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

1. 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

2. 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)

3. 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 & Biology5 (10): 245–249.

4. 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?

5. Phylogenetic three of bacteria (rRNA 16S) area: relative abundance of sequences

6. How to study the metagenome? metabolomics metaproteomics culture independenttechniques tostudymicrobialcommunities metatranscriptomics metagenomics total DNA isolationfromtheenvironmentalsample (soil, water, insectguts, human intestine, skin, saliva, etc) Whichmicrobes are in thesample? Analysis of microbialdiversity (sequencing of 16S rRNAlibraries) Sequencing of metagenome Construction of metagenomic libraries (host that can be cultured and genetically manipulable)

7. fragmentation vector + insert recombinant vectors Host: Escherichia coli Sequence Metagenomic library Functional analysis Construction of metagenomic libraries Environmental DNA

8. Selecting the appropiate protocol 1. Sampletype • Liquid, solid (soil, sediment, etc), faeces • From raw sample • After matrix/cell separation • Extraction of DNA or DNA/RNA together 2. DNA/RNA Extractionmethod • Short insert (phagemid or plasmid) • Large insert (fosmid of cosmid) • Mega-large insert (pBAC) 3. Vector type • Enzymatic • Physical 4. DNA digestion • Escherichia coli • Pseudomonas putida • Bacillus subtilis • Streptomyces • Pichia pastoris 5. Microbial host

9. Pyrosequencing “shotgun” (3Kb) Plasmid or fosmid isolation -Roche/454 FLX -Ilumina/Solexa End sequencing -Applied Biosystems SOLiD Pyrosequencing Discard vector seq DNA assembly in silico DNA assembly in silico DNA assembly in silico Sequencing of the metagenomic DNA Environmental sample B A Total DNA Direct sequencing Metagenomic library

10. Sequencing of the metagenomic DNA • Bioinformatic analysis: • gene annotation • genome and metabolism reconstruction of microbial communities, • comparation of microbial communities from different environments

11. 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

12. 16S rhodopsin 130 kb First time that a rhodopsin is discover in an uncultured bacteria (SAR86 group) (g-Proteobacteria) (protorhodopsin)

13. 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

14. 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)

15. 782 novel rhodopsin receptors from the Sargasso microorganisms • 13 subfamilies • 4 known (cultured organisms) • 9 from uncultured, 7 new

16. Labelling of cells (FISH): • yellow, Leptospirillum • green, other bacteria • blue, archaea 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) Sulfobacillus ssp. 1% Eucaryotes 4% Archaea 10% Leptospirillum gp III 10% Leptospirillum gp II 75%

17. 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.

18. Tringe et al., Science 2006 4. Comparative metagenomics of microbial communities • 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.

19. 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

20. 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)

21. 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 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.

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

23. 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… Biasin the known mechanisms of adaptation, most from cultured microorganisms Functional Metagenomic approach (culture independent)

24. OUTLINE 3. Construction of nickel resistant transgenic plants 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 4. Future: search for adaptation mechanisms in microorganisms from from rhizosphere and phyllosphere of Antartic plants, and from hypersaline environments

25. FeS2 S2- Fe2+ Acidithiobacillus ferrooxidans Acidithiobacillus ferrooxidans Leptospirillum ferrooxidans. SO42- Fe3+ +H H2SO4 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)

26. As 380 ppm Cr 380 ppm Cu 110 ppm Zn 220 ppm Ni 10 ppm Acid water and oxidation increase the solubility of other metals and metalloids Complex microbial communities. (High diversity of eukaryotes, but low diversity of bacteria and archaea in the planktonic phase)

27. 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)

