BIS2C. Biodiversity and the Tree of Life. 2014. L10. Studying Microbes

Slides by Jonathan Eisen for BIS2C at UC Davis Spring 2014

Lecture 9 continued

1


Different histories within one genome

2

Bacteria Archaea EukaryotesBacteria ArchaeaEukaryotes Bacteria

Nuclear Tree

Mitochondrial Tree

Nucleus

CPST

Bacteria ArchaeaEukaryotes BacteriaMITO

Chloroplast Tree


But ….

3


Model Has Limitations

N M

N M

N M

N M

N M

N M

Archaea

Eukarya

BacteriaLUCANM

NMNM

NM

NM

NM

Model like this is inconsistent with much of the data

C

C

C

C

C

C

4


Scattered distribution of chloroplasts

55


N MC

N MC

N MC

N MC

N MC

N MC


6

Hypothesis 1: Ancestral AND Loss


Cryptomonad

7


N MC

N M

N M

N M

N M

N M


8

Hypothesis 2: Diversification of Major Lineages !Symbiosis in Plantae Ancestor

N M

C

N M

C

N M

C

N M

C

Each lineage accumulates some unique properties, such as sequences of some of their genes (N, M or C genes).

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

Each lineage accumulates some unique properties, such as sequences of some of their genes (N, M or C genes).

N M

C

N M

C

N M

C

N M

C

N M

C


N MC


10

Hypothesis 2: Diversification of Major Lineages !Symbiosis in Plantae Ancestor

“Secondary Symbiosis” in other lineages


Model for “Secondary” Symbiosis

11


Symbiosis between two eukaryotic cells

12

N M

“Normal” eukaryote

Plantae representative with chloroplast

N M

C

Slides by Jonathan Eisen for BIS2C at UC Davis Spring 2014 13

N M

N M

C

Engulfment


NM

N M

C

Symbiont

Host

EndosymbiosisEndosymbiosis: when an organism (the host) bring another organism (the symbiont) inside of its cell.


NM

N M

C

Symbiont

Host

This is a “secondary” symbioses because the symbiont itself already was a host of other symbionts.

Endosymbiosis


NM

N

C

Symbiont

Host

Second mitochondria often lost


NM

C

Symbiont

Host

Second nucleus often lost


Secondary Symbioses of Euglenas

18


Excavates: Euglenids

• Have flagella. • Some are

photosynthetic, some always heterotrophic, and some can switch.

19

Movement in the euglenoid Eutreptia


Excavates: Euglenids

• Have flagella. • Some are

photosynthetic, some always heterotrophic, and some can switch.

19

Movement in the euglenoid Eutreptia

N M

N M

N M

N M

N M

N MC NMC NMC NMC N MC

N MC

N MC

N MC

N MC

Euglena Nuclear DNA tells us what its phylogenetic backbone is

20

Euglena plastid DNA says its plastid is related to those of chlorophytes

21


A lonely excavate ...

N M

22


N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

23

Engulfment of Chlorophyte


N M

N M

C

24

Engulfment of Chlorophyte


N M

N M

C

25

Endosymbiosis

N M

N M

N M

N M

N M


N MC

N MC

N MC

N MCPhylogenetic analysis of plastid DNA reveals that the eukaryote engulfed by euglena was a Chlorophyte


26

N M

N M

N M

N M

N M


N MC

N MC

N MC

N MCPhylogenetic analysis of plastid DNA reveals that the eukaryote engulfed by euglena was a Chlorophyte

Note - in some cases a “relic” nuclear genome of the symbiont is also still present and this can also be used to determine what type of organism the symbiont was


27


Secondary Symbioses of Diatoms

28


Stramenopiles: Diatoms

29

A colony of the diatom, Bacillaria paradoxa

•Unicellular, but many associate in filaments. •Have carotenoids and appear yellow or brown. •Excellent fossil record •Most are photoautotrophic •Responsible for 20% of all carbon fixation. •Oil, gas source


Stramenopiles: Diatoms

29

A colony of the diatom, Bacillaria paradoxa

•Unicellular, but many associate in filaments. •Have carotenoids and appear yellow or brown. •Excellent fossil record •Most are photoautotrophic •Responsible for 20% of all carbon fixation. •Oil, gas source

N M

N M

N M

N M

N M

Many lines of evidence indicate that it occurred in the common ancestor of the “Plantae” lineage. !One line of evidence for this is that all organisms on this branch have chloroplasts and the cells of these organisms resemble the “primary” symbiotic cell.


N MC

N MC

N MC

N MC

Diatom nuclear DNA tells us what its phylogenetic backbone is

30

Diatom plastid DNA says its plastid is related to those of red algae

31


A lonely stramenophile ...

