From populations to genomes: From populations to genomes:

Extensive genotypic diversity in a Extensive genotypic diversity in a natural bacterioplankton populationnatural bacterioplankton population

Janelle R. ThompsonJanelle R. ThompsonMassachusetts Institute of TechnologyMassachusetts Institute of Technology

OutlineOutlineIntroductionIntroduction

Microbial diversity and organization Microbial diversity and organization ““Vibrio spendidus” Vibrio spendidus” model systemmodel system

Plum Island Sound, MA and Barnegat Bay NJPlum Island Sound, MA and Barnegat Bay NJPopulation dynamics Population dynamics Genomic DiversityGenomic Diversity

Ecological and evolutionary considerationsEcological and evolutionary considerations

We live on a microbial worldWe live on a microbial world

Credit M.Polz70% Ocean!!

http://visibleearth.nasa.gov/

(Whitman et al. 1998)

4.15 - 6.40 x1030 organisms

353 - 546 Pg carbon

Estimates

Microbes are the most abundant living organisms on the Earth

Credit M.Polz70% Ocean!!

http://visibleearth.nasa.gov/

(Whitman et al. 1998)

4.15 - 6.40 x1030 organisms

353 - 546 Pg carbon

Microbes are the most abundant living organisms on the Earth

We live on a microbial worldWe live on a microbial world

- Diverse metabolism- Global processes

Estimates

Complex Microbial Communities

Microbial AssemblagesA. SEMB. Light microscopy with fluorescent-stain

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

A

B

Systemresponse

1 µm

Sanitation

P. Franks

Harmful algal blooms

Nutrient cycling

DeLong & Karl

Structure and Function Relationship?

Marine AssemblagesA. SEMB. Light microscopy with fluorescent-stain

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are needed to see this picture.

A

B

Community Diversity

DNA/RNA

Isolates

Evolutionary Relationships

Biomarker genese.g. 16S ribosomal RNA

(ribotypes)

~100% community

<0.1 to 10% community

Accessible only by molecular methods

Genetic diversity(e.g. 16S rRNA ribotypes)

How do they work together to mediate activities in the environment?

Individuals

Populations

Community

Community Organization?

Based on work by F. Cohan

Co-existing variationCo-existing variation

Selective SweepSelective Sweep

DiversificationDiversification

fitness advantagefitness advantage

Population theory informs search…Population theory informs search…

sequence clustersequence cluster

The challenge: identifying functional units in natural communities

Community Biomarker TreeCommunity Biomarker Tree

Organization of a bacterioplankton communityOrganization of a bacterioplankton communityPlum Island Sound, MAPlum Island Sound, MA

0.05 subst/site 0.01 subst/site0.1 subst/site

16SrRNA clone library16SrRNA clone library

Most of the ribotype Most of the ribotype diversity partitioned diversity partitioned into sequence clusters into sequence clusters with >99% identitywith >99% identity

=functional units?=functional units?

Acinas & Klepac-Ceraj, et al, Nature, 2004Acinas & Klepac-Ceraj, et al, Nature, 2004

Motivating Questions:Motivating Questions:

I) I) Can we identify populations in microbial Can we identify populations in microbial communities? communities?

II) How diverse are the individuals in II) How diverse are the individuals in microbial populations?microbial populations?

III) What are the evolutionary forces that III) What are the evolutionary forces that may drive the diversification and may drive the diversification and cohesion of natural populations?cohesion of natural populations?

A Test: Do sequence clusters have coherent environmental dynamics?

1 0

1 2

1 4

1 6

1 4 6 3 1 4 7 0 1 4 7 7 1 4 8 4

TIME

1 22

1

Environmental sequence clusters

I) Can we identify populations in I) Can we identify populations in microbial communities?microbial communities?

Cloning andSequencing

Vibrio-targetedPCR

16S 27F-680R

VIBRIOIDENTIFICATION

CDCE *

Vibrio specificcompetitive QPCR16S 567F-680R

VIBRIOQUANTIFICATION

Environmental DNA

Monthly SamplesBarnegat Bay Surface Water

Quantification and Identification of Quantification and Identification of VibriosVibrios

* Thompson et al., 2002, 2004

mapquest.com

4˚ to 27.5 ˚CBarnegat Bay, NJ

V.wodanis

V. splendidus

V. anguillarum

V. pectenicida

V. parahaemolyticusV. alginolyticusV. carchariea/harveyi

V. coralilyticus

V. shiloi

Thompson, et al, 20040.05 subst/site

V.logei/fischeri

16S rRNA

Survey of Vibrio DiversityBarnegat Bay, NJ

Culture-independent survey matched

collections of cultured strains.

