DNA Sequencing and the Modern Revolution in Studies of Microbial Diversity
1. 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
5. Outline
• Introduction: Diversity of microbes
• I: The Tree of Life
• II: Genome Sequencing
• III: Microbes in the Field
• IV: Metagenomics
5
Monday, November 26, 12
6. Introduction
Diversity of Microbes
6
Monday, November 26, 12
7. D. Diversity of form
Diversity of function
7
Monday, November 26, 12
17. Diversity of form II: complexity and size
17
Monday, November 26, 12
18. Fruiting bodies
Photo 26.24 Fruiting body of gliding bacterium Stigmatella aurantiaca. SEM. 18
Monday, November 26, 12
19. 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
Monday, November 26, 12
20. D. Diversity of form
Diversity of form:
microbial eukaryotes
20
Monday, November 26, 12
21. Part I:
The Tree of Life
21
Monday, November 26, 12
22. Darwin and a Single Tree of Life
George Richmond. Darwin Heirlooms Trust
Darwin Origin of Species 1859
Set stage for “tree thinking”
22
Monday, November 26, 12
23. Ernst Haeckel 1866
Plantae
Protista
Animalia
23
www.mblwhoilibrary.org
Monday, November 26, 12
24. Whittaker – Five Kingdoms 1969
Monera
Protista
Plantae
Fungi
Animalia
24
Monday, November 26, 12
25. 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
Monday, November 26, 12
29. 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
Monday, November 26, 12
30. 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
Monday, November 26, 12
31. Alignments
• Similar in
concept to lining
up bones from
different
species
31
Monday, November 26, 12
35. 26.23 Some Would Call It Hell; These Archaea Call It Home
35
Monday, November 26, 12
36. The Tree of Life
2006
36
adapted from Baldauf, et al., in Assembling the Tree of Life, 2004
Monday, November 26, 12
37. The Tree of Life
2006
37
adapted from Baldauf, et al., in Assembling the Tree of Life, 2004
Monday, November 26, 12
38. Why tree useful?
• Reclassification of many organisms, including diversity of
pathogens
Changes how to design treatments
• Interpret comparative data
Convergence vs. homology
38
Monday, November 26, 12
39. Part II:
Genome Sequencing
39
Monday, November 26, 12
53. Microbial genomes
From http://genomesonline.org
Monday, November 26, 12
54. 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
Monday, November 26, 12
56. 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
Monday, November 26, 12
66. 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
Monday, November 26, 12
70. • 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
Monday, November 26, 12
71. A. Method 1
Method 1:
Observe in the field
61
Monday, November 26, 12
96. 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
Monday, November 26, 12
98. Halophile adaptations
• Some stresses of high salt
Osmotic pressure on cells H20
Desiccation
69
Monday, November 26, 12
99. Halophile adaptations
• Some stresses of high salt
Osmotic pressure on cells H20
Desiccation
• Halophile adaptations
Increased osmolarity inside cell
Proteins H20
Carbohydrates
Salts
Membrane pumps
Desiccation resistance
70
Monday, November 26, 12
100. Halophile adaptations
• Some stresses of high salt
Osmotic pressure on cells
Desiccation
• Halophile adaptations
Increased osmolarity inside cell
Proteins
Carbohydrates
Salts - only done in extremely halophilic archaea
Membrane pumps
Desiccation resistance
71
Monday, November 26, 12
101. Halophile adaptations
• Some stresses of high salt
Osmotic pressure on cells
Desiccation
• Halophile adaptations
Increased osmolarity inside cell
Proteins
Carbohydrates
Salts - only done in extremely halophilic archaea
Membrane pumps
Desiccation resistance
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
Monday, November 26, 12
103. 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
Monday, November 26, 12
104. Method III:
CSI Microbiology
75
Monday, November 26, 12
105. Great Plate Count Anomaly
Culturing Microscopy
Count Count
76
Monday, November 26, 12
106. Great Plate Count Anomaly
Culturing Microscopy
Count <<<< Count
77
Monday, November 26, 12
107. Great Plate Count Anomaly
Problem because
appearance not
effective for “who
is out there?” or
“what are they
doing?”
Culturing Microscopy
Count <<<< Count
78
Monday, November 26, 12
108. Great Plate Count Anomaly
Solution?
Problem because
appearance not
effective for “who
is out there?” or
“what are they
doing?”
Culturing Microscopy
Count <<<< Count
79
Monday, November 26, 12
109. Great Plate Count Anomaly
Solution?
Problem because
appearance not
effective for “who
is out there?” or DNA
“what are they
doing?”
Culturing Microscopy
Count <<<< Count
80
Monday, November 26, 12
113. 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
Monday, November 26, 12
119. 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’
...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
Monday, November 26, 12
120. 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’...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
Monday, November 26, 12
121. Major phyla of bacteria and archaea (as of 2002)
No cultures
Some cultures
90
Monday, November 26, 12
122. Uses of rDNA PCR
Bohannan and
Hughes 2003
Hugenholtz 2002
91
Monday, November 26, 12
135. 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
Monday, November 26, 12
136. Wu et al. 2006 PLoS Biology 4: e188.
Monday, November 26, 12
137. Sharpshooter Shotgun Sequencing
shotgun
Collaboration with Nancy Moran’s
Wu et al. 2006 PLoS Biology 4: e188.
lab
Monday, November 26, 12