Microbes run the planet - Jonathan Eisen slides from #scifoo 2006
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![Page 1: Microbes run the planet - Jonathan Eisen slides from #scifoo 2006](https://reader036.fdocuments.net/reader036/viewer/2022062513/554e8e32b4c90573338b4c20/html5/thumbnails/1.jpg)
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
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But ….
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Great Plate Count Anomaly
Culturing Microscope
CountCount
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Great Plate Count Anomaly
Culturing Microscope
CountCount <<<<
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Environmental Microbiology Era I:Who is out There?
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rRNA Revolution
• Morphology and physiology evolve too rapidly
• Molecular systematics is the only way
• 16s rRNA is the choice
• Three domains discovered
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PCR Saves the Day
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Solving the Plate Count Anomaly
Culturing Microscope
CountCount
PCR
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Compare PCR Amplified rRNATo Those of Cultured Species
Eisen et al. 1992
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Majority of Microbes are “Uncultured”Numbers and Diversity
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Problems with rRNA PCR
• Doesn’t predict biology of organisms well
• Doesn’t work for viruses
• Not very quantitative
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Environmental Microbiology Era II:What are they Doing?
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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
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Phylogenetic Anchors
Beja et al. 2000
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Using a rRNA anchor allowed the
identification of a new form of phototrophy:
Proteorhodopsin
Beja et al. 2000
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Beja O, et.al., Science 2000 289:1902-6, Nature (2001) 411: 786-789
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Limits of Large Insert Approach
• Large insert libraries less random and less representative than small inserts
• Lower throughput
• Requires some thinking
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Enviornmental Microbiology Era III:Environmental Shotgun Sequencing
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Environmental Shotgun Sequencing
shotgunshotgun
sequencesequenceWarner Brothers, Inc.Warner Brothers, Inc.
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Assemble Fragments
sequencer sequencer outputoutput
assemble assemble fragmentsfragments
Closure &Closure &
AnnotationAnnotation
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• 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
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Co-Symbiosis?
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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
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Can Learn By “Black Box” Approach
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ABCDEFG
TUVWXYZ
Binning Much More Difficult in Complex Communities
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rRNA Phylotypes
Venter et al., 2004
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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
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Shotgun Sequencing Allows Use of Alternative Anchors (e.g., RecA)
Venter et al., 2004
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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
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Shotgun Sequencing Detects More Diversity than PCR-methods
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Biased Sampling of Genomes
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Biased Sampling of Genomes
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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
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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
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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
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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