V.wodanis

V. splendidus

V. anguillarum

V. pectenicida

V. parahaemolyticusV. alginolyticusV. carchariea/harveyi

V. coralilyticus

V. shiloi

Thompson, et al, 20040.05 subst/site

>99% ClustersAug 01Dec 01Feb 02Aug 02

V.logei/fischeri

16S rRNA

Survey of Vibrio DiversityBarnegat Bay, NJ

Sequence clusters change with season and recur in summer

Vibrio Dynamics

0.1

1

10

100

1000

10000

Jul-01 Oct-01 Jan-02 Apr-02 Jul-02

V.splendidus 1,2 V. pectinicida-likeV. logei/wodanis Detection Limit

Clone Libraries

V. pectenicida-like

V. logei/wodanis

cells/ml

V. splendidus-1,2

CV ~10 to 25%

Year-Round Vibrios (QPCR)

[Thompson, et al, AEM 2004]

0.1

1

10

100

1000

10000

Jul-01 Oct-01 Jan-02 Apr-02 Jul-02

Detection Limit V. parahaemolyticusCoral-assoc. & V. para. V. spp.

V. pectenicida-like

V. splendidus-1,2

V. logei/wodanis

cells/ml

Coral pathogen

V. parahaemolyticus

V. spp. Vibrio Dynamics

CV ~10 to 25%

Clone Libraries

Late-Summer Vibrios (QPCR)

[Thompson, et al, AEM 2004]

Temperature relationship: summer and year-round Temperature relationship: summer and year-round Vibrios Vibrios

Gulf Stream

Degrees C

1.1. PersistencePersistence2.2. CurrentsCurrents

Barnegat Bay, NJ

cells/ml

0.1

1

10

100

1000

10000

Jul-01 Oct-01 Jan-02 Apr-02 Jul-020

5

10

15

20

25

30

Late Summer Year-RoundBelow Detection TemperatureTEMPERATURETEMPERATURE

Diversity and dynamics of Vibrio ribotype clusters

√ Year-round and warm-water Year-round and warm-water VibrioVibrio ribotype clusters ribotype clusters

suggest differentiation with respect to seasonal suggest differentiation with respect to seasonal parameters (e.g. temperature).parameters (e.g. temperature).

√ Coherent environmental dynamics inCoherent environmental dynamics in V. parahaemolyticusV. parahaemolyticus-like cluster (>98% rRNA -like cluster (>98% rRNA

identity)identity)V. splendidus-V. splendidus-like cluster (>98% rRNA identity)like cluster (>98% rRNA identity)

--> Closer examination of --> Closer examination of V. splendidusV. splendidus

I) Can we identify populations I) Can we identify populations in microbial communities?in microbial communities?

Plum Island SoundIpswich, MA

V. splendidus (>99% 16S rRNA)

- one of 500+ microdiverse clusters [Acinas and Klepac-Ceraj, et al 2004]

- seasonal population dynamics?

- genomic diversity?

- Do some genotypes have different dynamics?

-1˚ to 16 ˚CII) How diverse are the individuals II) How diverse are the individuals in microbial populations?in microbial populations?

I) Can we identify populations I) Can we identify populations in microbial communities?in microbial communities?

Genomic profileGenome size

(PFGE)

Hsp60 sequences

16S rRNAsequences

DIVERSITY

EnvironmentalIsolates

CDCE*

Vibrio 16S rRNAQPCR

QUANTIFICATION

EnvironmentalDNA

Monthly SamplingSurface Water

Isolate and analyze strains in ribotype clusterIsolate and analyze strains in ribotype cluster

Internal standard

Identification: clusters

Higher resolution gene

* Thompson et al., AEM 2004

Vibrio selective media

GenomeFingerprint

DNA Digest Gel

V. splendidus dynamics Plum Island Sound, MA

QPCR

Isolation

Red = V. splendidus strains

[Thompson, et al, Science 2005]

V.splendidus ribotypes- Detected year round- Dominant isolate in summer- dynamics of genotypes (PFGE and Hsp60)?

- 333 strains isolated- 20 taxa of Vibrio and Photobacterium- 232 Vibrio splendidus

16S rRNA ribotypes

V. splendidus diversity

99%

Hsp60 sequences

Genome-typing (PFGE)87% uniqueN=206

60% uniqueN=232

AMOVA: Random distribution of sequence-types

Number of Hsp60 alleles: 141 (of 232 strains)Number of PFGE genotypes: 180 (of 206 strains)

Chao-1 estimator: 100 to 300 Hsp60 alleles per month

500 to 900 PFGE genotypes per monthat least 1,300 genomes overall

What is the estimated diversity of What is the estimated diversity of ““V. splendidusV. splendidus” genomes in the samples?” genomes in the samples?

