Metagenomics Bench and data analysis: concepts, historical milestones and next advances

Eduardo González-Pastor

TGAC Norwich, 2014Metagenomics: From the Bench to Data Analysis

Center of Astrobiology, MadridLaboratory of Molecular Adaptation

1. Introduction

• What is the metagenome?

• Why and how to study the metagenome?

2. Functional metagenomic approach to search for novel mechanisms of adaptation to extreme environments

Sequence

Functional analysis

• Metal and acid resistance mechanisms in microbial communities from the Rio Tinto (Spain)

OUTLINE

metagenome: the genomes of all the microorganisms (virus included) of an environmental sample, and it is studied using culture independent techniques

What is the metagenome?

“metagenomics”

Handelsman, J.; Rondon, M. R.; Brady, S. F.; Clardy, J.; Goodman, R. M. (1998). "Molecular biological access to the chemistry of unknown soil microbes: A new frontier for natural products". Chemistry & Biology 5 (10): 245–249.

Only a small percentage of the microorganisms can be cultured (around 1%) (Pace et al., 1985).

For instance, soil microbial communities could contain between 5,000 and 20,000 different species, but only few can be isolated and cultured (50-200)

The study of the metagenome provides culture independent information about the microorganisms of an environmental sample.

Why to study the metagenome?

Phylogenetic three of bacteria (rRNA 16S)

area: relative abundance of sequences

culture independent techniquesto study microbial communitiesculture independent techniquesto study microbial communities

How to study the metagenome?

metagenomicsmetagenomicsmetatranscriptomicsmetatranscriptomics

metabolomicsmetabolomicsmetaproteomicsmetaproteomics

Which microbes are in the sample?Analysis of microbial diversity

(sequencing of 16S rRNA libraries)

Which microbes are in the sample?Analysis of microbial diversity

(sequencing of 16S rRNA libraries)

Construction of metagenomic libraries(host that can be cultured

and genetically manipulable)

Construction of metagenomic libraries(host that can be cultured

and genetically manipulable)

Sequencing of metagenome Sequencing of metagenome

total DNA isolation from the environmental sample(soil, water, insect guts, human intestine, skin, saliva, etc)

total DNA isolation from the environmental sample(soil, water, insect guts, human intestine, skin, saliva, etc)

Construction of metagenomic libraries

Environmental DNA

insert

fragmentation

Host: Escherichia colirecombinant vectors

vector +

Metagenomic library

Sequence

Functional analysis

Selecting the appropiate protocol

• Liquid, solid (soil, sediment, etc), faeces

• From raw sample• After matrix/cell separation• Extraction of DNA or DNA/RNA together

• Enzymatic• Physical

• Short insert (phagemid or plasmid)• Large insert (fosmid of cosmid)• Mega-large insert (pBAC)

• Escherichia coli• Pseudomonas putida• Bacillus subtilis• Streptomyces• Pichia pastoris

Environmental sample

Total DNA Direct sequencing

Plasmid or fosmid isolation

Pyrosequencing

Discard vector seq

DNA assembly in silico

“shotgun”(3Kb)

End sequencing

DNA assembly in silico

Pyrosequencing

-Roche/454 FLX-Ilumina/Solexa

-Applied Biosystems SOLiD

DNA assembly in silico

A B

Sequencing of the metagenomic DNA

Metagenomic library

Bioinformatic analysis: • gene annotation• genome and metabolism reconstruction of microbial communities,• comparation of microbial communities from different environments

Sequencing of the metagenomic DNA

Beja et al, Science 2000

Bacteriorhodopsins

• Proton pumps localized in the cytoplasmic membrane of archaea

• Associated to retinal, a chromophore that changes its conformations when absorbs a photon. This induces a conformational change of the protein, and it is activated the proton pumping out of the cell. Then, the proton gradient is transformed in chemical energy

1. Rhodopsins in marine bacteria, a new group of phototrophs

First time that a rhodopsin is discover in an uncultured bacteria (SAR86 group) (-Proteobacteria) (protorhodopsin)

16S rhodopsin

130 kb

The bacterial protorhodopsin can be expressed in Escherichia coli, and it is functional

• binds to retinal (cells are red pigmented)• works as a proton pump activated by light

Venter et al., Science 2004

Microorganisms were collected from the Sargasso sea

Metagenomic DNA is fractionated and libraries are constructed with inserts from 2-6 kbp

