Genómica y ecología de las comunidades bacterianas de Cuatrociénegas, Coahuila

13 noviembre 2012 Gabriela Olmedo

Cinvestav Unidad Irapuato

Gabriela Olmedo Departamento de Ingeniería Genética���CINVESTAV Irapuato

Microbial communities

Dispersal and community assembly “Everything is everywhere and the environment selects” Bass-Becking, 1934; O'Malley, 2007

Environmental distribution of prokaryotic taxa

Soil and freshwater habitats are the least restrictive environments as they harbor the highest number of prokaryotic taxa and species

Environmental distribution of prokaryotic taxa. Tamames et al. BMC Microbiology 2010

Should we be concerned about taxa ….or about traits?

Freshwater Soil

Gut

Phyla

Environments

Microbioma

•  Cuáles son las áreas que transformarán la investigación biomédica en la siguiente década?

•  La investigación sobre el Microbioma humano (El total de las especies microbianas que habitan en el cuerpo humano)

Nuestro lado Microbiano

Biodiversidad microbiana

•  Organismos unicelulares •  Aproximadamente 5 millones de trillones de trillones en

la Tierra (¿quien las contaría?) •  Afectan todos los procesos ecológicos desde

decaimiento de la materia orgánica hasta la producción de oxígeno.

•  Los nuevos inventarios microbianos nos dan una nueva visión de los ecosistemas y cómo responden a estrés… como pesticidas, especies invasivas, cambio climático…

Today’s talk: Bacillus and Astrobiology

Cuatro Ciénegas Geology, dunes, desert, ponds, and stromatolites

Diversity: Individual genomes and metagenomes Genomics, phylogeny, clues to ecology (Bacillus coahuilensis, Bacillus m3-13) Comparative genomics of the Bacilli

Communities How is the high diversity of the Bacillus spp. in sediment communities explained? Bacillus assembly: competition determines structure (Gangs in Microbial Communities) Ecological species concept Ecophylogenetics

Microbial Stowaways: Inimitable Survivors or Hopeless Pioneers? Janet L. Siefert, Valeria Souza Luis Eguiarte and Gabriela Olmedo-Alvarez

ASTROBIOLOGY Volume 12, Number 7, 2012

Polizones Microbianos: Sobrevivientes inigualables o pioneros sin esperanza?

The resiliency of prokaryotic life has provided colonization across the globe and in the recesses of Earth’s most extreme environments.

Horizontal gene transfer provides access to a global bank of genetic resources that creates diversity and allows real-time adaptive potential to the clonal prokaryotic world.

What is the likelihood that this Earth-based strategy could provide survival and adaptive potential, in the case of microbial stowaways off Earth?

Bacillus, a spore former •  Bacilli are ubiquitous

bacteria •  Spores are resistance to

radiation, disecation: great survival strategy

•  Spores complicate understanding the Bacillus ecology

Sporulation

Germination

Vegetative cycle

Heat resistant spore

Bacillus subtilis life cycle

Concern about biological contamination?

•  Potential for spacecraft contaminants to interfere with life-detection protocols and the evolution of the planet’s putative life itself

•  DNA can persist in certain terrestrial environments for at least thousands of years (Willerslev et al., 2004)

•  On Mars, DNA would be subjected to environmental challenges such as: high UV radiation, desiccation, low temperatures, low pressure, and oxidants in martian soil

•  These factors are expected to damage DNA by inducing formation of pyrimidine dimers, bulky adducts, interstrand crosslinks, and breaks in the phosphodiester backbone

•  Any of these processes that damage DNA can result in the loss of its biological function, that is, its capacity to serve as a template for replication or transcription.

Bacillus subtilis Spores on Artificial Meteorites Survive Hypervelocity Atmospheric Entry:

Implications for Lithopanspermia

Patricia Fajardo-Cavazos, Lindsey Link, H. Jay Melosh, and Wayne L. Nicholson. Astrobiology. December

2005, 5(6): 726-736.

