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Metagenomics Bench and data analysis: concepts, historical milestones and next advances Center of Astrobiology, Madrid Laboratory of Molecular Adaptation Eduardo González-Pastor TGAC Norwich, 2014 Metagenomics: From the Bench to Data Analysis OUTLINE 1. Introduction • What is the metagenome? • Why and how to study the metagenome? Sequence Functional analysis 2. Functional metagenomic approach to search for novel mechanisms of adaptation to extreme environments • Metal and acid resistance mechanisms in microbial communities from the Rio Tinto (Spain) What is the metagenome? metagenome: the genomes of all the microorganisms (virus included) of an environmental sample, and it is studied using culture independent techniques “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. Why to study the metagenome? 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. Phylogenetic three of bacteria (rRNA 16S) area: relative abundance of sequences How to study the metagenome? metabolomics metaproteomics culture independent techniques to study microbial communities metagenomics metatranscriptomics total DNA isolation from the environmental sample (soil, water, insect guts, human intestine, skin, saliva, etc) 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) Sequencing of metagenome Construction of metagenomic libraries Environmental DNA fragmentation vector + recombinant vectors insert Host: Escherichia coli Sequence Metagenomic library 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 • • • Short insert (phagemid or plasmid) Large insert (fosmid of cosmid) Mega-large insert (pBAC) • • Enzymatic Physical • • • • • Escherichia coli Pseudomonas putida Bacillus subtilis Streptomyces Pichia pastoris Sequencing of the metagenomic DNA Environmental sample A Metagenomic library Total DNA Direct sequencing Pyrosequencing “shotgun” (3Kb) End sequencing B Plasmid or fosmid isolation -Roche/454 FLX -Ilumina/Solexa Pyrosequencing -Applied Biosystems SOLiD Discard vector seq DNA assembly in silico DNA assembly in silico DNA assembly in silico Sequencing of the metagenomic DNA Bioinformatic analysis: • gene annotation • genome and metabolism reconstruction of microbial communities, • comparation of microbial communities from different environments 1. Rhodopsins in marine bacteria, a new group of phototrophs 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 First time that a rhodopsin is discover in an uncultured bacteria (SAR86 group) (g-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 2. Sequencing of the microbial communities from the Sargasso sea 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) 782 novel rhodopsin receptors from the Sargasso microorganisms 13 subfamilies • 4 known (cultured organisms) • 9 from uncultured, 7 new 3. Genome reconstruction of microorganisms from acid mine drainage Tyson et al., Nature, 2004 • Acid mine drainage: process in which water, oxygen and chemolithotrophic microorganisms interact with sulfide minerals producing very acidic solutions • 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) Eucaryotes 4%Sulfobacillus ssp. 1% Archaea 10% Leptospirillum gp III 10% Leptospirillum gp II 75% Labelling of cells (FISH): • yellow, Leptospirillum • green, other bacteria • blue, archaea 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. 4. Comparative metagenomics of microbial communities 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. 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 bacteriorhodopsin Transport of proline/glycine betaine cellobiose phosphorilase photosynthesis Polyketide synthesis (antibiotics) COGs: Cluster of orthologous groups of proteins KEGG: Kyoto Encyclopedia of genes and genomes (high order cellular processes) Functional metagenomics: search of genes expressing a function • 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 b -lactamases in a remote Alaskan soil Heather K Allen1,2, Luke A Moe1, Jitsupang Rodbumrer1,3, Andra Gaarder1 and Jo Handelsman1 2. Functional metagenomic approach to search for novel mechanisms of adaptation to extreme environments Study of life in extreme environments Which are the limits of life? 