(“shotgun” sequencing, pairwise-end sequencing)

• Weatherbird II: 1.66 million sequences (1.36 Gbp)

• Sorcerer II: 325,561 sequences (265 Mbp)

1800 species o phylotypes (148 new)

2. Sequencing of the microbial communities from the Sargasso sea

782 novel rhodopsin receptors from the Sargasso microorganisms

13 subfamilies• 4 known (cultured organisms)• 9 from uncultured, 7 new

Tyson et al., Nature, 2004

• Bacterial biofilms floating on acidic water from Richmond Mine (Iron Mountain, California)(pH 0-1 and high concentration of toxic metals Fe, Zn, Cu y As)

Leptospirillum gp III 10%

Archaea 10%Eucaryotes 4%Sulfobacillus ssp. 1%

Leptospirillum gp II 75%

Labelling of cells (FISH):• yellow, Leptospirillum • green, other bacteria• blue, archaea

• Acid mine drainage: process in which water, oxygen and chemolithotrophic microorganisms interact with sulfide minerals producing very acidic solutions

3. Genome reconstruction of microorganisms from acid mine drainage

Sequence of the microorganisms from the biofilms of the acidic waters, and reconstruction of the metabolism

Reconstruction of the complete genome sequence of the two most abundant microorganisms: Leptospirillum and Ferroplasma, both of them obtein energy from iron oxidation.

The sequence data allowed to create a model of the biogeochemichal cycles ruled by the microorganisms in this environment.

Tringe et al., Science 2006

• Comparison of unassembled sequence data obtained from shotgun sequencing DNA isolated from different environments.

• Quantitative gene content analysis (abundance or absence) reveals habitat specific fingerprints that reflects known characteristics of the sampled environment

• Identification of genes or metabolic pathways specific for a particular environment.

4. Comparative metagenomics of microbial communities

Comparison of 8 libraries: 3 from Sargasso sea, 3 from Whale fall (cemetery of whales, deep sea), 1 from farm soil and 1 from acid mine drainage

Comparison of libraries from soils, whale corpses and Sargasso sea

COGs: Cluster of orthologous groups of proteinsKEGG: Kyoto Encyclopedia of genes and genomes (high order cellular processes)

bacteriorhodopsin

cellobiose phosphorilase

Transport of proline/glycine betaine

photosynthesis

Polyketide synthesis (antibiotics)

• Screening of metagenomic libraries to search for a particular function (resistance to some compounds, fluorescence, etc).

• Many compounds like antibiotics, quorum sensing inhibitors or inducers, enzymes of commercial interest, pigments, etc, have been discovered.

The ISME Journal, 9 October 2008; Functional metagenomics reveals diverse

 -lactamases in a remote Alaskan soil

Heather K Allen1,2, Luke A Moe1, Jitsupang Rodbumrer1,3, Andra Gaarder1 and Jo Handelsman1

Functional metagenomics: search of genes expressing a function

2. Functional metagenomic approach to search for novel mechanisms of adaptation to extreme environments

Bias in the known mechanisms of adaptation,most from cultured microorganisms

Study of life in extreme environmentsWhich are the limits of life?

Functional Metagenomic approach(culture independent)

Search for novel molecular mechanisms of adaptation of the microorganisms to extreme conditions (toxic metales, acidic pH, low and high temperatures, high radiation and high

salt concentrations)

Biotechnological aplications, bioremediation, biomining…

OUTLINE

1. Search for metal resistance genes in microorganisms from the Río Tinto

• Nickel resistance genes from rhizosphere communities

2. Search for acid pH resistance genes in microorganisms from the Río Tinto

3. Construction of nickel resistant transgenic plants

4. Future: search for adaptation mechanisms in microorganisms from from rhizosphere and phyllosphere of Antartic plants, and from hypersaline environments

Río Tinto

S2-

SO42-

Acidithiobacillus ferrooxidans

Acidithiobacillus ferrooxidansLeptospirillum ferrooxidans.

