibitors

Life in the Atacama 2004 Workshop4Carnegie Mellon Detecting Macroscopic Life Is Unambiguous?

Life in the Atacama 2004 Workshop5Carnegie Mellon Endolithic Microorganims  Friedmann & Ocampo (1976) report blue-green algae (cyanobacteria) in rocks from the Dry Valley area of the Ross Desert, Antarctica.  SEM and CLSM images of the communities in the fissure of a granite rock and 1-2 mm below the surface of sandstone. Ascaso & Wierzchos, Int. Microbiol. 5: (2002)

Life in the Atacama 2004 Workshop6Carnegie Mellon Detection and Enumeration via Cultivation Dilution and PlatingMost Probable Number (MPN) Analysis

Life in the Atacama 2004 Workshop7Carnegie Mellon Cultivation of Microorganisms on Plates  Cultivation of bacteria from a field at a wood-treating plant  Left, an area contaminated with creosote and pentachlorophenol  Right, a nearby uncontaminated region

Life in the Atacama 2004 Workshop8Carnegie Mellon Detection without Cultivation Amann et al., Microbiol. Rev. 59: (1995) Estimates of culturability from various media  Seawater %  Freshwater0.25%  Lake0.1-1%  Estuary0.1-3%  Activat. sludge1-15%  Sediments0.25%  Soil0.3%

Life in the Atacama 2004 Workshop9Carnegie Mellon The Polymerase Chain Reaction (PCR)  PCR can be used to selectively amplify a defined segment of a DNA molecule by one million-fold or more  Sensitivity can be increased by carrying out a total genomic pre-amplification using Phi29 DNA polymerase and random hexanucleotide primers  A homogeneous PCR product can be gel purified and sequenced directly  A PCR product mixture can be cloned into a vector plasmid, introduced into a bacterial host strain (transformation) and recombinant bacteria that contain unique PCR products obtained by dilution and plating onto selective media

Life in the Atacama 2004 Workshop10Carnegie Mellon Molecular Phylogeny Casamayor et al., Environ. Microbiol. 4: (2002)  Relatedness of microorganisms based on a comparison of the sequences of their small subunit (SSU) ribosomal RNA genes  Rationale: DNA › RNA › Protein  Genetic code is universal  Ribosome translates the code  Large and small subunits  3 rRNAs + 75 proteins

Life in the Atacama 2004 Workshop11Carnegie Mellon Phylogenetic Trees Woese, Microbiol. Rev. 51: (1987)  Eubacteria and archaea are prokaryotes  Eubacterial branch includes chloroplasts and mitochondria  Diversity comes from microorganisms

Life in the Atacama 2004 Workshop12Carnegie Mellon Phylotypes in Clone Libraries  Samples of beacon sandstone from McMurdo Dry Valleys  Microscopic examination to identify two community types  Extraction of total community genomic DNA  PCR amplification and construction of rDNA clone libraries  DNA sequence determination and phylogenetic analysis of 672 lichen community clones and 480 cyanobacterial clones de la Torre, Goebel, Friedmann & Pace, Appl. Environ. Microbiol. 69: (2003)

Life in the Atacama 2004 Workshop13Carnegie Mellon Atacama Rocks from the Spring, 2003 Trip Sample NameGrowth on Culture Media DNA Extract PCR Product Sequence MatchPercent N’s in Sequence Synechocystis ATCC Blue-Green+ culture+ BSynechocystis 94.7% 0.2 % Small rockAll negative- rock- A,B,Euk Green rock+ R2A, LA Min - Blue-Green - rock- A,B,Euk Pond 6” Purple fluoresc. + R2A, LA Min +/- Blue-Green + rock+ A,B - Euk 16.8% A-no match 23% 13.5% Pond 6” Green stuff +R2A, LA Min + Blue-Green + rock+B - A,Euk 18.9%20% Pond 20’ Dark specks All negative+ rock+ A,B - Euk 19.7% A-Halophile 20.1% 9.3% 2A (slope)-R2A, LA Min +/- Blue-Green + rock-A,B,Euk 9-LA Min, B-G- rock - ø29 Pol -A,B,Euk +B Streptococcus 96.9%0

Life in the Atacama 2004 Workshop14Carnegie Mellon Atacama Soil Samples: Sites and Profiles Navarro-Gonzalez et al. Science, 302: (2003)

Life in the Atacama 2004 Workshop15Carnegie Mellon Bacteria in Atacama Desert Soils  Results from Navarro-Gonzalez et al.  Six nearby samples (≈ 2m radius) collected with a sterile scoop from the upper 10-cm soil layer and composited in polyethylene bags (500 g total weight)  Total culturable heterotrophic bacteria by dilution and plating on Difco plate count agar at 3 strengths  1st dilution contains 0.01 g soil (10 2 cfu/g is MDL)  Counts ranged from nd (≤10 2 ) to 2.2 x 10 6 CFU/g-soil)  Diversity from SSU rDNA PCR clones  6 TU per 75 clones to 26 TU/75 clones  35 TU per 75 clones in coastal sample  Yungay area (S 24º 4’ 9.6”) was the most arid studied and contained no detectable bacteria by dilution plating on PCA, no recoverable DNA and no PCR products  A few colonies were detected by MPN analysis with 1-10 g soil  Genotypes are those typically found in arid soils AT02-22 (S 28º 7’ 4.5”) AT02-23 (S 27º 1’ 17.4”)

Life in the Atacama 2004 Workshop16Carnegie Mellon Where Are the Bacteria in Atacama Soils?  Key finding was presence of bacteria in every soil sample tested  Need guidance on strategy for robotic search for soil bacteria  Spatial distribution of microbes in the near-surface soil layer can be distinguished by collecting core samples and sectioning  Multiple nearby samples can reveal extent of local heterogeneity

Life in the Atacama 2004 Workshop17Carnegie Mellon Biologic Ground Truth-Conclusions  Levels of analysis  Level 1. Visible analysis of macroscopic targets (field)  Level 2. Microscopic imaging of target rocks/soils (field)  Level 3. Laboratory analysis of target rocks/soils  Sample acquisition for lab analysis  Sample log (sample no., GPS coordinates, local environmental data)  Photodocumentation  Asceptic 10-cm sample cores (coincident with imaged area)  Rocks (actual field samples)  Laboratory analyses  Microscopic  Light  EM  Geochemistry (N, P, TOC, moisture content)  Culture-based  Chemoautotrophs (R2A, FA Min)  Photoautotrophs (B-G + nitrate)  Chemoautotrophs (ammonia oxidizers)  Direct PCR clone libraries  Decision--Do we pursue Level 3 analyses?  Level 3.1. Single cores. Yes/No. Spatial distribution. Limited diversity.  Level 3.2. Multiple nearby cores. Spatial distribution. Local heterogeneity. Diversity.  Level 3.3. West-to-East traverse following a precipitation gradient. Microbial Ecology.