N M

32

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

C

N M

Engulfment of a red algae

33


N M

N M

C

34


N M

N M

C

35

N M

N M

N M

N M

N M


N MC

N MC

N MC

N MCPhylogenetic analysis of plastid DNA reveals that the eukaryote engulfed by diatoms was a red algae


36


Secondary Symbioses of Others

37


N M

N M

N M

N M

N M


N MC

N MC

N MC

N MC

Many other secondary endosymbioses

Apicomplexans

Dinoflagellates

Amoebozoans

38


N M

N M

N M

N M

N M


N MC

N MC

N MC

N MC

Many other secondary endosymbioses

Apicomplexans

Dinoflagellates

Amoebozoans

38


Still Can’t Fit Model to Some Eukaryotes

39


Dinoflagellate Kryptoperidinium foliaceum

http://onlinelibrary.wiley.com/doi/10.1111/j.1550-7408.2007.00245.x/full40

http://onlinelibrary.wiley.com/doi/10.1111/j.1550-7408.2007.00245.x/full


•All are multicellular; some get very large (e.g., giant kelp). •The carotenoid fucoxanthin imparts the brown color. •Almost exclusively marine.

Stramenopiles: Brown Algae

41

A community of brown algae: The marine kelp forest


•All are multicellular; some get very large (e.g., giant kelp). •The carotenoid fucoxanthin imparts the brown color. •Almost exclusively marine.

Stramenopiles: Brown Algae

41

A community of brown algae: The marine kelp forest


N M

42

Tertiary Symbioses?



N M

43

N MN M

C

Tertiary Symbioses?


Euglenoid


N M

Engulfment

44

N MN M

C


N M

45

N MN M

C

Host

Symbiont

Endosymbsiosis

This is a “tertiary” symbiosis because the symbiont itself already underwent a secondary symbiosis.


N M

N M

N M

N M

N M


N MC

N MC

N MC

N MC

46

Brown Algae

Tertiary Endosymbsiosis


N M

N M

N M

N M

N M


N MC

N MC

N MC

N MC

46

Brown Algae

Tertiary Endosymbsiosis


Plants and Animals Get Many Functions from Symbionts

• Endosymbioses (only really work with eukaryotic cells as hosts) !Legumes with nitrogen fixing bacteria !Aphids with amino acid synthesizing

bacteria !Tubeworms with chemosynthetic bacteria !Lichens - fungi with algae or cyanobacteria !100s more

• Other symbioses !Cellulose digestion in the guts of termintes,

ruminants 47


Lecture 10

!

Lecture 10 !

Extremophiles and Methods for Studying Microbes

!!

BIS 002C Biodiversity & the Tree of Life

Spring 2014 !

Prof. Jonathan Eisen48


Where we are going and where we have been

• Previous Lecture: !9: Acquisitions and Mergers

• Current Lecture: !10: Extremophiles

• Next Lecture: !11: Symbioses

49


Lecture 9 Wrap Up

• Lateral gene transfer

50


Case 1: Antibiotic Resistance

51


Antibiotic Resistance Evolves Rapidly

52


• http://www.niaid.nih.gov/SiteCollectionImages/topics/antimicrobialresistance/3geneTransfer.gif

53

Antibiotic Resistance Can Transfer Between Species


Case 2: E. coli

54


E. coli genome comparison

55

substantial variation in gene content among members of the same species have beenreported in other lineages of bacteria and archaea. Thus, the diminishing number ofcore orthologous genes is simply an extension of something happening among closerelatives.

How do such extensive differences in gene content among close relatives originate?One of the most important clues comes from comparing the genome structures of re-lated species. (A graphical method for aligning circular genomes is introduced in Box7.1; see Figs. 7.8 and 7.9.) In comparing E. coli K12 and O157:H7, the genes that areshared between the two strains not only are highly conserved at the sequence level; but

176 Part I I • THE ORIGIN AND DIVERSIFICATION OF LIFE

Graphical Alignment for Comparing Circular Genomes Using Dotplots

Comparing the arrangement of genomes is a critical tool for un-derstanding how they evolve. This enables scientists to identifyand characterize genome rearrangements (e.g., inversions andtranslocations) and to search for patterns and associations thatmay explain how and why certain events occur. For example,differences in gene order between species are frequently at siteswhere repetitive DNA is found, which suggests that recombina-tion at the repetitive DNA may have led to rearrangements. Oneof the more useful methods compares two genomes on an x–yplot, a procedure commonly referred to as a dotplot.