Inactivation of strain 168 spore viability in chromosomal DNA isolated from spores exposed

on aluminum coupons to simulated martian conditions.

Spore viability

rpoB template functionality

Paradoxical DNA Repair and Peroxide Resistance Gene Conservation in Bacillus

pumilus SAFR-032

•  Bacillus spores are notoriously resistant to: UV radiation, γ-radiation, H2O2, desiccation, chemical disinfection, or starvation

•  B. pumilus SAFR-032 survives standard decontamination procedures of the Jet Propulsion Lab spacecraft assembly facility

•  Both spores and vegetative cells exhibit elevated resistance to UV radiation and H2O2 compared to other Bacillus species.

Gioia J et al PLoS ONE 2007

Resistance of B. pumilus SAFR-032 spores to UV radiation and H2O2

Gioia J et al PLoS ONE 2007

B. pumilus SAFR-032

B. subtilis 168 B. pumilus KL 196

Radiation dose J/m2

% s

urvi

val

Sur

viva

l N/ N

o

Concern about biological contamination

•  Potential for spacecraft contaminants to interfere with life-detection protocols and the evolution of the planet’s putative life itself.

•  DNA can persist in certain terrestrial environments for at least thousands of years (Willerslev et al., 2004).

Sporulation

Germination

Vegetative cycle

Biofilm Bacillus subtilis

Sporulation

Germination

Vegetative cycle

Matrix producer

Competent Swarming

Swimming Canibal

(DNA uptake)

(eDNA release)

DNA plays an important role in the community ecology of the Bacillus spp.

DNA is the favorite P substrate for most of the cultured Bacillus

K2PO4 CaPO4 DNA RNA

Origin of terrestrial gypsum dunes—Implications for Martian gypsum-rich dunes of Olympia Undae

•  Estancia, White Sands, Guadalupe and Cuatrociénegas Dune Fields: among the largest known aeolian gypsum sand-dune accumulations on Earth and occupy closed-drainage basins within the Rio Grande Rift

•  High sedimentation rates of lacustrine gypsum occur in topographic depressions within the closed basins.

•  The gypsum accumulations result from long-term, complex, interaction between tectonism, climate, and a hydrologic cycle

•  As on Earth, gypsum-rich dune fields apparently are rare on Mars. Gypsum has been identified only within the Olympia Undae Dune Field which encircles a portion of the Martian north polar residual ice cap

•  Gypsum-rich source sediment could have been formed by confined groundwater or surface water activity and later transported by the wind

Szynkiewicz, A. et al. Geomorphology 121 (2010) 69–83

Estancia, White Sands, Guadalupe and Cuatrociénegas Dune Fields

Cuatro Ciénegas: dunas desde el espacio

Cuatro Ciénegas, Coahuila, México

Cuatro Ciénegas Dunes

Tapetes microbianos donde aparentemente no hay nada

Cuatro Ciénegas: One of the most biologically rich and diverse ecoregions

in the world

An interior basin containing hundreds of spring-fed pools in a desert environment

Half of the 20 species of fish, and 23 of the 34 species of freshwater mollusks are endemic (i.e. Cichlasoma minckleyi)

Some of these fish and mollusk species are restricted to individual pools of only a few square meters in size.

Stromatolites: Oldest evidence of microbial life

NATURE|Vol 448|30 August 2007

Some of the ponds in CC have stromatolites

Classical trophic chain in a pond

Phosphates nitrates

Phosphorous

Nitrogen

Carbon

Sediment community at Churince

Bacillus spore

Microbial mats

Today’s talk: Bacillus and Astrobiology

Cuatro Ciénegas Geology, dunes, desert, ponds, and stromatolites

Diversity: Individual genomes and metagenomes Genomics, phylogeny, clues to ecology (Bacillus coahuilensis, Bacillus m3-13) Comparative genomics of the Bacilli

Communities How is the high diversity of the Bacillus spp. in sediment communities explained? Bacillus assembly: competition determines structure (Gangs in Microbial Communities) Ecological species concept Ecophylogenetics

Different scales in Microbial Ecology

Individual Population Community Ecology Physiology:

Phenotype Demographics: Birth, death, immigration,etc.

