Olmedo CuatroCienegasEMA2013
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Transcript of Olmedo CuatroCienegasEMA2013
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
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
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
Matrix producer
Competent Swarming
Swimming Canibal
(DNA uptake)
(eDNA release)
DNA plays an important role in the community ecology of the Bacillus spp.
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
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.
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
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
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.
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
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
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
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
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
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.
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!!!!