Dotplots let people use their visual pattern-recognitionskills to identify similarities. Their power and simplicityhave made them a valuable analytical tool in fields beyondbiology, including electrical engineering and computer sci-

ence. Let us illustrate the method using some text-based ex-amples. Figure 7.8A plots a familiar quotation against itself.The central diagonal line is the axis of identity. The outlyingpoints represent text that repeats. A quick examination candistinguish a pattern that is repeated in its entirety (Fig.7.8B) from one with some unique elements (Fig. 7.8C).

Because most bacterial and archaeal chromosomes are cir-cular, a chromosome must first be “opened” before laying it outon the x- or y-axis. Although the circle can be linearized at anypoint, it is preferable to open each chromosome at its origin ofreplication (Fig. 7.9A). One linearized chromosome is thenaligned along the x-axis with the origin of replication placed atthe graphical origin. The other chromosome is similarlyarranged along the y-axis. The two chromosomes are com-

MG1655 (K-12)nonpathogenic

EDL933 (0157:H7)enterohemorrhagic

585

514 204

1932996

1346

1623CFT073uropathogenic

FIGURE 7.7. Number of shared proteins be-tween strains of Escherichia coli. Note thelarge number of genes found in one strainbut not the others (seen in the outer portionsof each circle).

be

A B C

to

not

or

be

to

to be or not to be

edcbaedcba

a b c d e a b c d e

ed

zy

bc

aedcba

a b c d e a b c y z d e

FIGURE 7.8. Dotplots of repeating text.

Box 7.1

169-194_Evo_Ch07.qxd:13937_C05.qxd 12/15/08 11:05 AM Page 176


Binary fission

56


Binary fission

57


Binary fission

57


Binary fission

57


Binary fission

57


Binary fission

57


Binary fission

57


Mutations happen…

Mutation = heritable change in the genome (i.e., some change in DNA bases)

58


Mutations happen…


58


Mutations happen…


58


Mutations happen…


58


Mutations happen…


58


Mutations happen…


58


Differential reproduction

59


Recombination in bacteria and archaea

DNA gets passed from one cell to another

60



61

This movement of DNA from one lineage to another, is known as lateral (or horizontal) gene transfer.


Vertical Transmission Continues


Sexual recombination in eukaryotes

63

In eukaryotes, the variants produced by mutation can “recombine” via sex

meiosis meiosis

fertilization


Lateral gene transfer

64


Lecture 10 Outline

• Methods for studying microbes: ! Extremophiles as an example ! Field observations ! Culturing ! CSI Microbiology

65


Lecture 10 Outline


66

105°C CH3

CO, 80°CH2S, pH 0, 95°C High salt

CO2 4°Clow pH

!67

Diversity: The Unusual

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)

• Will use extremophiles as an example

• The principles here apply to any bacteria, archaea or eukaryotic microbes

!68


Lecture 10 Outline


69

Field Observations an Important Tool

!70


!70


!70


!70


!70


!70


!70


!71


!71


!71


!71


!71


!71


!71

Observe Via Microscopy

!72

! Can look at organisms in a microscope !

! Can observe behaviors and responses to stimuli !

! Can try to identify them by appearance

Method 1: Observe in the Field

• For bacteria and archaea appearance is not very helpful in identifying organisms

• For some microbial eukaryotes it is more useful because of the synapomorphies outlined in Ch 27, Lecture

• In many cases, there is not enough material to work with for field observed microbes (e.g., a few cells in a pond water sample)

• Difficult to determine what is going on inside cells

!73


Lecture 10 Outline


74

Method 2: Culturing

• Culturing (or cultivation) is the growth of microorganisms in controlled or defined conditions.

• A pure culture (which is the ideal if possible) is one in which only one type of microbe is present

!75

General approach to culturing

!! Collect field sample ! Provide specific growth conditions

" Energy " Electrons " Carbon " Other conditions (e.g., O2, temperature, salt, etc)

! Dilution/passaging until one obtains a “pure” sample with just a single clone

!76

Method 2: Culturing

!77

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.