Interspecific interactions that shape community structure and function

Genomics Individual genomes/genes

Comparative genomic analysis to assess variation

Metagenomics: Genetic potential of collective members of a community

Adapted from Little et al. Microb. Rev. 08

Microbial Ecology at Cuatro Ciénegas

Individual Population Community Ecology Physiology:

Phenotype Bacillus

Demographics: Birth, death, immigration,etc. Bacillus

Interspecific interactions Bacillus

Genomics Individual genomes Bacillus

Comparative genomic Bacillus

Metagenomics: Poza Azul & Poza Roja mats Stromatolites & oncolites

Adapted from Little et al. Microb. Rev. 08

Genómica y Metagenómica

El DNA tiene un código universal de 4 letras (bases)

A

C

G

T

Los genes son la información codificada en el DNA

Cada proteína es codificada por un gen

Proteína

Gene

hemoglobina

hemoglobina

insulina

insulina

colágena

colágena

DNA

Las 4 bases dictan el orden de los aminoácidos, por lo que si conocemos la secuencia de bases podemos deducir la codificación de proteínas

A

C

C

G T

T

C

G

A

Leu

Aminoácido

Val

Arg

5’ ACCGTTCGA….

5’ ACC/GTT/CGA….

Leu Val Arg

¿Qué es el genoma?

El cuerpo está formado por aprox. 100 billones de células

Cada célula tiene un juego completo de instrucciones (DNA) para hacer un ser humano

Este juego de instrucciones en forma de DNA es tu GENOMA.

Los humanos tienen 46 cromosomas (23 pares), ese es su genoma

El DNA está empaquetado entre proteínas formando cromosomas.

Una carpa (pez) tiene 104 y una bacteria sólo 1 cromosoma

Iniciativa de los 10,000 genomas animales (Abril 2009)

¿El arca de Noé? Incluye genomas de especies extintas como lobo de Tasmania

Todos se vale… Metagenoma de un parabrisas

Diversidad de insectos en un trayecto dado…

Colecta todos los insectos que quedaron “embarrados”, saca su DNA y secuéncialo

Kosakovsky Pond et al. 06

¿Puedes distinguirlas?

Código de barras de DNA para descubrir y distinguir especies

E. coli Acetobacter Campylobacter

Los microbios dominan al planeta

•  Invisibles a nuestra vista y aun muchos imposibles de cultivar…

•  Cómo caracterizarlos? Cómo conocer su diversidad?

•  Algunas bacterias pueden cultivarse y se puede secuenciar un gen que nos sirva para identificarla (16S)

•  Menos del 1% de los microbios pueden cultivarse

•  Con la metagenómica no es necesario cultivarlos

¿Qué elemento usarías para comparar estos medios de transporte?

El RNA ribosomal 16S está conservado en TODAS las bacterias…es “la rueda”

de la Biología Molecualr Tiene segmentos idénticos, pero otros variables que permiten compararlos

Al determinar las distancias genéticas podemos deducir las relaciones evolutivas entre los organismos

16s rRNA y la diversidad microbiana

•  La mayoría de las revisiones filogenéticas han sido basadas sobre el estudio de los genes 16s rRNA usando PCR con iniciadores o primers específicos para posiciones altamente conservadas en estos genes.

•  Más de 60,000 secuencias de genes de 16s rRNA han sido reportadas ( Cole, et al. 2003).

•  La inferencia filogenética con estos genes puede revelar que tipo de organismos microbianos estan presentes en una muestra.

