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

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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.