!78

!“Who is out there?”

via Culturing

!79


Woese Tree of Life

80


Woese Tree of Life

80


Woese Tree of Life

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Woese Tree of Life

80


Woese Tree of Life

80


Woese Tree of Life

80

rRNA rRNArRNA


Woese Tree of Life

80

rRNA rRNArRNA


Woese Tree of Life

80

rRNA rRNArRNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG


Woese Tree of Life

80

rRNA rRNArRNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG


Woese Tree of Life

80

rRNA rRNArRNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG

Taxa Characters! S ACUGCACCUAUCGUUCG!! E ACUCCAGCUAUCGAUCG!! C ACCCCAGCUCUCGCUCG


Woese Tree of Life

80

rRNA rRNArRNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG

Taxa Characters! S ACUGCACCUAUCGUUCG! R ACUCCACCUAUCGUUCG! E ACUCCAGCUAUCGAUCG! F ACUCCAGGUAUCGAUCG! C ACCCCAGCUCUCGCUCG! W ACCCCAGCUCUGGCUCG



Woese Tree of Life

80

rRNA rRNArRNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG



EukaryotesBacteria Archaebacteria

!“What Are They Doing?”

via Culturing

!81

Example 1: Halophiles

!82

Determining “Optimal Growth Conditions” in the lab

• Culture specific type (usually referred to as a strain)

• Take single clone of that organism

• “Inoculate” multiple flasks that have different conditions ! 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M Salt ! 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C ...

• Measure concentration of cells in each condition over time.

• Change in concentration over time = growth rate

!83

TextText

!8433

!8433

!8433

!8433

Grow starter culture

!8433


Set up some flasks with growth media

!8433



Add a small portion of the starter culture to flasks

!8433




!8433




1 2 3 4 Use different flasks for different conditions

!8433





1M 2M 3M 4M

!8433





1M 2M 3M 4M

Monitor growth over time

!8433





1M 2M 3M 4M


1 2 3 41M 2M 3M 4M1h 1h 1h 1h

!8433





1M 2M 3M 4M


1 2 3 41M 2M 3M 4M1h 1h 1h 1h

1 2 3 41M 2M 3M 4M2h 2h 2h 2h

!8433





1M 2M 3M 4M


1 2 3 41M 2M 3M 4M1h 1h 1h 1h

1 2 3 41M 2M 3M 4M2h 2h 2h 2h

1 2 3 41M 2M 3M 4M3h 3h 3h 3h

Plot Growth vs. Time for Each Condition

!85

0.0

20.0

40.0

60.0

80.0

0h 1h 2h 3h

1M 2M 3M 4M

!86

0.0

12.5

25.0

37.5

50.0

1M 2M 3M 4M

Growth Rate

Calculate and Plot Growth Rate vs. Conditions

Optimal salt concentration for different species

!87

• Some stresses of high salt ! Osmotic pressure on cells ! Desiccation

Halophile adaptations

!88

H20


• Halophile adaptations ! Increased osmolarity inside cell

" Proteins " Carbohydrates " Salts

! Membrane pumps ! Desiccation resistance


!89

H20

H20



" Proteins " Carbohydrates " Salts - only done in extremely halophilic archaea



!90



" Proteins " Carbohydrates " Salts - only done in extremely halophilic archaea



!91

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

Extreme halophiles are a monophyletic group

!92

Example 2: Thermophiles

!93

!9433

!9433

!9433


!9433



!9433




!9433




!9433





!9433





20° 30° 40° 50°

!9433





20° 30° 40° 50°


!9433





20° 30° 40° 50°


1 2 3 420° 30° 40° 50°1h 1h 1h 1h

!9433





20° 30° 40° 50°


1 2 3 420° 30° 40° 50°1h 1h 1h 1h

1 2 3 420° 30° 40° 50°2h 2h 2h 2h

!9433





20° 30° 40° 50°


1 2 3 420° 30° 40° 50°1h 1h 1h 1h

1 2 3 420° 30° 40° 50°2h 2h 2h 2h

1 2 3 420° 30° 40° 50°3h 3h 3h 3h

!95

0.0

20.0

40.0

60.0

80.0

0h 1h 2h 3h

20° 30° 40° 50°

Plot Growth vs. Time for Each Condition

!96

0.0

12.5

25.0

37.5

50.0

20° 30° 40° 50°

Growth Rate

Calculate and Plot Growth Rate vs. Conditions

Optimal growth temperature (OGT) for Different Species

!97

Temperature limits

!98

Temperature limits

Mesophile = Optimum at moderate temps Thermophile = Optimum at 45-80°C Hyperthermophile = Optimum at > 80°C

!99

Temperature limits

A > B >> E!100

Mesophile = Optimum at moderate temps Thermophile = Optimum at 45-80°C Hyperthermophile = Optimum at > 80°C

Thermophiles found throughout the bacteria and archaea

!101

Thermophile Adaptations

!102

Stresses of High Temperature

Examples of common adaptations

Denatures proteins, RNA and DNA

Make proteins more stable

Speeds up reactions Slow down enzyme rates

Liquifies membranes Decrease fluidity of membranes

\

!103

!104

Uses of extremophiles

Type of environment

Examples Example of mechanism of survival

Practical Uses

High temp (thermophiles)

