Biologic Ground TruthBiologic Ground Truth
Life in the Atacama 2004Science & Technology Workshop
Ned MinkleyCenter for Biotechnology and
Environmental Processes
Carnegie Mellon University
Life in the Atacama 2004 Workshop 2 Carnegie Mellon
Detecting Life in Extreme Environments
Detecting Life in Extreme Environments
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Photographs from the November, 2003 trip to the Atacama
Life in the Atacama 2004 Workshop 3 Carnegie Mellon
Life Detection Methods for Microorganisms
Life Detection Methods for Microorganisms
MethodsMacroscopic vs. MicroscopicMicroscopes
Light TEM, SEM
Cultivation Semi-solid surfaces (petri plates) Liquid media (tubes, flasks)
Detection without Cultivation PCR amplification of 16S rDNA Molecular Phylogeny
Endolithic BiotaAtacama Desert RocksAtacama Desert Soils
Limitations
Preparation artifacts
Extraction (e.g. biofilm)Choice of MediaExtraction; inhibitors
Life in the Atacama 2004 Workshop 4 Carnegie Mellon
Detecting Macroscopic Life Is Unambiguous?
Detecting Macroscopic Life Is Unambiguous?
Life in the Atacama 2004 Workshop 5 Carnegie Mellon
Endolithic MicroorganimsEndolithic 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. 5:215-222 (2002)
Life in the Atacama 2004 Workshop 6 Carnegie Mellon
Detection and Enumeration via CultivationDetection and Enumeration via Cultivation
Dilution and Plating Most Probable Number (MPN) Analysis
Life in the Atacama 2004 Workshop 7 Carnegie Mellon
Cultivation of Microorganisms on Plates
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 Workshop 8 Carnegie Mellon
Detection without CultivationDetection without Cultivation
Amann et al., Microbiol. Rev. 59:143-169 (1995)
Estimates of culturability from various media
Seawater 0.001-0.1%
Freshwater 0.25%
Lake 0.1-1%
Estuary 0.1-3%
Activat. sludge 1-15%
Sediments 0.25%
Soil 0.3%
Life in the Atacama 2004 Workshop 9 Carnegie Mellon
The Polymerase Chain Reaction (PCR)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 Workshop 10 Carnegie Mellon
Molecular PhylogenyMolecular Phylogeny
Casamayor et al., Environ. Microbiol. 4:338-348 (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 codeLarge and small subunits
3 rRNAs + 75 proteins
Life in the Atacama 2004 Workshop 11 Carnegie Mellon
Phylogenetic TreesPhylogenetic Trees
Woese, Microbiol. Rev. 51:221-271 (1987)
Eubacteria and archaea are prokaryotes
Eubacterial branch includes chloroplasts and mitochondria
Diversity comes from microorganisms
Life in the Atacama 2004 Workshop 12 Carnegie Mellon
Phylotypes in Clone LibrariesPhylotypes in Clone Libraries
Samples of beacon sandstone from McMurdo Dry ValleysMicroscopic examination to identify two community typesExtraction of total community genomic DNAPCR amplification and construction of rDNA clone librariesDNA sequence determination and phylogenetic analysis of 672lichen community clones and 480 cyanobacterial clones
de la Torre, Goebel, Friedmann & Pace, Appl. Environ. Microbiol. 69: 3858-3867 (2003)
Life in the Atacama 2004 Workshop 13 Carnegie Mellon
Atacama Rocks from the Spring, 2003 TripAtacama Rocks from the Spring, 2003 Trip
Sample Name Growth on Culture Media
DNA Extract
PCR Product
Sequence Match Percent N’s in Sequence
Synechocystis ATCC 27150
+ Blue-Green + culture + B Synechocystis sp. @ 94.7%
0.2 %
Small rock All negative - rock - A,B,Euk
Green rock + R2A, LA Min
- Blue-Green
- rock - A,B,Euk
Pond #2 @ 6”
Purple fluoresc.
+ R2A, LA Min
+/- Blue-Green
+ rock + A,B
- Euk
B-Phylum @ 16.8%
A-no match
23%
13.5%
Pond #2 @ 6”
Green stuff
+R2A, LA Min
+ Blue-Green
+ rock +B
- A,Euk
Phylum @ 18.9% 20%
Pond #2 @ 20’
Dark specks
All negative + rock + A,B
- Euk
B-Phylum @ 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 sp.
@ 96.9% 0
Life in the Atacama 2004 Workshop 14 Carnegie Mellon
Atacama Soil Samples: Sites and ProfilesAtacama Soil Samples: Sites and Profiles
Navarro-Gonzalez et al. Science, 302:1018-1021 (2003)
Life in the Atacama 2004 Workshop 15 Carnegie Mellon
Bacteria in Atacama Desert SoilsBacteria 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 (102 cfu/g is MDL)
Counts ranged from nd (≤102) to 2.2 x 106 CFU/g-soil)
Diversity from SSU rDNA PCR clones6 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 soilsAT02-22 (S 28º 7’ 4.5”)
AT02-23 (S 27º 1’ 17.4”)
Life in the Atacama 2004 Workshop 16 Carnegie Mellon
Where Are the Bacteria in Atacama Soils?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 Workshop 17 Carnegie Mellon
Biologic Ground Truth-ConclusionsBiologic 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
LightEM
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.