QPCR estimation of “V. splendidus” population size: - summer months is 640 to 1,890 cells/ml

Genome concentration* = (population size/diversity)

- each month 2 to 15 cells/ml share identical Hsp60 alleles - on average <1 cell/ml identical genome*

*Conservative estimate:- Chao-1 predicts minimum diversity (richness)- “culture biases” would underestimate diversity (richness)

What is the estimated diversity of What is the estimated diversity of ““V. splendidusV. splendidus” genomes in the samples?” genomes in the samples?

* Based on the Chao-1

10001000Genomes*Genomes*

100100Hsp60 types*Hsp60 types*

1116S rRNA16S rRNA

clustercluster

1 ml

What is the estimated diversity of What is the estimated diversity of ““V. splendidusV. splendidus” genomes in the samples?” genomes in the samples?

Abundance:103 cells/ml

Diversity:103 genome types/ml

Genome sizes (4.5 to 5.6 Mb)

~1000 genes

Isolates paired by identical Hsp60 sequences

Some diversification is due to large-scale genome changes

Size variation among “V. splendidus” genomes

Relationships of Hsp60 sequences

spectrum of observed diversity

How are genomes differentiated?How are genomes differentiated?

What drives genome diversification?What drives genome diversification?

Elements In Elements Out

DuplicationHorizontal gene transfer-homologous recombination-mobile genetic elements:- phage-related gene clusters- >1% ORFs are integrases or

transposases in strain 12B01

Gene Loss

Dynamic genome size & contentDynamic genome size & content

GENOME

What drives population cohesion?What drives population cohesion?

Considerations:Considerations:

(A) Frequency of recombination increases with sequence similarity(B) “Microdiverse” organisms may have access a shared genetic pool.

(A) (B)

Are microbial populations genetically cohesive Are microbial populations genetically cohesive via biospecies-like evolution?via biospecies-like evolution?

genetransfer amongclosely related strains

Fraser, Science 2007

Mechanisms for co-existing diversityMechanisms for co-existing diversity

1)1) Genomic variants represent ecologically distinct Genomic variants represent ecologically distinct populations consistent with niche theory.populations consistent with niche theory.

2)2) Genome variation does not confer a time-averaged fitness Genome variation does not confer a time-averaged fitness advantage in a stochastic environment.advantage in a stochastic environment.• Variation is neutral Variation is neutral • Variation is contextually-neutral i.e. it may be under selection in Variation is contextually-neutral i.e. it may be under selection in

alternate unknown environmentsalternate unknown environments• Variation affects fitness; is maintained by balancing selection Variation affects fitness; is maintained by balancing selection

(e.g. kill the winner, environmental heterogeneity)(e.g. kill the winner, environmental heterogeneity)

---> no single genotype may gain a lasting growth advantage.---> no single genotype may gain a lasting growth advantage.

ConclusionsConclusions

I) Can we identify populations I) Can we identify populations in microbial communities?in microbial communities?

II) How diverse are individuals in II) How diverse are individuals in microbial populations?microbial populations?

III) What are the evolutionary III) What are the evolutionary forces that may drive the forces that may drive the diversification and cohesion of diversification and cohesion of natural populations?natural populations?

ConclusionsConclusions

I) Can we identify populations I) Can we identify populations in microbial communities?in microbial communities?

Microdiverse ribotype clustersMicrodiverse ribotype clustersCoherent environmental dynamicsCoherent environmental dynamics

II) How diverse are individuals in II) How diverse are individuals in microbial populations?microbial populations?

III) What are the evolutionary III) What are the evolutionary forces that may drive the forces that may drive the diversification and cohesion of diversification and cohesion of natural populations?natural populations?

II) How diverse are individuals in II) How diverse are individuals in microbial populations?microbial populations?

ConclusionsConclusions

I) Can we identify populations I) Can we identify populations in microbial communities?in microbial communities?

V. splendidusV. splendidus10103 3 genome types/10genome types/1033 cells ml cells ml-1-1 heterogeneity up to ~1000 genesheterogeneity up to ~1000 genes

Microdiverse ribotype clustersMicrodiverse ribotype clustersCoherent environmental dynamicsCoherent environmental dynamics

III) What are the evolutionary III) What are the evolutionary forces that may drive the forces that may drive the diversification and cohesion of diversification and cohesion of natural populations?natural populations?

II) How diverse are individuals in II) How diverse are individuals in microbial populations?microbial populations?

ConclusionsConclusions

I) Can we identify populations I) Can we identify populations in microbial communities?in microbial communities?

V. splendidusV. splendidus10103 3 genome types/10genome types/1033 cells ml cells ml-1-1 heterogeneity up to ~1000 genesheterogeneity up to ~1000 genes

Microdiverse ribotype clustersMicrodiverse ribotype clustersCoherent environmental dynamicsCoherent environmental dynamics

- Diversity within a population: balance Diversity within a population: balance of HGT & cohesion by recombination.of HGT & cohesion by recombination.