Deep sea vents, hotsprings

Amino acid changes

Heat stable enzymes

Low temp (psychrophile)

Antarctic ocean, glaciers

Antifreeze proteins

Enhancing cold tolerance of crops

High pressure (barophile)

Deep sea vents, hotsprings

Solute changes Industrial processes

High salt (halophiles

Evaporating pools

Incr. internal osmolarity

Soy sauce production

High pH (alkaliphiles)

Soda lakes Transporters Detergents

Low pH (acidophiles)

Mine tailings Transporters Bioremediation

Desiccation (xerophiles)

Deserts Spore formation Freeze-drying additives

High radiation (radiophiles)

Nuclear reactor waste sites

Absorption, repair damage

Bioremediation, space travel

Novozymes in Davis

!105


Lecture 10 Outline


106

Great Plate Count Anomaly

!107

Culturing Microscopy


!108


CountCount


!109

<<<<


!110


CountCount


!111

Problem because appearance not

effective for “who is out there?” or “what are they

doing?”

<<<<


CountCount


!112



doing?”

<<<<


CountCount

Solution?


!113



doing?”

<<<<


CountCount

Solution?

DNA

Collect from environment

Analysis of uncultured microbes

!114



!115

Deep Sea Ecosystems

!116

Deep Sea Ecosystems

!117

Polymerase Chain Reaction- PCR

!118


Woese Tree of Life

119


Woese Tree of Life

119


Woese Tree of Life

119

DNA DNADNA


Woese Tree of Life

119

DNA DNADNA


Woese Tree of Life

119

DNA DNADNA


Woese Tree of Life

119

DNA DNADNA


Woese Tree of Life

119

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG


Woese Tree of Life

119

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG


Woese Tree of Life

119

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG



Woese Tree of Life

119

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG




Woese Tree of Life

119

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG




Woese Tree of Life

119

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG





Woese Tree of Life

119

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG





Woese Tree of Life

119

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG





!120

NOTES 3419

A. pisum P

A. piswn S Tx. nivea

L awaaa symLL equizenata syrCud orbgcdar s,ym

rs. gesgosterorn - I/\ -- V IN. gonorrhoeae

B. Uhar.opkiuns sym

5% C. magncisca sym

Tns. sp. L-12

A. tnefaciens

R. ricketsil

FIG. 4. Unrooted phylogenetic tree showing the position of the S. velum symbionts in relation to that of other Proteobacteria species onthe basis of 16S rRNA gene sequences. The tree was constructed from evolutionary distances in Table 1. Members of the alpha and betasubclasses of the Proteobacteria are bracketed; all others are of the gamma subclass. Chemoautotrophic symbionts (sym) are listed inboldface type. Full species names listed in Table 1. Scale bar represents percent similarity.

dicted size bands for S. velum genomic DNA (Fig. 3A): AvaIand BclI, 1,080 bp; EcoNI, 1,109 bp; and Nco and Stul, 998bp (data not shown). We suggest that this technique isgenerally useful for the confirmation of the presence ofPCR-generated sequences in cells with multiple types ofDNA.The restriction patterns of 16S rRNA coding regions for

DNA extracted from S. velum gills were identical for all nineclams examined; representative results are shown in Fig. 3.This, along with the lack of variability in the partial sequenceof 16S rDNA for three individuals, suggests that there is asingle dominant bacterial species within S. velum and thatthe host-symbiont association is species specific. This resultis in agreement with the findings of Distel et al. (12) forlamellibranch bivalve and tubeworm chemoautotrophic sym-bionts.

Single bands were evident for all enzymes predicted to cutoutside or near the ends of the gene such as AvaI, Bcll,EcoNI, PvuII, XhoI (Fig. 3), and NcoI (band size, 9,600 bp;data not shown). Some of these enzymes generated restric-tion fragments larger than that of a typical bacterial ribo-somal operon (which includes the 5S, 16S, and 23S rRNAgenes [-5 kb]), indicating that the single bands observedwere not generated by double cuts within multiple operons.Furthermore, only two bands were observed for enzymespredicted to cut near the middle of the 16S rRNA gene suchas EcoRI (Fig. 3B) and StuI (bands of 4,400 and 19,500 bp;data not shown). Thus, all enzymes in all animals generatedpatterns consistent with the presence of only one copy of the16S rRNA gene in the symbiont genome (Fig. 3). However,

it should be noted that a large duplication of the regioncontaining the rRNA operon with no subsequent changes atany of the nine restriction sites could escape detection bythis analysis.These results suggest that the symbiont genome contains