Amplificación del 16S ribosomal

Comparación de secuencias

Aislado 1 Aislado 2 Aislado 3 Aislado 4 Aislado 5 Aislado 6 Aislado 7 Aislado 8

Evolutionary history of bacteria through whole genome sequencing

Gene

n= 3,600

n= 4,000

n= 5,000

1 Phylogeny based on a single conserved gene (16S)

2 Phylogeny based on a few or all conserved genes (phylo genomics)

3 Phylogeny based on all genes (alignment-free Phylogeny)

Strain A

Strain B

Strain C

1 2 3 4 5

Ecological function information in complete genomes

Gen1 Gen2 Gen3 Gen4 Gen5 Gen6 Gen7 Gen n

n= 3,600

n= 4,000

n= 5,000

Conserved genes (core) (central metabolic functions: replication, glycolysis, etc.)

Non conserved genes Niche revealing: antibiotic production and resistance, resistencia a antibióticos, particular carbohydrate utilization, etc.)

Strain A

Strain B

Strain C

genomicaislands prophages

Transposons

Core Genome Super integrons

Transposons Insertion sequences

plsasmids

Cornet y Chandler, 2004

Genetic diversity in bacteria is the result of mutations and gene transfer

Evolutionary history of bacteria through whole genome sequencing

Gene

n= 3,600

n= 4,000

n= 5,000

1 Phylogeny based on a single conserved gene (16S)

2 Phylogeny based on a few or all conserved genes (phylo genomics)

3 Phylogeny based on all genes (alignment-free Phylogeny)

Strain A

Strain B

Strain C

1 2 3 4 5

Ponds and rivers at CC come in different colors and shapes

Pozas Azules

Rio Mezquites

Poza Roja

Pozas Azules II Stromatolite Oncolite

Mat Mat

Fotótrofos oxigénicos

Fotótrofos oxigénicos

Fotótrofos anoxigénicos

Sulfooxidantes

Sulforreductores Thermodesulfobacteria

Microbial mats

Cyanobacteria

Diatomeas

Alpha/Gamma Proteobacteria

Archaeoglobus sp

Deltaproteobacteria

Thiocapsa thiopedia

A B

Microbial Mats in Cuatrociénegas

Pozas Azules

Poza Roja

Pozas Azules

Diversity Analysis of the mats

Isolated strains: 16S amplification and sequencing (Aprox. 340 strains)

Purifed DNA

16S amplification and sequencing

454 sequencing (Metagenome)

Taxonomic diversity in the Pozas Azules Mat and the Poza Roja mat

SPECIAL NUMBER, CUATROCIENEGAS Astrobiology Aug 2012: Comparative Metagenomics of Two Microbial Mats at Cuatro Ciénegas Basin II: Community Structure and Composition in Oligotrophic Environments Peimbert et al. (and Bonilla et al.)

Map is constructed using 123 KEGG pathways (Kanehisa et al., 2008). Color lines imply the presence of the path, gray lines implies that no reads were affiliated to the path.

a b

c

Collective metabolism of microbes in Red pool 2

P. syringae pv tomato

P. fluorescenses PfO-1

Pathways observed in Red pool 2

Core and Pangenome concept

Touchon et al. PLoS Genetics 09 Genomes of 20 E. coli strains

Bacillus core and pangenome

3640 3.35

5691 5.08

3500 3.63 3498 3.54

5117 5.23

4105 4.21 4152 4.22

4066 4.20

4096 4.30

5134 5.30 5234 5.41

5603 5.22 5287 5.22 5311 5.22

Proteins Length (Mbp)

Bacillus coahuilensis

Bacillus sp. NRRL B-14911

Oceanobacillus iheyensis HTE831 Geobacillus kaustophilus HTA426

Bacillus thuringiensis konkukian

Bacillus subtilis str. 168 Bacillus licheniformis ATCC 14580

Bacillus halodurans C-125

Bacillus clausii KSM-K16

Bacillus cereus E33L Bacillus cereus ATCC 14579

Bacillus cereus ATCC 10987 Bacillus anthracis str. Sterne Bacillus anthracis str. Ames

Core= 815 genes

Pan= 74,300

Alcaraz et al. PNAS 2008

Difficulty with 16S rRNA phylogenies….