Fe2+

Fe3+ +H

H2SO4

FeS2

• Tinto river flows through the Iberian Pyrite Belt (FeS2), southwestern Spain

• Natural environment (not the result of mining) of at least 2.000.000 years old

• Acid mine drainage (AMD): natural process in which water, oxygen and chemolitothophic microorganisms interact with the pyrite to produce oxidized iron and highly acidic solutions (average pH=2.3)

1. Search for metal resistance genes in microorganisms from the Río Tinto

Acid water and oxidation increase the solubility of other metals and metalloids

As 380 ppm

Cu 110 ppm

Cr 380 ppm

Zn 220 ppm

Ni 10 ppm

Complex microbial communities. (High diversity of eukaryotes, but

low diversity of bacteria and archaea in the planktonic phase)

Metagenomic libraries• planktonic phase: highly enriched in toxic metals, very low pH, low bacterial diversity (less than ten species)

• rhizosphere from the endemic heather, Erica andevalensis: less enriched in heavy metals, pH ~ 4-5, high bacterial diversity (root exudates are enriched in nutrients)

0.1

1H3C12

F7H7

E5Uncultured acidobacterium (AF200698)1A3Acidobacterium capsulatum (D26171)1H5C81A1

1F61B3

Uncultured acidobacterium (AB192240)1D31F3

1E2E1

Uncultured planctomycete (AF465657)F6

G10Uncultured candidate bacterium TM7 (AY225653)

1c1F12

Acidiphilium acidophilum (D86511)G3G1Acidocella sp. X91797

1B1H8Rhodopila globiformis M59066

C4B1

Bacterium Ellin 340 (AF498722)1G5Enterobacter dissolvens (Z96079)

B4Conexibacter woesei (AJ440237)

1C3Mycobacterium florentinum (AJ616230)

B9Acidimicrobium ferroxidans (U75647)

H10D9C6F5Uncultured actinomycetales bacterium (X92708)F1

F3C9

D121C5

100

Acidobacteria (26,2%)

Tm7 (1,2%)

-proteobacteria (18%)

Actinobacteria (46,4%)

-proteobacteria (1%)

Bacterial diversity in rhizosphere(16S RNA, 1450 bp)

Mirete et al. Appl. Env. Microbiol, 2007

Construction of metagenomic libraries

Environmental DNA

insert: 1-10 Kb

partial Sau3AI digestion

Host: Escherichia colirecombinant vectors

Rhizosphere:750.000 recombinantsAverage size insert: 2 Kb1,4 Gbp ~350 bact. genomes

Planktonic:30.000 recombinantsAverage size insert: 2.5 kb75 Mbp ~19 bact. genomes

AMPLIFICATION SCREENING

vectorpBluescript SKIIBam HI digested

+

SelectionConfirm resistance

Retransformation(to discard chromosomal

mutations)

Pool

Individual clones

PlasmidDNA isolation

Digestion(independent clones)

Sequence

Annotation

Identification of the genes involved in the resistance phenotype

Subcloning In vitro mutagenesistransposon

Screening of metagenomic libraries

Screening of nickel resistant genes in niquel 2 mM (toxic concentration

for the E. coli host)

13 clones with different DNA fragments inserted

LB-Nickel 2 mM

pSM1

pSM2

pSM3

pSM4

pSM5

pSM6

pSM7

pSM8

pSM9

pSM10

pSM11

pSM12

pSM13

pSKII +

0 10-1 10-2 10-3 10-4

1.1. Nickel resistance genes from rhizosphere communities

• Mirete et al. Appl. Env. Microbiol, 2007• Gonzalez-Pastor & Mirete, Metagenomics: methods and protocols, 2010

Salvador Mirete, Carolina G. de Figueras

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

DH5 pSM1 pSM2 pSM3 pSM4 pSM5 pSM6 pSM7 pSM8 pSM9 pSM10 pSM11 pSM12 pSM13

Ni concentration(mg/g dry weight)(ICP-MS)

Intracellular nickel concentration in the resistant clones

Control

pSM5

pSM12

0 -1 -2 -3 -4

pSM12

ORF2 ORF1261 aa 229 aa

pSM5

ORF1 ORF2178 aa 298 aa

ORF 1: ABC transporter, membrane subunit (48%)

ORF 2: ABC transporter, ATPase subunit (57%)

ORF 1: ABC transporter, ATPase subunit (43%)

ORF 2: ABC transporter, membrane subunit (36%)

0

1.000

2.000

3.000

4.000

5.000

6.000

7.000

8.000

9.000

DH5 pSM5 pSM12

Active transport of nickel?Ni concentration(mg/g dry weight)