but a single rRNA operon. Bacterial rRNA operons (rm),which include the 5S, 16S, and 23S rRNA genes, varyconsiderably in number among bacteria. In contrast tofree-living species of Proteobacteria, which have 4 to 7 rmloci (18), only one copy has been detected in other endosym-bionts including both the primary (P) and secondary (S)symbionts of the pea aphid, Acyrthosiphon pisum (33) (in-cluded in Fig. 4). Multiple rRNA operons have generallybeen thought necessary to support a high rate of rRNAsynthesis in rapidly dividing cells (3, 22). Unterman andBaumann (32) suggested that the aphid symbionts thereforegrow slowly, with doubling times of 2 days to parallel thegrowth rate of the aphid host. They further speculated thatthe single rRNA operon in the aphid symbiont genome is aconsequence of the adaptation to a symbiotic existence,which necessitates a slow growth rate. Although the divisionrate of S. velum symbionts is not known, it is unlikely thatthey grow slowly, since they must produce all of the biomassfor their invertebrate host. Studies of rn copy number andgrowth rates of endosymbionts and their free-living relativesfrom a variety of phylogenetic groups may help resolve thesignificance of rRNA operon redundancy.

Phylogenetic analysis of the S. velum symbionts. Phyloge-netic analysis was conducted using the Genetic Data Envi-ronment program (Steve Smith, Harvard Genome Laborato-

VOL. 174, 1992

JOURNAL OF BACTERIOLOGY, May 1992, p. 3416-3421 Vol. 174, No. 100021-9193/92/103416-06$02.00/0Copyright © 1992, American Society for Microbiology

Phylogenetic Relationships of Chemoautotrophic BacterialSymbionts of Solemya velum Say (Mollusca: Bivalvia) Determined

by 16S rRNA Gene Sequence AnalysisJONATHAN 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 symbiontsintracellularly within its gills. These symbionts were characterized through sequencing of polymerase chainreaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velumgenomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. Thelack of variability in the 16S sequence and hybridization patterns within and between individual S. velumorganisms suggested that one species of symbiont is dominant within and specific for this host species.Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophiccluster of the gamma subdivision of the eubacterial group Proteobacteria.

Procaryote-eucaryote associations in which marine inver-tebrates harbor chemoautotrophic bacteria as endosym-bionts appear to be widespread in marine habitats such asdeep-sea hydrothermal vents and coastal sediments (8, 15).In such symbioses, the procaryotes utilize the energy re-leased by the oxidation of reduced inorganic substrates, suchas hydrogen sulfide, to fix carbon dioxide via the Calvin-Benson cycle (7, 13). The hosts appear to derive nutritionfrom their endosymbionts and in turn provide the symbiontssimultaneous access to the substrates from anoxic and oxicenvironments which are necessary for energy generation.Maintenance of such intracellular symbionts presents anovel metazoan acquisition of procaryotic energy generationand autotrophic carbon fixation.While the existence of chemoautotroph-invertebrate sym-

bioses is now generally accepted, little is actually knownabout the symbionts observed in the tissues of any of thehosts because none have been cultured. Comparison ofrRNA sequences has greatly facilitated the identification ofbacteria, including unculturable microorganisms, and theelucidation of their natural relationships (38). Phylogeneticanalysis of 16S rRNA sequences enabled Distel et al. (12) toestablish that the chemoautotrophic symbionts of the hydro-thermal vent tubeworm and five species of bivalves of thesubclass Lamellibranchia are related and cluster in thegamma subdivision of the Proteobacteria (formerly purplephotosynthetic bacteria), one of the 11 major groups ofeubacteria (30).

In this investigation we sought to establish the phyloge-netic relationships and the species specificities of the sym-bionts of the protobranch bivalve Solemya velum Say, anAtlantic coast clam which has been studied as a shallow-water model of invertebrate-chemoautotroph associations(7, 9, 10). The phylogenetic placement of the S. velumsymbionts, to date limited to sequence analysis of the 5SrRNA, indicates that these symbionts also fall in the Proteo-bacteria gamma subdivision (31). However, the small size of

* Corresponding author.t Present address: Department of Biological Sciences, Stanford

University, Stanford, CA 94305.

the 5S rRNA molecule (-120 bp) precludes resolution thatcan be attained with larger molecules such as 16S rRNA(-1,550 bp) (16). Species of the genus Solemya are, to date,the only bivalves of the subclass Protobranchia in whichchemoautotrophic symbiosis has been documented. Theprotobranchs represent an important component of studiesof chemoautotrophic symbioses, since they may be theclosest living group to the ancestral bivalve condition, be-cause they dominate the deep sea and are present along agradient from the deep sea bottom to the shore (1).PCR amplification. We used the polymerase chain reaction