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… Bias in the known mechanisms of adaptation, most from cultured microorganisms Functional Metagenomic approach (culture independent) 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 1. Search for metal resistance genes in microorganisms from the Río Tinto Río Tinto • 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) FeS2 Fe2+ Acidithiobacillus ferrooxidans Leptospirillum ferrooxidans. S2Acidithiobacillus ferrooxidans SO42- Fe3+ +H H2SO4 Acid water and oxidation increase the solubility of other metals and metalloids As 380 ppm Cr 380 ppm Cu Zn 110 ppm 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) 1H3 C12 F7 H7 E5 Uncultured acidobacterium (AF200698) 1A3 Acidobacterium capsulatum (D26171) 1H5 C8 1A1 1F6 1B3 Uncultured acidobacterium (AB192240) 1D3 1F3 1E2 E1 Uncultured planctomycete (AF465657) F6 G10 Uncultured candidate bacterium TM7 (AY225653) 1c1 F12 Acidiphilium acidophilum (D86511) G3 G1 Acidocella sp. X91797 1B1 H8 Rhodopila globiformis M59066 C4 B1 Bacterium Ellin 340 (AF498722) 100 1G5 Enterobacter dissolvens (Z96079) Bacterial diversity in rhizosphere (16S RNA, 1450 bp) Acidobacteria (26,2%) Tm7 (1,2%) a-proteobacteria (18%) g-proteobacteria (1%) B4 Conexibacter woesei (AJ440237) 1C3 Mycobacterium florentinum (AJ616230) B9 Acidimicrobium ferroxidans (U75647) H10 D9 C6 F5 Uncultured actinomycetales bacterium (X92708) F1 Actinobacteria (46,4%) F3 C9 D12 1C5 0.1 Mirete et al. Appl. Env. Microbiol, 2007 Construction of metagenomic libraries Environmental DNA partial Sau3AI digestion vector pBluescript SKII Bam HI digested + recombinant vectors insert: 1-10 Kb Host: Escherichia coli Rhizosphere: 750.000 recombinants Average size insert: 2 Kb 1,4 Gbp ~350 bact. genomes SCREENING AMPLIFICATION Planktonic: 30.000 recombinants Average size insert: 2.5 kb 75 Mbp ~19 bact. genomes Screening of metagenomic libraries Pool Plasmid DNA isolation Individual clones Retransformation (to discard chromosomal mutations) Confirm resistance Selection Digestion (independent clones) Identification of the genes involved in the resistance phenotype Subcloning In vitro mutagenesis transposon Sequence Annotation 1.1. Nickel resistance genes from rhizosphere communities 0 10-1 10-2 10-3 10-4 pSM1 pSM2 Screening of nickel resistant genes in niquel 2 mM (toxic concentration for the E. coli host) pSM3 pSM4 pSM5 pSM6 pSM7 13 clones with different DNA fragments inserted pSM8 pSM9 pSM10 pSM11 pSM12 pSM13 pSKII + LB-Nickel 2 mM Salvador Mirete, Carolina G. de Figueras • Mirete et al. Appl. Env. Microbiol, 2007 • Gonzalez-Pastor & Mirete, Metagenomics: methods and protocols, 2010 Intracellular nickel concentration in the resistant clones 16,000 14,000 12,000 10,000 8,000 6,000 4,000 2,000 0 DH5 Ni concentration (mg/g dry weight) (ICP-MS) pSM1 pSM2 pSM3 pSM4 pSM5 pSM6 pSM7 pSM8 pSM9 pSM10 pSM11 pSM12 pSM13 Active transport of nickel? Ni concentration (mg/g dry weight) 9.000 Control 8.000 7.000 6.000 pSM5 5.000 4.000 3.000 pSM12 2.000 1.000 0 DH5 0 -1 -2 -3 pSM5 pSM12 -4 pSM5 pSM12 ORF2 261 aa ORF1 229 aa ORF1 178 aa ORF2 298 aa ORF 1: ABC transporter, membrane subunit (48%) ORF 1: ABC transporter, ATPase subunit (43%) ORF 2: ABC transporter, ATPase subunit (57%) ORF 2: ABC transporter, membrane subunit (36%) ABC transporters (ATP Binding Cassette) First description of this type of ABC transporter related to metal export but not import Resistance by intracellular protection Ni concentration (mg/g dry weight) 16.000 14.000 DH5a (pBluescript) Control -1 -2 -3 10.000 8.000 DH5a (pSM11) 0 12.000 6.000 4.000 -4 2.000 0 DH5 pSM11 pSM11 253 aa 74 aa serine O-acetyltransferase (SAT) (51%) SAT is involved in nickel resistance 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) ORFs organization of other nickel resistant clones pSM1 Unknown, and hypothetical pSM2 Protein of unknown function DUF195 COG1322: Uncharacterized protein conserved in bacteria pSM3 Hypothetical pSM4 DnaA protein pSM6 Conserved hypothetical protein pSM7 Acyl-CoA sterol acyltransferase (fungi) pSM8 hypothetical protein Cphamn1DRAFT_2587 VrlI-like protein pSM9 penicillin binding protein 1A Tfp pilus assembly protein, ATPase PilM pSM10 similar to Amino acid transporters Apolipoprotein N-acyltransferase pSM13 Conjugal transporter protein TraA 0,5 Kb Mirete et al. Appl. Env. Microbiol, 2007 Gonzalez-Pastor & Mirete, Metagenomics: methods and protocols, 2010 2. Search for acid pH resistance genes in microorganisms from the Río Tinto Screening by acid shock (pH 1.8) in liquid medium (2 h) E. coli DH10B (control -) 1AA A B C D E 1AA Libraries rhizosphere planktonic E. coli DH10B (Control) Dilution 10-3 in LB (pH 1.8 ) Incubation at 37ºC with shaking (2 h) Plating in LB agar-Ap-Xgal DNA digestion 15 independent clones María Eugenia Guazzaroni Guazzaroni et al. Env. Microbiol, 2012 100.000 100 10.000 10 1.000 1 0,1 0.100 0,01 0.010 0,001 T: 0 h 10-3 10-3 10-5 10-7 10-5 T: 1 h D3 D2 B2 A6 A5 1AA-13 Clon A2 DH10B pSKII+ ( negative control) 10-7 1AA-12 D1 B1 A3 A2 A1 1AA-11 1AA-10 1AA-8 0.001 DH10B Survival at pH 1.8 (log) Percent Percent Survival at pH 1.8 (log) % Survival 1h 10-7 T: 0 h 10-3 10-3 10-5 10-7 10-5 T: 1 h Guazzaroni et al. Env. Microbiol, 2012 DNA protection Clon B1 Glycosyl hydrolase BNR repeat-containing protein Ferritin DPS family protein * 2,855 bp 25% survival at pH 1.8 (1h) DPS: DNA Protecting protein under Starved conditions Some DPS proteins nonspecifically bind DNA, protecting it from cleavage caused by reactive oxygen species. Guazzaroni et al. Env. Microbiol, 2012 A chaperon involved in acid pH resistance ATP-dependent Clp ATP-dependent Clp protease, protease, ATP-binding proteolytic subunit ClpP subunit ClpX Clon B2 1,701 bp * 32 % survival at pH 1.8 (1h) 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. Guazzaroni et al. Env. Microbiol, 2012 ORFs organization of other acid pH resistant clones A1 Unknown Unknown 4-hydroxy-3-methylbut-2-enyl diphosphate reductase multi-sensor hybrid histidine kinase * 2 Kb * A2 A5 2,4 Kb * PhoH family protein * Alkyl hydroperoxide Amino acid-binding ACT domain-containing protein reductase 1,9 Kb Hypothetical protein D1 stringent response LexA repressor Repressor of genes in the cellular SOS response to DNA damage (non-active heterodimers?) 1,4 Kb RNA-binding protein Hypothetical protein D3 * 1,3 Kb DNA-binding protein HU Unknown Gp45 protein 1AA10 * 2 Kb Unknown 1AA12 1AA13 Hypothetical protein 1,9 Kb * Integrase family protein * Involvement of HU in DNA repair. Plays a positive role in translation of RpoS. Unknown 1,7 Kb 0.2 Kb Guazzaroni et al. Env. Microbiol, 2012 Percent Survival (log) Test of the ORFs involved in acid pH resistance in E. coli, also in Pseudomonas putida and Bacillus subtilis 100.000 100 10 10.000 1.0001 0,1 0.100 0,01 0.010 Percent Survival (log) RNAbinding protein ACT domaincontaining protein HU protein ClpP protease E. coli DH10B LexA repressor HP No homology HP -pSKII + ≈500 copies per cell -pH 1.8 (60 m) 0,001 0.001 100.000 100 10.000 10 1 1.000 0,1 0.100 0,01 0.010 P. putida KT2440 -pSEVA 15-20 copies per cell -pH 3.8 (10 m) 0,001 0.001 100.000 100 Percent Survival (log) (-) Dps protein 10.000 10 1.000 1 B. subtilis PY79 0.100 0,1 -Gene inserted in chromosome, promoter induction with ITPG 0,01 0.010 -pH 4.0 (10 m) 0,001 0.001 3. Construction of nickel resistant transgenic plants Cloning in pCAMBIA3500 to transform in Arabidopsis thaliana nickelR T-border (left) • • CaMV polyA phosphinothricin CaMV 35S 2x CaMV 35S CaMV polyA T-border (right) 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 Carolina González de Figueras Salvador Mirete 3. Construction of nickel resistant transgenic plants 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? Ni concentration (mg/g dry weight) 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 DH5 pSM6 pSM7 3. Construction of nickel resistant transgenic plants Wt Wt pSM6 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 acid pH resistant transgenic plants Ferritin Dps family protein B1 * ORF4 RNA-binding protein D3 * ORF5 A5 Amino acid-binding ACT domain-containing protein * 5 individual genes were selected for cloning in pCAMBIA3500 vector ORF9 DNA-binding protein HU 1AA10 * ORF14 ATP-dependent Clp protease, proteolytic subunit ClpP B2 * ORF23 M Eugenia Guazzaroni Carolina González de Figueras 4. Search for adaptation mechanisms in microorganisms from rhizosphere and phyllosphere of Antartic plants 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 hypersaline environments (collaboration Ramón Rosselló-Móra) Imagen aérea de los lagos en Bratina Island Hipersaline antarctic ponds (Bratina Island) Salt flats Añana (Spain) Coast Salt flats Boyeruca (Chile), Es Trenc (Mallorca) Rhizosphere and 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) CONCLUSIONS 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 The team…… Carolina González de Figueras M. Eugenia Guazzaroni Salvador Mirete Castañeda Verónica Morgante Maria Lamprecht Olga Zafra Collaborators from CAB Manuel Gómez Marina Postigo M. Paz Martín