Tough Times in Death Valley Soils: Geochemical Stressors and Diversification of the Bacillus

subtilis-B. licheniformis Clade

Sarah KopacPhD candidate

Wesleyan UniversitySKopac@wesleyan.edu

April 2014

Bacteria have evolved to inhabit virtually all terrestrial

habitats

Wu et al. 2009

Deinococcus radiodurans

Vibrio fischeri

Bacteroides fragilis

What characteristics of the environment cause bacteria to speciate?

• What environmental factors cause speciation?

• Are some environmental dimensions more difficult to adapt to than others?

• Can we predict which parts of the environment are evolutionarily relevant to bacteria that colonize Spacecraft and/or extra-terrestrial bodies?

Ecologically distinct groups exist within species

South-facing

North-facing

Koeppel et al. 2008. PNAS 105:2504.

Ecologically distinct groups exist within species

South-facing

North-facing

Koeppel et al. 2008. PNAS 105:2504.

Ecotype Simulation analysis of three protein-coding genes in Bacillus simplex.

Note: All strains are identical for 16S rRNA!= one recognized species

Ecotype Simulation:Rejects a priori criteria.Identifies species clusters based on analysis of population dynamics.

Death Valley National Park

Soil conductivity, boron and copper levels vary over a

transect

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4 transects x 20+ levels x 3 replicates

Soil parameters• pH• Electrical conductivity

(salinity)• Lime estimate• % organic matter• Nitrate:N• Phosphorus• Potassium

• Zinc• Iron• Manganese• Copper• Boron• Texture

(sand/clay/silt)

588 strains were isolated from the soil

• Screened for Bacillus subtilis-licheniformis• gyrA gene sequenced from each

Roughly thirty putative ecotypes have been demarcated from 588

strains

B. subtilissubclade

B. licheniformissubclade

Ecotypes differ in their associations with iron

1

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

1

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1

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111

1

1

1

1

…as well as four additional soil dimensions

1

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

1

11

1

1

1

1

1

1

111

1

1

1

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ParameterP

value

Iron 0.002

Phosphorus 0.0083

NO3-N 0.016

Soil pH 0.032

% clay 0.0897

Copper tolerance also differs across strains

Growth experiments show boron tolerance is ecotype-specific

p=1E-6 Brackets=95% CIEcotype A Ecotype B Ecotype C

In summary…• Closely related ecotypes differ in their ecologies– Associations with iron, phosphorous, NO3-N, pH, % clay

• Strains and/or ecotypes differ in their tolerance for boron and copper

• Adaptations to other parameters (salinity, lime, organic matter, potassium, zinc, manganese) might be more ancient, and therefore more difficult

Further questions

• Is there a genetic basis to differences in growth tolerance?

• Which combinations of environmental dimensions are most commonly associated with speciation?

• Are resource-based pressures also influencing speciation in this system?

Thesis committeeFrederick CohanAnnie BurkeDanny KrizancMichael Singer

Undergraduates (Krizanc)Diego CalderonCarlos FransiscoLing KeAaron PlaveWei Wang

Undergraduates (Cohan)

Alexa BoselSophie BreitbartJon ChabonClaire ConwayShyam DesaiWesley HoMelanie KorenMfundi MakamaJanine PetitoJess SherryNoor TellBrianne WeimannGreg Wong

MA/BAs and PhDs

Stephanie AracenaRob ClarkClaire FournierMenherit GoodwynMichelle TiptonJane Weidenbeck

CollaboratersAlex RooneyHeather KlineJohannes Sikorski

p=0.761171

Ecotypes have similar growth at 0mM boron