ABC transporters (ATP Binding Cassette)

First description of this type of ABC transporter related to metal export but not import

DH5 (pBluescript) Control

DH5 (pSM11)

Resistance by intracellular protection

pSM11

253 aa 74 aa

serine O-acetyltransferase (SAT) (51%)

0 -1 -2 -3 -40

2.000

4.000

6.000

8.000

10.000

12.000

14.000

16.000

DH5 pSM11

Ni concentration(mg/g dry weight)

SAT is involved in nickelresistance in plants (Thlaspi)

SAT overexpression in plant cells increases the intracellular leves of reduced glutathione (GSH), which protects against the oxidative stress produced by Ni (Freeman et al., AEM, 2005)

0,5 Kb

pSM2

pSM3

pSM4

pSM8

pSM9

pSM10

pSM1

pSM13

Protein of unknown function DUF195COG1322: Uncharacterized protein conserved in bacteria

Hypothetical

DnaA protein

Unknown, and hypothetical

Conserved hypothetical protein

hypothetical protein Cphamn1DRAFT_2587VrlI-like protein

penicillin binding protein 1ATfp pilus assembly protein, ATPase PilM

similar to Amino acid transportersApolipoprotein N-acyltransferase

Conjugal transporter protein TraA

pSM7

pSM6

Acyl-CoA sterol acyltransferase (fungi)

Mirete et al. Appl. Env. Microbiol, 2007Gonzalez-Pastor & Mirete, Metagenomics: methods and protocols, 2010

ORFs organization of other nickel resistant clones

E. coli DH10B (control -)

1AA

Libraries

DNA digestion

Dilution 10-3 in LB (pH 1.8 )

Incubation at 37ºC with shaking (2 h)

Plating in LB agar-Ap-Xgal

E. coli DH10B (Control)

A B C D E 1AA

Screening by acid shock (pH 1.8) in liquid medium (2 h)

María Eugenia Guazzaroni

rhizosphereplanktonic

2. Search for acid pH resistance genes in microorganisms from the Río Tinto

15 independent clones

Guazzaroni et al. Env. Microbiol, 2012

100

10

1

0,1

0,01

0,001Perc

ent S

urvi

val a

t pH

1.8

(log

)

DH10B pSKII+ ( negative control) Clon A2

T: 0 h T: 0 hT: 1 h T: 1 h

10-

3

10-

5

10-

7

10-

3

10-

5

10-

7

10-

3

10-

5

10-

7

10-

3

10-

5

10-

7

Guazzaroni et al. Env. Microbiol, 2012

DPS: DNA Protecting protein under Starved conditions

Some DPS proteins nonspecifically bind DNA, protecting it from cleavage caused by reactive oxygen species.

Clon B1 2,855 bp

DNA protection

25% survival at pH 1.8 (1h)

Glycosyl hydrolase BNR repeat-containing protein

Ferritin DPS family protein

*

Guazzaroni et al. Env. Microbiol, 2012

ClpPX: a two component protease involved in removing heat-damaged proteins (heat shock). Not previously reported to be involved in acid pH tolerance

• ClpP is the proteolytic subunit• ClpX is the ATP-binding subunit and works as a molecular chaperone.

A chaperon involved in acid pH resistance

32 % survival at pH 1.8 (1h)

1,701 bp

ATP-dependent Clp protease, ATP-binding

subunit ClpX

Clon B2 *

ATP-dependent Clp protease, proteolytic subunit ClpP

Guazzaroni et al. Env. Microbiol, 2012

2,4 KbA1 *

2 Kb

4-hydroxy-3-methylbut-2-enyl diphosphate reductase

multi-sensor hybrid histidine kinase

A2 ** stringent response

PhoH family protein

Alkyl hydroperoxide

reductaseA5 *

Amino acid-binding ACT domain-containing protein

1,9 Kb

RNA-binding protein Hypothetical protein

D3 * 1,3 Kb

Hypothetical protein

LexA repressorD1 1,4 Kb Repressor of genes in the cellular SOS response

to DNA damage (non-active heterodimers?)

2 Kb

DNA-binding protein HU Gp45 protein

1AA10 *Involvement of HU in DNA repair.

Plays a positive role in translation of RpoS.

