microbes run the planet - jonathan eisen slides from #scifoo 2006
DESCRIPTION
Presentation by Jonathan Eisen from SciFoo meeting in 2006TRANSCRIPT
Microbes Can Grow On Anything
• Energy– Light– Organic and inorganic chemicals
• Carbon– Organic degradation– Inorganic “fixation”
• CO2, CO, CH4
• Contol global cycling of most nutrients– N, S, P, – Can manipulate just about every form
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ExtremophilyType Conditions
Temperature Thermophiles,Psychorphile
pH Alkaliphiles,Acidiphiles
Pressure Barophile
Salt Halophiles
Radiation Radiophiles
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How Survive at 100°C
• Change amino acid composition of all proteins
• Change composition of membranes• Add enzymes to repair heat specific damage
(e.g., deamination of DNA)• Changing which metals are used as
cofactors in biological processes• Cell wall coatings
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How Survive at High Salt
• High salt will cause water to want to flow out of cell
• Compensate by increasing solute concentrations in cell
• Many organisms use different solutes• Extreme halophiles fill up inside of cell with salts
also• Enzymes from these organisms work well in
industrial applications where salts are present
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How Survive Desiccation?
• Spore formation
• Increase solute concentration
• Starvation tolerance
• Repair desiccation damage
But ….
Great Plate Count Anomaly
Culturing Microscope
CountCount
Great Plate Count Anomaly
Culturing Microscope
CountCount <<<<
Environmental Microbiology Era I:Who is out There?
rRNA Revolution
• Morphology and physiology evolve too rapidly
• Molecular systematics is the only way
• 16s rRNA is the choice
• Three domains discovered
PCR Saves the Day
Solving the Plate Count Anomaly
Culturing Microscope
CountCount
PCR
Compare PCR Amplified rRNATo Those of Cultured Species
Eisen et al. 1992
Majority of Microbes are “Uncultured”Numbers and Diversity
Problems with rRNA PCR
• Doesn’t predict biology of organisms well
• Doesn’t work for viruses
• Not very quantitative
Environmental Microbiology Era II:What are they Doing?
Metagenomics by Large Inserts
• Isolate, by filtration, all microbes in a sample
• Extract total DNA in very large pieces
• Clone those pieces as BACs into E.coli to get enough.
• ID BACs of interest (e.g., containing rRNA)
• Sequence and analyze the BACs like a bacterial genome
Sample
Filterconcentrate
ExtractDNA
CloneInto BACs
SequenceGeneList
Phylogenetic Anchors
Beja et al. 2000
Using a rRNA anchor allowed the
identification of a new form of phototrophy:
Proteorhodopsin
Beja et al. 2000
Beja O, et.al., Science 2000 289:1902-6, Nature (2001) 411: 786-789
Limits of Large Insert Approach
• Large insert libraries less random and less representative than small inserts
• Lower throughput
• Requires some thinking
Enviornmental Microbiology Era III:Environmental Shotgun Sequencing
Environmental Shotgun Sequencing
shotgunshotgun
sequencesequenceWarner Brothers, Inc.Warner Brothers, Inc.
Assemble Fragments
sequencer sequencer outputoutput
assemble assemble fragmentsfragments
Closure &Closure &
AnnotationAnnotation
• Sap feeding insects
Glassy-winged Sharpshooter
• Carriers of Xylella fastidiosa that causes Pierce’s disease of grapevines
• There are >20000 sharpshooter species, within which intracellular symbiotic bacteria are wildspread
Baumannia cicadellinicola genome project:1° symbionts of the Glassy-winged Sharpshooter
Co-Symbiosis?
Sargasso Sea Shotgun Sequencing
shotgunshotgun
sequencesequence
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Analysis led by Venter Institute. Eisen lab contributions by Dongying Wu, Martin Wu, Jonathan Badger
Can Learn By “Black Box” Approach
ABCDEFG
TUVWXYZ
Binning Much More Difficult in Complex Communities
rRNA Phylotypes
Venter et al., 2004
taxonomic content per SHOTGUN 16S
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
GS-02
GS-03
GS-04
GS-05
GS-06
GS-07
GS-08
GS-09
GS-10
GS-11
GS-12
GS-13
GS-14
GS-15
GS-16
GS-17
GS-18
GS-19
GS-20
GS-21
GS-22
GS-23
GS-25
GS-26
GS-27
GS-28
GS-29
GS-30
GS-31
GS-32
GS-33
GS-34
GS-35
GS-36
Station
Shotgun Sequencing Allows Use of Alternative Anchors (e.g., RecA)
Venter et al., 2004
Sargasso Phylotypes
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
AlphaproteobacteriaBetaproteobacteriaGammaproteobacteriaEpsilonproteobacteria
Deltaproteobacteria
CyanobacteriaFirmicutes
Actinobacteria
Chlorobi
CFB
ChloroflexiSpirochaetesFusobacteria
Deinococcus-Thermus
EuryarchaeotaCrenarchaeota
Major Phylogenetic Group
Weighted % of Clones
EFG
EFTu
HSP70
RecA
RpoB
rRNA
Other Markers Give Similar Phylotpyes
Shotgun Sequencing Detects More Diversity than PCR-methods
Biased Sampling of Genomes
Biased Sampling of Genomes
Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Proteobacteria
Chlorobi
FirmicutesFusobacteria Actinobacteria
Cyanobacteria
Chlamydia
Spriochaetes
Deinococcus-Thermus
Aquificae
Thermotogae
TM6OS-K
Termite GroupOP8
Marine GroupAWS3
OP9
NKB19
OP3
OP10
TM7
OP1OP11
Nitrospira
SynergistesDeferribacteres
Thermudesulfobacteria
Chrysiogenetes
Thermomicrobia
Dictyoglomus
Coprothmermobacter
• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
• Solution: sequence more phyla