28. 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) 1G5 Enterobacter dissolvens (Z96079) 100 B4 Conexibacter woesei (AJ440237) 1C3 Mycobacterium florentinum (AJ616230) B9 Acidimicrobium ferroxidans (U75647) H10 D9 C6 F5 Uncultured actinomycetales bacterium (X92708) F1 F3 C9 D12 1C5 0.1 Bacterial diversity in rhizosphere (16S RNA, 1450 bp) Acidobacteria (26,2%) Tm7 (1,2%) a-proteobacteria (18%) g-proteobacteria (1%) Actinobacteria (46,4%) Mirete et al. Appl. Env. Microbiol, 2007

29. partialSau3AI digestion vector pBluescript SKII Bam HI digested + insert: 1-10 Kb recombinant vectors Host: Escherichia coli Rhizosphere: 750.000 recombinants Average size insert: 2 Kb 1,4 Gbp ~350 bact. genomes Planktonic: 30.000 recombinants Average size insert: 2.5 kb 75 Mbp ~19 bact. genomes AMPLIFICATION SCREENING Construction of metagenomic libraries Environmental DNA

30. Pool Plasmid DNA isolation Retransformation (to discard chromosomal mutations) Individual clones Confirm resistance Digestion (independent clones) Identification of the genes involved in the resistance phenotype Sequence In vitro mutagenesis transposon Annotation Subcloning Screening of metagenomic libraries Selection

31. - 1 - 2 - 3 - 4 0 10 10 10 10 pSM1 Screening of nickel resistant genes in niquel 2 mM (toxic concentration for the E. coli host) pSM2 pSM3 pSM4 pSM5 pSM6 pSM7 pSM8 pSM9 pSM10 pSM11 pSM12 pSM13 pSKII + LB-Nickel 2 mM 1.1. Nickel resistance genes from rhizosphere communities 13 clones with different DNA fragments inserted • Mirete et al. Appl. Env. Microbiol, 2007 • Gonzalez-Pastor & Mirete, Metagenomics: • methods and protocols, 2010 Salvador Mirete, Carolina G. de Figueras

32. Intracellular nickel concentration in the resistant clones Ni concentration (mg/g dry weight) (ICP-MS)

33. pSM5 pSM12 ORF2 ORF1 261 aa 229 aa ORF1 ORF2 178 aa 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%) Active transport of nickel? Ni concentration (mg/g dry weight) Control pSM5 pSM12 0 -1 -2 -3 -4

34. ABC transporters (ATP Binding Cassette) First description of this type of ABC transporter related to metal export but not import

35. serine O-acetyltransferase (SAT) (51%) 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) SAT is involved in nickel resistance in plants (Thlaspi) pSM11 Resistance by intracellular protection Ni concentration (mg/g dry weight) DH5a (pBluescript) Control DH5a (pSM11) 0 -1 -2 -3 -4 253 aa 74 aa

36. pSM1 pSM2 pSM3 pSM4 pSM6 pSM7 pSM8 pSM9 pSM10 pSM13 ORFs organization of other nickel resistant clones Unknown, and hypothetical Protein of unknown function DUF195 COG1322: Uncharacterized protein conserved in bacteria Hypothetical DnaA protein Conserved hypothetical protein Acyl-CoA sterol acyltransferase (fungi) hypothetical protein Cphamn1DRAFT_2587 VrlI-like protein penicillin binding protein 1A Tfp pilus assembly protein, ATPase PilM similar to Amino acid transporters Apolipoprotein N-acyltransferase Conjugal transporter protein TraA 0,5 Kb Mirete et al. Appl. Env. Microbiol, 2007 Gonzalez-Pastor & Mirete, Metagenomics: methods and protocols, 2010

37. A B C D E 1AA 2. Search for acid pH resistance genes in microorganisms from the Río Tinto Libraries rhizosphere planktonic E. coli DH10B (Control) Screening by acid shock (pH 1.8) in liquid medium (2 h) Dilution 10-3 in LB (pH 1.8) Incubation at 37ºC with shaking (2 h) 1AA E. coli DH10B (control -) Plating in LB agar-Ap-Xgal DNA digestion 15 independent clones María Eugenia Guazzaroni Guazzaroni et al. Env. Microbiol, 2012

