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