DNA Sequencing and the Modern Revolution in Studies of Microbial Diversity

DNA Sequencing
and the
Modern Revolution in
Studies of Microbial Diversity

Jonathan A. Eisen
UC Davis
Talk at Calacademy
December 17, 2010
1
Monday, November 26, 12

Social Networking in Science


Bacterial evolve


Outline

• Introduction: Diversity of microbes
• I: The Tree of Life
• II: Genome Sequencing
• III: Microbes in the Field
• IV: Metagenomics

5

Introduction

Diversity of Microbes

6

D. Diversity of form

Diversity of function

7

Many major pathogens are bacteria


Bacteria and archaea are key commensals of many eukaryotes


Extreme conditions are dominated by bacteria and archaea


Microbes run global cycles


Photosynthetic Organisms Changed Earth’s Atmosphere

The first photosynthetic cells were similar to cyanobacteria.


6.15 Metabolic Pathways

13


Diversity of form: prokaryotes

14

More shape diversity

15

Diversity of form II: complexity and size

17

Fruiting bodies

Photo 26.24 Fruiting body of gliding bacterium Stigmatella aurantiaca. SEM. 18

Diversity of form III: biofilms
Attraction of
Free-swimming Signal other organisms
prokaryotes molecules

Binding to surface Matrix

Signal
molecules

Single-species biofilm
Irreversible attachment

Growth and division,
formation of matrix
Mature biofilm
19


Diversity of form:
microbial eukaryotes

20

Part I:

The Tree of Life

21

Darwin and a Single Tree of Life

George Richmond. Darwin Heirlooms Trust

Darwin Origin of Species 1859

Set stage for “tree thinking”

22

Ernst Haeckel 1866

Plantae
Protista
Animalia

23
www.mblwhoilibrary.org

Whittaker – Five Kingdoms 1969

Monera
Protista
Plantae
Fungi
Animalia
24

The Microbe Problem

Most trees of life did not deal with microbes very
well

Trees were not based on comparing homologous
traits between all organisms


Carl Woese

http://mcb.illinois.edu/faculty/profile/1204
26

12.3 From Gene to Protein

27

The Ribosome

28

rRNA Systematics

• All cellular organisms have ribosomes

• All have homologous subunits of the ribosomes including specific
ribosomal proteins and ribosomal RNAs (i.e., these are universally
homologous genes)

• Woese determined the sequences of ribosomal RNAs from different
species

• The sequences are highly similar but have some variation

• Each position in a rRNA can be considered a distinct character trait

• Each position has multiple possible character states (A, C, U, G)

29

Alignments

• Method of assigning
homology to
individual residues
in different
sequences
• Allows one to have
multiple traits within
individual genes
• Each column in
alignment = a
different character
• Each residue
(ACTG) = state

30

Alignments

• Similar in
concept to lining
up bones from
different
species

31

Woese 1987 - rRNA

Microbiological Reviews 51:221
32

4.7 Eukaryotic Cells (Part 1)

33

4.4 A Prokaryotic Cell

34

26.23 Some Would Call It Hell; These Archaea Call It Home

35

The Tree of Life
2006

36
adapted from Baldauf, et al., in Assembling the Tree of Life, 2004

The Tree of Life
2006

37
adapted from Baldauf, et al., in Assembling the Tree of Life, 2004

Why tree useful?

• Reclassification of many organisms, including diversity of
pathogens
Changes how to design treatments
• Interpret comparative data
Convergence vs. homology

38

Part II:

Genome Sequencing

39

Fleischmann et al.
1995

Whole Genome Shotgun Sequencing



Warner Brothers, Inc.



shotgun




shotgun

sequence


Assemble Fragments


Assemble Fragments

sequencer output


Assemble Fragments

sequencer output

assemble
fragments


Assemble Fragments

sequencer output

assemble
fragments

Closure &

Annotation


Microbial genomes

From http://genomesonline.org

General Steps in Analysis of
Complete Genomes
• Identiﬁcation/prediction of genes
• Characterization of gene features
• Characterization of genome features
• Prediction of gene function
• Prediction of pathways
• Integration with known biological data
• Comparative genomics

44


Vibrio cholerae Metabolism


Genome Sequences Have
Revolutionized Microbiology
• Predictions of metabolic processes
• Better vaccine and drug design
• New insights into mechanisms of evolution
• Genomes serve as template for functional
studies
• New enzymes and materials for engineering
and synthetic biology

Genome Size


Genome
Structure:
More
Variable
than Once


Figure 7.6 - Gene content


Figure 7.7 - Gene content E. coli


Figure 7.10 - K12 vs O157H7


Lateral Transfer

from Doolittle, 1999

from Lerat et al

A. Studying microbes

Part III:

Microbes in the field

55

How to study microbes

• Key questions about microbes in environment:
Who are they? (i.e., what kinds of microbes are they)
What are they doing? (i.e., what functions and
processes do they possess)

56

Figure 26.24 Extreme Halophiles

58

Deep Sea Ecosystems

59

• For any particular environment, there are many different
ways one could go about characterizing the microbes there
• 1. Observe directly in the field
• 2. Grow in the laboratory
• 3. CSI Microbiology (collect & analyze DNA from field)

60

A. Method 1

Method 1:
Observe in the field

61

Field Observations an Important Tool

62


63


64

B. Method 2

Method 2:
Culturing

65

Method 2: Culturing

66

Examples of Benefits of Culturing:

• Allows one to connect processes and properties to single
types of organisms

• Enhances ability to do experiments from genetics, to
physiology to genomics

• Provides possibility of large volumes of uniform material for
study

• Can supplement appearance based classification with
other types of data. Many types are useful, though the
standard is analysis of rRNA sequences.