- Vast genomic variation may be - Vast genomic variation may be contextually neutral or adaptivecontextually neutral or adaptive

III) What are the evolutionary III) What are the evolutionary forces that may drive the forces that may drive the diversification and cohesion of diversification and cohesion of natural populations?natural populations?

CollaboratorsCollaborators

Dr. Martin PolzDr. Martin Polz

Polz Lab: Sarah PacochaPolz Lab: Sarah Pacocha

Vanja Klepac-Ceraj Vanja Klepac-Ceraj

Chanathip PharinoChanathip Pharino

Dana HuntDana Hunt

Jennifer BenoitJennifer Benoit

Ramahi Sarma-RupavtarmRamahi Sarma-Rupavtarm

Dr. Luisa MarcelinoDr. Luisa Marcelino

Dr. Aoy Tomita-MitchellDr. Aoy Tomita-Mitchell

Dr. Ee Lin Lim (Temple University) Dr. Ee Lin Lim (Temple University)

Dr. Daniel Distel (Ocean Genome Legacy, New England Biolabs)Dr. Daniel Distel (Ocean Genome Legacy, New England Biolabs)

Dr. William Thilly (MIT)Dr. William Thilly (MIT)

AcknowledgementsAcknowledgements

FundingFundingNational Science FoundationNational Science FoundationSeagrant Seagrant Department of Energy Joint Genome Department of Energy Joint Genome InstituteInstitute

Questions?Questions?

EnvironmentEnvironment UnitsUnits Richness Richness (model)(model) ReferenceReference

Human GI tractHuman GI tract

- per individual- per individual

99% 16S rRNA99% 16S rRNA 164 to 332 (chao1) 164 to 332 (chao1) Eckburg et al. Eckburg et al. Science 2005Science 2005

Sargasso seawaterSargasso seawater

- 10- 103 3 LL

100%16S rRNA 100%16S rRNA

94% rpoA94% rpoA

1,412 (observed)1,412 (observed)

~1000 (chao1)~1000 (chao1)

Venter et al., Venter et al., Science 2004Science 2004

Plum Island Sound Plum Island Sound seawaterseawater - 1L - 1L

100% 16S rRNA100% 16S rRNA

99% 16S rRNA99% 16S rRNA

1633 (chao1)1633 (chao1)

520 (chao1)520 (chao1)

Acinas and Acinas and Klepac-Ceraj et Klepac-Ceraj et al., Nature 2004al., Nature 2004

Hypersaline Hypersaline

microbial matmicrobial mat

100% 16S rRNA100% 16S rRNA

99-100% 16S rRNA99-100% 16S rRNA

1,336 (observed)1,336 (observed)

>10>104 4 (chao1 or ACE)(chao1 or ACE)

Ley R. E. et al., Ley R. E. et al., AEM 2006AEM 2006

Salt marsh Salt marsh sedimentsediment - 5g - 5g

99% 16S rRNA99% 16S rRNA 2411 ± 542 2411 ± 542

(pareto distribution)(pareto distribution)

Hong et al., Hong et al.,

PNAS 2006PNAS 2006

Marine sedimentMarine sediment

Forest soilForest soil

genomesgenomes

genomesgenomes

1.1 x 101.1 x 104 4

60006000

(DNA reassociation)(DNA reassociation)

Torsvik et al. Torsvik et al.

J. Biotech 1998J. Biotech 1998

SoilSoil - 10g - 10g genomesgenomes 8.3 x 108.3 x 1066

(DNA reassociation & (DNA reassociation & power law distribution)power law distribution)

Gans et al. Gans et al. Science 2005Science 2005

1.1. How diverse are natural microbial How diverse are natural microbial communities?communities?

Biological speciesBiological species Evolutionary speciesEvolutionary species

Ecological speciesEcological species

SpeciesSpeciespluralismpluralism

Units of BiologyUnits of Biology

Reproductive Isolation

Ecological niche

Single lineage

(Multiple definitions relevant!)

Three Three E. coliE. coli strains share <40% of total protein genes in genomes strains share <40% of total protein genes in genomes

Welch et al. (2002)

• strains from different environments

Would strains co-exist in nature outside a human host?

Core genome:- shared by all

(e.g., housekeeping)

Flexible genome:- strain specific

(e.g., pathogenicity islands, integrons)

Are co-occurring genomes with same “ribotype” ecologically-equivalent?

Stackebrandt and Goebel, 1994

16S rRNAsimilarity

Genome similarity

Challenge: To identify ecologically-differentiated populations

Correlation of ribotype to genome similarity

99% 16S rRNA

70% DNA-DNA hybridization