(PCR) (28) to amplify 16S rRNA coding regions from amixture of procaryotic and eucaryotic DNA extracted fromthe symbiont-containing gills of S. velum. S. velum werecollected from eelgrass beds near Woods Hole, Mass., andplaced in filtered (passed through filters with a pore size of0.2 ,um) seawater to cleanse body surfaces prior to dissec-tion. The gills, which contain -109 bacterial symbionts per g(wet weight), and feet, in which symbionts have not beenobserved (7), were dissected, frozen in liquid nitrogen, andstored at -85° C. Frozen tissue was homogenized in lysisbuffer, and DNA was isolated by using hexadecyltrimethy-lammonium bromide (4). DNA from Escherichia coli JM109,prepared by the miniprep method (4), was used as a positivecontrol.

Amplification of 16S rRNA genes by PCR was carried outessentially by the method of Weisburg et al. (34) usingeubacterial universal primers and 200 ng of template DNA.DNA products (Fig. 1) amplified from S. velum gill tissue(lane 1) and from the positive-control E. coli (lane 4) wereprominent single bands of approximately 1,500 bp. Amplifi-cation was not detected when DNA template was not added(lane 2), nor when DNA from S. velum foot tissue was usedas the template (lane 3).The strong amplification from gill tissue DNA and lack of

amplification from foot tissue DNA (Fig. 1) supports theconclusions from studies of enzyme activity, electron mi-croscopy (9), and 5S rRNA sequences (31) that the bacteriaare abundant within, and specific to, the gill tissue. Thisconclusion was further supported by lack of hybridization of

3416



!121


Woese Tree of Life

122


Woese Tree of Life

122


Woese Tree of Life

122


Woese Tree of Life

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DNA DNADNA


Woese Tree of Life

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DNA DNADNA


Woese Tree of Life

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DNA DNADNA


Woese Tree of Life

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DNA DNADNA


Woese Tree of Life

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DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG


Woese Tree of Life

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DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG


Woese Tree of Life

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DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG



Woese Tree of Life

122

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG




Woese Tree of Life

122

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG




Woese Tree of Life

122

DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG





Woese Tree of Life

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DNA DNADNA

ACUGC ACCUAU CGUUCG

ACUCC AGCUAU CGAUCG

ACCCC AGCUCU CGCUCG




Key Finding 1: Major phyla of bacteria and archaea (as of 2002)

No cultures

Some cultures!123

Key Finding #2: Biogeography

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Key Finding #3: Microbiomes

125

Censored

Censored

!126

• Endosymbioses continued

• Lateral gene transfer

• Symbioses

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Lecture 11 Outline

Symbioses

• Symbiosis is an intimate association between at least two different organisms in which at least one of them benefits

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Classes of symbiosis

Organism

Class of symbiosis A B

Mutualism + +

Commensalism + 0

Parasitism + -

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Organism


Mutualism + +

Commensalism + 0

Parasitism + -

!130

• Eukaryotes as a group are somewhat metabolically limited in their capabilities

• Eukaryotes appear less able to “acquire” metabolic processes from other species via lateral gene transfer

• However, eukaryotes are remarkably adept at “acquiring” capabilities by engaging in symbioses with bacteria and archaea

• This may be related to their propensity for phagocytosis

!131

Deep Sea Ecosystems

!132

Deep Sea Ecosystems

!133

• Mutualistic symbioses involving bacteria and archaea abound in eukaryotes and take many forms

• Digestive ! Ruminants ! Cellulolytic insects

• Defensive

• Behavioral ! Squid light organs

• Autotrophic ! Photosynthetic ! Chemosynthetic in deep sea

• Nutritional ! Aphids ! Nitrogen fixation in legumes

!134


Organism


Mutualism + +

Commensalism + 0

Parasitism + -

!135


Organism


Mutualism + +

Commensalism + 0

Parasitism + -

!136

II. Some terms

• Pathogens are infectious agents that cause a disease (can be considered a subclass of parasites)

• Pathogenicity = ability to enter a host and cause disease

• Virulence = degree of pathogenicity

• Note - not all parasites are pathogens but all pathogens are parasites

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No archaeal pathogens

• Lots of types of pathogens ! Bacteria that infect eukaryotes ! Viruses that infect eukaryotes, archaea and

bacteria ! Eukaryotes that infect other eukaryotes

• No known archaeal pathogens of any organism ! No clear explanation of why ! If you discover one, you will become famous

(well, among scientists)

!138

Symbioses

• Endosymbiosis is a symbiosis (could be mutualism, commensalism or parasitism) in which one of the organisms live inside the cells of the other

!139

Lateral Gene Transfer

!140


Case 1: Antibiotic Resistance

141


Antibiotic Resistance Evolves Rapidly

142


• http://www.niaid.nih.gov/SiteCollectionImages/topics/antimicrobialresistance/3geneTransfer.gif

143

Antibiotic Resistance Can Transfer Between Species


Case 2: E. coli

144


substantial variation in gene content among members of the same species have beenreported in other lineages of bacteria and archaea. Thus, the diminishing number ofcore orthologous genes is simply an extension of something happening among closerelatives.