67

Optimal salt concentration for different species

68

Halophile adaptations

• Some stresses of high salt
Osmotic pressure on cells H20

Desiccation

69


Osmotic pressure on cells H20

Desiccation
• Halophile adaptations
Increased osmolarity inside cell
Proteins H20

Carbohydrates

Salts

Membrane pumps
Desiccation resistance

70


Osmotic pressure on cells
Desiccation
Proteins

Carbohydrates

Salts - only done in extremely halophilic archaea

Membrane pumps

71


Osmotic pressure on cells
Desiccation
Proteins

Carbohydrates

Salts - only done in extremely halophilic archaea

Membrane pumps
High internal salt requires ALL cellular components to be
adapted to salt, charge. For example, all proteins must
change surface charge and other properties.
72

Extreme halophiles are a monophyletic group

73

Uses of extremophiles
Type of Examples Example of Practical Uses
environment mechanism of
survival
High temp Deep sea vents, Amino acid Heat stable enzymes
(thermophiles) hotsprings changes
Low temp Antarctic ocean, Antifreeze Enhancing cold
(psychrophile) glaciers proteins tolerance of crops
High pressure Deep sea vents, Solute changes Industrial processes
(barophile) hotsprings
High salt Evaporating pools Incr. internal Soy sauce production
(halophiles osmolarity
High pH Soda lakes Transporters Detergents
(alkaliphiles)
Low pH Mine tailings Transporters Bioremediation
(acidophiles)
Desiccation Deserts Spore formation Freeze-drying
(xerophiles) additives
High radiation Nuclear reactor Absorption, repair Bioremediation,
(radiophiles) waste sites damage space travel
74

Method III:
CSI Microbiology

75

Great Plate Count Anomaly

Culturing Microscopy

Count Count
76



Count <<<< Count
77


Problem because
appearance not
effective for “who
is out there?” or
“what are they
doing?”


Count <<<< Count
78


Solution?
Problem because
appearance not
is out there?” or
“what are they
doing?”


Count <<<< Count
79


Solution?
Problem because
appearance not
is out there?” or DNA
“what are they
doing?”


Count <<<< Count
80

Analysis of uncultured microbes

Collect from
environment
81

Polymerase Chain Reaction- PCR

82

PCR and phylogenetic analysis of rRNA genes

DNA
extraction PCR

Makes lots of Sequence
PCR copies of the rRNA genes
rRNA genes
in sample

rRNA1
5’
...TACAGTATAGGTGG
Phylogenetic tree Sequence alignment = Data matrix AGCTAGCGATCGATC
GA... 3’
rRNA1 Yeast
rRNA1 A C A C A C

Yeast T A C A G T

E. coli A G A C A G

E. coli Humans Humans T A T A G T

83

Deep Sea Ecosystems

84

Chemosymbionts


1992 NOTES 3419

A. pisum P

A. piswn S Tx. nivea

L awaaa sym
L equizenata syr
L
Cud orbgcdar s,ym

rs. gesgosterorn - / I -- V
I
N. gonorrhoeae
B. Uhar.opkiuns sym

5% C. magncisca sym
Tns. sp. L-12

A. tnefaciens
JOURNAL OF BACTERIOLOGY, May 1992, p. 3416-3421 Vol. 174, No. 10
0021-9193/92/103416-06$02.00/0
Copyright © 1992, American Society for Microbiology

R. ricketsil Phylogenetic Relationships of Chemoautotrophic Bacterial
Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined
by 16S rRNA Gene Sequence Analysis
JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`*
Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2
Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
Received 4 November 1991/Accepted 9 March 1992

The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts 86
Unrooted phylogenetic tree showing the position of the S. velum symbionts in relation to that of other Proteobacteria species on
intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain
reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum
only
genomic DNA restriction fragments. The symbionts appeared to have one copy of the 16S rRNA gene. The
evolutionary distances in Table 1. Members of the alpha and beta
variability lack of in the 16S sequence and hybridization patterns within and between individual S. velum


Collect from
environment
87


DNA
extraction PCR

rRNA genes
in sample

rRNA1
5’
...ACACACATAGGTG
Phylogenetic tree Sequence alignment = Data matrix GAGCTAGCGATCGAT
CGA... 3’
rRNA1 rRNA2
rRNA1 A C A C A C

rRNA2 T A C A G T rRNA2
5’
E. coli A G A C A G ...TACAGTATAGGTGG
E. coli Humans Humans T A T A G T AGCTAGCGATCGATC
GA... 3’
Yeast Yeast T A C A G T