Bacillus from the Gulf of Mexico

16S rRNA won’t give you resolution beyond genus…

B. coahuilensis

B. m3-13 Gulf of Mexico

Phylogenomics of the Bacillus with a concatenated set of core genes (all 815)

Filogenia de los Bacillus usando los genes del core (815 genes)

Phylogenetic  Reconstruction  by  Maximum  Likelyhood  (ML)  

Robust Phylogeny of the Bacillus with all core genes (815)

Filogenia de los Bacillus usando los genes del core (815 genes)

Phylogenetic  Reconstruction  by  Maximum  Likelyhood  (ML)  

Extremophiles

subtilis and related

cereus group

Water Bacillus

Ecological function information in complete genomes

Gen1 Gen2 Gen3 Gen4 Gen5 Gen6 Gen7 Gen n

n= 3,600

n= 4,000

n= 5,000

Conserved genes (core) (central metabolic functions: replication, glycolysis, etc.)

Non conserved genes Niche revealing: antibiotic production and resistance,, particular carbohydrate utilization Horizontal gene transfer

Strain A

Strain B

Strain C

Pangenome Vs. Core genome

present absent

Core

B. subtilis & related

B. subtilis

B. subtilis & related + cereus

Gene categories based on conservation

water & extremophiles

cereus group

subtilis & related

200 sporulation and competence genes from Bacillus subtilis

Plasticity of proteins for signal transduction and

for sensing environmental

cues

Phosphorelay

Spore coat

Germination

Sporulation cascade (spo0A and sigHEGFK…)

Competence genes

Alcaraz et al. BMC Genomics 2010

Genome differences between m3-13 and B. coahuilensis.

*P limitation adaptations?

Sulfoquinovose genes: membrane sulfolipid replacement?

sqd operon from Synechococcus and B. coahuilensis

3D modeling to the Arabidopsis protein homologue

Cuatro Ciénegas Geology, desert, ponds and stromatolites

Diversity: Individual genomes and metagenomes Bacillus coahuilensis, Bacillus m3-13 Genomics, phylogeny, metagenomics Comparative genomics of the Bacilli

Communities How is the Bacillus diversity explained? Bacillus assembly, competition determines structure of the communities Emerging patterns and Self-organization

Churince system Dissecation lagoon

Intermediate lagoon

•  Approx. 2 Km long •  Extremely low P

concentration •  High concentration of sulfates,

calcium, and magnesium •  Clay sediment (CaSO4)

Sampling for Bacillus

Enrich for Bacillus spp. (80oC)

Plating on Marine medium

Surface sediment 1 cm2,1-2 mm deep

0 -1

Δ 80oC, 20min

0 -1

Churince (intermediate lagoon) Sampling, 2007

RESULTS:

Water Few thermoresistant colonies

Sediment Approx. 103 CFU/ml

Conclusion: thermotolerant bacteria (mostly Bacillus) prefer sediment

1.  Plating of water and sediment on Marine medium 2.  Superficial (1-2 mm) sediment samples: area of 1-2 cm2

Diversity of heat resistant isolates among different samplig sites in Churince: Random sampling is an

unsatisfactory answer

Water Sediment Soil

All in Marine Medium

Phylogenetic diversity of thermoresistant isolates from sediment communities (16S rRNA gene)

Small sample: recover potentially interacting bacteria

How is diversity generated and maintained?

B. cereus

B. horikoshii

B. megaterium

B. marisflavi

B. subtilis

B. aquimaris

B. endophyticus B. sp NRRL B 14911

Corynebacterium

B. pumilus

Phylogenetic reconstruction based on 16S rRNA (ML)

Is community structure influenced by antagonic interactions?

isolate “A” is overlaid on agar

Growth of isolate “B” is inhibited

Isolate “C” is NOT inhibited

Isolate “D” inhibits growth of “A”

Is community structure influenced by antagonic interactions? If this was the case within and across compare interference within-site and across-site interactions would be different.