How do such extensive differences in gene content among close relatives originate?One of the most important clues comes from comparing the genome structures of re-lated species. (A graphical method for aligning circular genomes is introduced in Box7.1; see Figs. 7.8 and 7.9.) In comparing E. coli K12 and O157:H7, the genes that areshared between the two strains not only are highly conserved at the sequence level; but

176 Part I I • THE ORIGIN AND DIVERSIFICATION OF LIFE

Graphical Alignment for Comparing Circular Genomes Using Dotplots

Comparing the arrangement of genomes is a critical tool for un-derstanding how they evolve. This enables scientists to identifyand characterize genome rearrangements (e.g., inversions andtranslocations) and to search for patterns and associations thatmay explain how and why certain events occur. For example,differences in gene order between species are frequently at siteswhere repetitive DNA is found, which suggests that recombina-tion at the repetitive DNA may have led to rearrangements. Oneof the more useful methods compares two genomes on an x–yplot, a procedure commonly referred to as a dotplot.

Dotplots let people use their visual pattern-recognitionskills to identify similarities. Their power and simplicityhave made them a valuable analytical tool in fields beyondbiology, including electrical engineering and computer sci-

ence. Let us illustrate the method using some text-based ex-amples. Figure 7.8A plots a familiar quotation against itself.The central diagonal line is the axis of identity. The outlyingpoints represent text that repeats. A quick examination candistinguish a pattern that is repeated in its entirety (Fig.7.8B) from one with some unique elements (Fig. 7.8C).

Because most bacterial and archaeal chromosomes are cir-cular, a chromosome must first be “opened” before laying it outon the x- or y-axis. Although the circle can be linearized at anypoint, it is preferable to open each chromosome at its origin ofreplication (Fig. 7.9A). One linearized chromosome is thenaligned along the x-axis with the origin of replication placed atthe graphical origin. The other chromosome is similarlyarranged along the y-axis. The two chromosomes are com-

MG1655 (K-12)nonpathogenic

EDL933 (0157:H7)enterohemorrhagic

585

514 204

1932996

1346

1623CFT073uropathogenic

FIGURE 7.7. Number of shared proteins be-tween strains of Escherichia coli. Note thelarge number of genes found in one strainbut not the others (seen in the outer portionsof each circle).

be

A B C

to

not

or

be

to

to be or not to be

edcbaedcba

a b c d e a b c d e

ed

zy

bc

aedcba

a b c d e a b c y z d e

FIGURE 7.8. Dotplots of repeating text.

Box 7.1

169-194_Evo_Ch07.qxd:13937_C05.qxd 12/15/08 11:05 AM Page 176

Transmission of traits in bacteria and archaea

• Trait transmission in bacteria and archaea is simpler in some ways and more complex in others than in eukaryotes.

• Sexual reproduction with crossing over and gamete fusion does not occur in bacteria and archaea.

• Two main features to discuss: ! Binary fission (clonality) ! Lateral gene transfer

!146

!147

Binary fission

!148

!148

Binary fission

149

Binary fission generates a cell lineage “tree” = analogous to a phylogenetic tree

Binary fission

149


Binary fission

149


Binary fission

149


Binary fission

149


Binary fission

149


Mutations happen…


150

Mutations happen…


150

Mutations happen…


150

Mutations happen…


150

Mutations happen…


150

Mutations happen…


150


151


151

Not all variants reproduce equally well

Note - equivalent processes happen in eukaryotes

152


152

Binary fission is a form of asexual reproduction


152


Leads to “clonality”


152


Also known as “vertical transmission”Leads to “clonality”


153


The recipient can mix the new DNA with its own 154



This movement of DNA from one lineage to another, is known as lateral (or horizontal) gene transfer.

Normal vertical transmission cont.

155

The recipient can mix the new DNA with its own





Lateral inheritance I: Competence

157


Lateral inheritance II: Conjugation

158


Lateral inheritance III: Transduction

159