88


DNA
extraction PCR

rRNA genes
in sample

rRNA1
5’...ACACACATAGGTGGAGCTA
GCGATCGATCGA... 3’
Phylogenetic tree Sequence alignment = Data matrix
rRNA2
rRNA1 rRNA2
rRNA1 A C A C A C 5’..TACAGTATAGGTGGAGCTAG
CGACGATCGA... 3’
rRNA4
rRNA3 rRNA2 T A C A G T
rRNA3
rRNA3 C A C T G T 5’...ACGGCAAAATAGGTGGATT
E. coli Humans rRNA4 C A C A G T CTAGCGATATAGA... 3’

Yeast E. coli A G A C A G rRNA4
5’...ACGGCCCGATAGGTGGATT
Humans T A T A G T CTAGCGCCATAGA... 3’
Yeast T A C A G T
89

Major phyla of bacteria and archaea (as of 2002)

No cultures

Some cultures
90

Uses of rDNA PCR
Bohannan and
Hughes 2003

Hugenholtz 2002

91


Censored

Censored

93

Part IV:

Metagenomics

95

4.

Microbes in the world I:
rRNA PCR

Perna et al. 2003

Metagenomics

shotgun

clone


Novel Form of Phototrophy

Beja et al. 2000


clone assembled with the appropriate orientation and separation, as unpublished data), and local gene orde
expected for a low rate of mispairing error (tracking and chimaeric conserved (Supplementary Fig. S7). The
clones). represent a nearly complete genome of

Acid Mine Drainage 2004
The first step in assignment of scaffolds to organism types was to uncultured Ferroplasma species distinct
this as Ferroplasma type II. The dominan
was unexpected before the genomic analy
We assigned the roughly 3£ coverage
Leptospirillum group III on the basis of rRN
up to 31 kb, totalling 2.66 Mb). Comparis
those assigned to Leptospirillum group
sequence divergence and only locally co
firming that the scaffolds belong to a rel
Leptospirillum group II. A partial 16S rR
Sulfobacillus thermosulfidooxidans was
assembled reads, suggesting very low cov
any Sulfobacillus scaffolds .2 kb were a
grouped with the Leptospirillum group II
We compared the 3£ coverage, low Gþ
4.12 Mb) to the fer1 genome in order to
types (Supplementary Fig. S6). Scaffold
identity to fer1 were assigned to an enviro
I genome (170 scaffolds up to 47 kb in
1.48 Mb of sequence). The remaining
scaffolds are tentatively assigned to G-pla
in this bin (62 kb) contains the G-plasma
scaffolds assigned to G-plasma comprise
partial 16S rRNA gene sequence from A-pl
unassembled reads, suggesting low covera
scaffolds from A-plasma .2 kb would be
bin. Although eukaryotes are present in th
in low abundance in the biofilm studied.
eukaryotes have been detected.
As independent evidence that the Lep
Ferroplasma type II genomes are nearly co
complement of transfer RNA synthetases
Figure 1 The pink biofilm. a, Photograph of the biofilm in the Richmond mine (hand An almost complete set of these genes
included for scale). b, FISH image of a. Probes targeting bacteria (EUBmix; fluorescein Leptospirillum group III. The G-plasma bin
isothiocyanate (green)) and archaea (ARC915; Cy5 (blue)) were used in combination with a set of tRNA synthetases, consistent with in
probe targeting the Leptospirillum genus (LF655; Cy3 (red)). Overlap of red and green scaffolds. In addition, we established
(yellow) indicates Leptospirillum cells and shows the dominance of Leptospirillum. group II, Leptospirillum group III, Ferrop
c, Relative microbial abundances determined using quantitative FISH counts. type II and G-plasma bins contained onl
Monday, November 26, 12 2 ©2004 Nature Publishing Group NATURE | doi:10.1038/n

Metagenomics Challenge


Metagenomics Challenge

Who is out there?
What are they doing?


Glassy Winged Sharpshooter

• Feeds on xylem sap
• Vector for Pierce’s
Disease
• Potential bioterror agent
• Collaboration with Nancy
Moran to sequence
symbiont genomes
• Funded by NSF
• Published in PLOS
Biology 2006


Wu et al. 2006 PLoS Biology 4: e188.

Sharpshooter Shotgun Sequencing

shotgun

Collaboration with Nancy Moran’s
Wu et al. 2006 PLoS Biology 4: e188.
lab

Binning challenge

A T
B U
C V
D W
E X
F Y
G No reference genome? What do you do? Z
Phylogeny ....

CFB Phyla


Sulcia makes vitamins and cofactors

Baumannia makes amino acids

Wu et al. 2006 PLoS Biology 4: e188. 110

Part V:

Knowing What We Don’t Know

111

DNA Sequencing and the Modern Revolution in Studies of Microbial Diversity

Recomendados

Recomendados

Más contenido relacionado

Más de Jonathan Eisen

Más de Jonathan Eisen (20)

DNA Sequencing and the Modern Revolution in Studies of Microbial Diversity