1AA12 *Hypothetical protein

1,9 Kb

1,7 KbIntegrase family protein

1AA13 *0.2 Kb

ORFs organization of other acid pH resistant clones

Unknown Unknown

Unknown

Unknown

Unknown

Guazzaroni et al. Env. Microbiol, 2012

-pSKII + ≈500 copies per cell

-pH 1.8 (60 m)

E. coli DH10B

-Gene inserted in chromosome, promoter induction with ITPG

-pH 4.0 (10 m)

B. subtilis PY79

P. putida KT2440

-pSEVA 15-20 copies per cell

-pH 3.8 (10 m)

Dps protein

RNA-binding protein

ACT domain-containing protein

No homology

HU protein

LexA repressor

ClpP protease

HP

HP

(-)100

10

1

0,1

0,01

0,001

100

10

1

0,1

0,01

0,001

Perc

ent S

urvi

val (

log)

100

10

1

0,1

0,01

0,001

Perc

ent S

urvi

val (

log)

Perc

ent S

urvi

val (

log)

Test of the ORFs involved in acid pH resistance in E. coli, also in Pseudomonas putida and Bacillus subtilis

Cloning in pCAMBIA3500 to transform in Arabidopsis thaliana

• Replication origin of Agrobacterium tumefaciens• T-DNA from Agrobacterium:

– Three copies of 35S promoter from Cauliflower Mosaic Virus (CaMV35S), one to transcribe the phosphinothricin gene (herbicide to select the transgenic plants), and two copies to transcribe the gene to be cloned.

– Trancriptional terminator, CaMV polyA

T-border(left)

T-border(right)

CaMVpolyA

CaMVpolyA

CaMV35S

2x CaMV35S

nickelR

phosphinothricin

Carolina González de FiguerasSalvador Mirete

3. Construction of nickel resistant transgenic plants

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

DH5 pSM6 pSM7

pSM6: Conserved hypothetical protein

pSM7: Acyl-CoA sterol acyltransferase (fungi). This enzyme solubilizes the sterol from the membrane, and is accumulated in the cytoplasm.

Could the Ni resistance be explained by changes in membrane permeability?

3. Construction of nickel resistant transgenic plants

Ni concentration(mg/g dry weight)

Wt

pSM6

Wt

pSM7

3rd generation of plants transformed with two genes involved in metal resistance genes from pSM6 and pSM7 plasmids (125ug/ml Ni) (18 days)

3. Construction of nickel resistant transgenic plants

Ferritin Dps family protein

B1 *ORF4

RNA-binding proteinD3 *

ORF5

A5 *

Amino acid-binding ACT domain-containing

protein

ORF9

B2 *

ATP-dependent Clp protease, proteolytic subunit ClpP

ORF23

DNA-binding protein HU

1AA10 *ORF14

5 individual genes were selected for cloning in pCAMBIA3500 vector

3. Construction of acid pH resistant transgenic plants

M Eugenia GuazzaroniCarolina González de Figueras

Colobanthus quitensis Deschampsia antartica

• Microbial diversity from rhizosphere and phyllosphere

• Metagenomics: - sequence - funtional (genes involved in cold and radiation adaptacion)

Verónica Morgante

4. Search for adaptation mechanisms in microorganisms from rhizosphere and phyllosphere of Antartic plants

Salt flatsAñana (Spain)

Coast Salt flatsBoyeruca (Chile),

Es Trenc (Mallorca)

Hipersaline antarctic ponds (Bratina Island)

Rhizosphereand phyllosphere

Salicornia

Calonecris diomedea (nostril salt glands)

• Microbial and viral diversity

• Functional diversit: salt resistance, UV radiation resistance, low temperatures, etc (functional metagenomics, sequencing, and metatranscriptomic in experiments with mesocosms)

4. Search for adaptation mechanisms in microorganisms from hypersaline environments (collaboration Ramón Rosselló-Móra)

Small insert metagenomic libraries have been useful to retrieve genes involved in resistance to toxic metals and acidic pH.

- genes previously described (chaperons, transporters, DNA binding proteins…)

- hypothetical and unknown genes not previously assigned to be resistant to these conditions, and now they can be annotated

CONCLUSIONS

Carolina González de FiguerasM. Eugenia GuazzaroniSalvador Mirete CastañedaVerónica MorganteMaria LamprechtOlga Zafra

Manuel GómezMarina PostigoM. Paz Martín

The team……

Collaborators from CAB