38. 100 10 1 0,1 0,01 0,001 Percent Survival at pH 1.8 (log) Clon A2 DH10B pSKII+ ( negative control) 10-3 10-3 10-3 10-3 10-7 10-5 10-7 10-5 10-7 10-5 10-7 10-5 T: 0 h T: 1 h T: 0 h T: 1 h Guazzaroni et al. Env. Microbiol, 2012

39. DNA protection Clon B1 2,855 bp Ferritin DPS family protein Glycosyl hydrolase BNR repeat-containing protein * 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

40. A chaperon involved in acid pH resistance ATP-dependent Clp protease, ATP-binding subunit ClpX ATP-dependent Clp protease, proteolytic subunit ClpP 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

41. Repressor of genes in the cellular SOS response to DNA damage (non-active heterodimers?) ORFs organization of other acid pH resistant clones 0.2 Kb Unknown Unknown A1 2,4 Kb * multi-sensor hybrid histidine kinase 4-hydroxy-3-methylbut-2-enyl diphosphate reductase stringent response 2 Kb A2 * * Alkyl hydroperoxide reductase Involvement of HU in DNA repair. Plays a positive role in translation of RpoS. Amino acid-binding ACT domain-containing protein PhoH family protein A5 1,9 Kb * Hypothetical protein LexA repressor D1 1,4 Kb RNA-binding protein Hypothetical protein D3 1,3 Kb * DNA-binding protein HU Gp45 protein Unknown 1AA10 2 Kb * Hypothetical protein Unknown 1AA12 1,9 Kb * Unknown Integrase family protein 1AA13 1,7 Kb * Guazzaroni et al. Env. Microbiol, 2012

42. Test of the ORFs involved in acid pH resistance in E. coli, also in Pseudomonas putida and Bacillus subtilis RNA-binding protein ACT domain-containing protein Dps protein HU protein ClpP protease (-) E. coli DH10B 100 10 1 0,1 0,01 0,001 LexA repressor HP -pSKII + ≈500 copies per cell -pH 1.8 (60 m) No homology HP Percent Survival (log) 100 10 1 0,1 0,01 0,001 P. putida KT2440 -pSEVA 15-20 copies per cell -pH 3.8 (10 m) Percent Survival (log) 100 10 1 0,1 0,01 0,001 B. subtilis PY79 Percent Survival (log) -Gene inserted in chromosome, promoter induction with ITPG -pH 4.0 (10 m)

43. nickelR T-border (left) CaMV polyA CaMV 35S 2x CaMV 35S CaMV polyA T-border (right) phosphinothricin 3. Construction of nickel resistant transgenic plants 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 Carolina González de Figueras Salvador Mirete

44. 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)

45. Wt Wt pSM7 pSM6 3. Construction of nickel resistant transgenic plants 3rd generation of plants transformed with two genes involved in metal resistance genes from pSM6 and pSM7 plasmids (125ug/ml Ni) (18 days)

46. 3. Construction of acid pH resistant transgenic plants Ferritin Dps family protein B1 * ORF4 RNA-binding protein D3 * 5 individual genes were selected for cloning in pCAMBIA3500 vector ORF5 Amino acid-binding ACT domain-containing protein A5 * ORF9 DNA-binding protein HU 1AA10 * ORF14 ATP-dependent Clp protease, proteolytic subunit ClpP B2 * ORF23 M Eugenia Guazzaroni Carolina González de Figueras

47. 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

48. 4. Search for adaptation mechanisms in microorganisms from hypersaline environments (collaboration Ramón Rosselló-Móra) Salt flats Añana (Spain) Coast Salt flats Boyeruca (Chile), Es Trenc (Mallorca) Rhizosphere and phyllosphere Salicornia Hipersaline antarctic ponds (Bratina Island) 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)

49. 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

50. 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