78 x 78 matrix (6,084 interactions tested)

Assembly of microbial communities is influenced by antagonism

Antagonism within-site

Antagonism across-site

Antagonic isolates

B. subtilis

B. pumilus

50% of the isolates are antagonic

Antagonism has an important taxonomic component

The potential for interference competition is much lower within natural populations than between them

B. aquimaris

Pérez et al.,The ISME J∫ Nov 2012

B. cereus

B. NRRL14911

B. horikoshii

B. alkalophiluss

Staphilococcus

Actinobacteria

Antagonist interactions can be described by network graphs

Bacillus eats Bacillus eats Bacillus…?

X

Y

Z

Feed forward loop

A very simple three species food web is illustrated in Fig. A. Species Z is prey only, Y is both a predator or consumer of Z while being prey to X, and X is the top consumer in the network.

Feed forward loops in food webs

Antagonistic traits select for resistant genotypes, thereby influencing community assembly

Fifteen antagonistic isolates produce a bacteriocin-like substance, suggesting that this is the direct mechanism for competition.

Competition for resources may still influence the interactions observed

Different ecological strategies may determine the position that the Bacillus spp. groups ocupy in the food-chain like network. Are ther trade-offs of antagonism?

At what scale does antagonism operate?

Alejandra Moreno-Letelier, Gabriela Olmedo, Luis E. Eguiarte and Valeria Souza. Divergence of Firmicutes form the Cuatro Cienegas Basin, Mexico: a true window to the Precambrian ocean. Astrobiology 12(7):674-684 Mariana Peimbert, Luis David Alcaraz, German Bonilla-Rosso, Gabriela Olmedo-Alvarez, Felipe García-Oliva, Lorenzo Segovia, Luis E. Eguiarte, and Valeria Souza. Comparative metagenomics of two microbial mats at Cuatro Ciénegas Basin I: Ancient lessons on how to cope with an environment under severe nutrient stress. Astrobiology. 12(7): 648-658 German Bonilla-Rosso, Mariana Peimbert, Luis David Alcaraz, Ismael Hernandez, Luis E. Eguiarte, Gabriela Olmedo-Alvarez and Valeria Souza. Comparative metagenomics of two microbial mats at Cuatro Ciénegas Basin II: Community Structure and Composition in Oligotrophic Environments. Astrobiology. 12(7): 659-673 Janet L. Siefert,1 Valeria Souza,2 Luis Eguiarte,2 and Gabriela Olmedo Microbial Stowaways: Inimitable Survivors or Hopeless Pioneers? (Essay). Astrobiology. 12(7): 710-715.659-673

Departamento Ing. Genética: José Antonio González Diana Fabiola Díaz Francisco Varinia López Africa Islas Ismael Hernández Zulema Gómez Lolis Torres Paul Decenas Ernesto Vázquez

Instituto de Ecología: Valeria Souza Luis Eguiarte Luis David Alcaraz

CIECO Felipe García

Langebio: Luis Herrera Estrella Unidad Monterrey Moisés Santillán Román U. Zapián

U Minnesota: Mike Travisano

Wilfrid Lauriel U: Gabriel Moreno

Cinvestav UNAM

U Arizona: James Elser

U del papaloapan, Oax: Beatriz Carely

UAQ: Juan Campos Guillén Julio Cruz

UNL: Susana de la Torre Héctor Arocha

Financing for these work came from: Proyecto Multidisciplinario de Cinvestav (G. Olmedo)

Proyecto de grupo Metagenomas de Conacyt (Valeria Souza)

Anagonists secrete a putative

bacteriocine

Antagonist on cellophan, over night

Marine medium, 37oC

Dilutions of sensitive isolate Dilutions from sensitive isolate culture Remove celophan

Thank you, Summer students!!!!

Antagonistic traits select for resistant genotypes, thereby influencing

community assembly

Within

Across