Community genomics and microbial diversity NM

MICROBIAL COMMUNITY GENOMICS Nov 16 2006 Reading: Venter et al Science 2004 Tyson et al Nature 2004 Microbial community of biomat in a hypersaline pond T. Kawaguchi, University of South Carolina, USA 1 History of approaches to microbial diversity • Presequencing era: bacterial species characterized on the basis of biochemical properties (stains/metabolism) and morphology (cell wall, shape) – Microscopy came first – Need to culture as prerequisite for associating biochemical properties with morphological ones • • • • • Ribosomal RNA sequencing: revolutionized views of “prokaryotic” relationships and gave basis for current view of universal tree of life (Carl Woese, 1980’s) Small subunit rDNA large scale sequencing (Ribosomal Database Project) using PCR, improved accuracy of the universal tree (late 1980’s-present) Environmental analyses to bacterial diversity (1990’s) – In situ hybridization of rRNA to relate sequence to distribution Comparative Genomics: revealed massive movement of foreign genes into lineages of organisms over evolutionary time, development of advanced methods for recognizing functional homology Community genomics / “meta-genomics”: merging genomics and inferences about functional properties with evolutionary and ecological framework Microbiology and cultivability, some background • Roots of microbiology are in microscopy (Antonie von Leeuwenhoek, 1680’s -->) • Culturing offered a way to establish causality of a microbial process such as disease • Robert Koch’s postulate for demonstrating disease causality required culturing. • Most knowledge of microbial processes was established on the basis of experiments with cultured organisms. • Most study was focused on the role of microbes in disease. • But several lines of evidence suggested that a large portion of the microbial world did not grow in lab cultures. Reference: Handelsman J. Microbiol Mol Biol Rev. 2004. 68:669-85. “Metagenomics: application of genomics to uncultured microorganisms.” 2 “Great Plate Count Anomaly” • Discrepancy between counts of bacterial cells with microscopy and numbers of colonies that grew on lab media. • Molecular parallel -- Studies comparing 16S rDNA sequence diversity from PCR or in situ screens to diversity as revealed by culture methods show that 99.8% of environmental bacteria do not grow in standard culture conditions – Many 16S rRNA sequences from environmental samples are not found in cultured organisms from those same sources. • – True for soil, marine, symbiotic, other communities – Primary obstacle in the study of bacterial communities – Obstacle to the harvest of useful genes for catalyzing biochemical reactions or for medicines General consensus: <1% of prokaryotes is cultivable using standard lab methods. Some early ( pre-1990) results of bacterial molecular phylogenetics based on rRNA – Recognition of the Archaea as distinct group – Previously recognized higher taxa completely reorganized, many were heterogeneous – Small genome bacteria recognized as derived rather than primitive (Mycoplasmas/Mollicutes, Rickettsia) – Confirmation of symbiotic origin of mitochondria and chloroplasts 3 mitochondria chloroplasts Relating sequence to morphology and location • In situ hybridization – Oligomer with tag for visualization used as probe to bind specifically with target characterizing organism known from sequence – rRNA most commonly used (abundant in cell) – Multiple oligos with different colored tags for different organisms. • In situ PCR – Can be used for very low copy numbers of template – E.g., some pathogens in host tissue 4 Environmental in situ hybridization Different fluorescent dyes attached to specific oligomers Complementary to rRNA of different taxa Reveals distribution and abundance of different groups of bacteria Sludge from wastewater plant in Munich R. Amann et al. 1996. J Bacteriology Environmental microbiology • What is the genomic basis of processes in natural microbial communities? – Bacteria important in ecological, medical, industrial processes – Diverse biochemical capabilities with big differences among species – Many communities are complex mixture of interdependent organisms How can we study the function and ecology of noncultivable microorganisms? 5 Community Genomics = Metagenomics = Environmental Genomics =Study of genomes of microorganisms sampled directly from the environment, without laboratory culture Rationale: Microorganisms are the reservoir of most gene diversity and metabolic capabilities. Most microorganisms have not been cultured and cannot easily be cultured. Metagenomics offers a way to study genetic features and capabilities of microbes without culturing them. Goals of “metagenomics” • Two goals – Understanding of microbial community diversity and functioning • How do bacterial communities affect ecosystem processes? – Medical, environmental applications • How much recombination is occurring in natural populations? – Discovery of novel biocatalyst genes for applications 6 Potential uses of microbe-encoded products & processes • Bioremediation • Energy production • Pesticides for use in agriculture including GM organisms • Industrial production of compounds • Production of drugs, such as anti-tumor compounds or anti-microbials • Probiotics 1. Extract DNA from environmental sample 2. Construct library conventional small insert (<10kb) library 3. Screen Sequence and look for genes with functions of interest Limits search to genes Limitations: with homology to functionally characterized genes (from models) large insert (cosmid or BAC) library (up to 200 kb), allows sampling of whole operons Perform functional screens: directly test for some biochemical property in the cloning host Possible problems with efficient transcription of the cloned fragment, translation, secretion of the product, correct chaperones for folding of the product 7 Marine bacteria PCR-based approaches have revealed that 99% of marine microbes cannot be readily cultured in the lab. Gene sequencing from marine DNA has revealed undiscovered genes that imply new metabolic strategies: Proteorhodopsin = rhodopsin-based photosynthesis in bacteria located on a 130 kb BAC from ocean sample recognized based on similarity to archaeal rhodopsins Same basic structure as animal eye opsins Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea Beja et al. Science 2000 Expressed in E. coli (red) structure Symbionts providing defense: Polyketides produced by symbionts of beetles and sponges Biosynthesis is encoded in a 75kb acquired chromosome fragment Used as anti-tumor drugs Similar compounds made by fungi, easier to produce with bacterial genes J Piel 2002 PNAS 99: 14002 8 Metagenomics based on assembling small inserts • Simple communities – Possible to assemble large pieces of individual genomes Insect symbiont examples, Tyson et al example • Complex communities – More difficult to assemble large parts individual genomes – Is information useful? Sharpshooters harbor two obligate symbionts in their bacteriomes provide the host with nutrients not in diet bacteriomes Sharpshooter: Cuerna sayi D Takiya Candidatus “Baumannia cicadellinicola” (Gammaproteobacteria) Candidatus “Sulcia muelleri” (Bacteroidetes) Moran et al. 2003 Environ. Microbiol. Moran et al. 2005 Appl. Environ. Microbiol. 9 Baumannia cicadellinicola Sulcia muelleri from Homalodisca lacerta (Cicadellinae) 5 µm P. Tran, Univ of Arizona 400 Glassy-Winged Sharpshooters from lemon grove in Riverside, California Dissect out bacteriomes Filters to remove most host nuclei Extract DNA Small and medium insert libraries Assemble, sort with blast, GC content Host, other contaminants Wolbachia reads Sulcia muelleri Partial genome Baumannia cicadellinicola Full genome D. Wu, J Eisen, N. Moran et al. 2006 PLoS Biology 10 From genomic sequences of two H. coagulata symbionts Baumannia cicadellinicola (complete genome sequence) MAKES VITAMINS Includes pathways for 12 vitamins (84 genes) One amino acid pathway (His) Sulcia muelleri (partial genome sequence, 146 kb) MAKES AMINO ACIDS Part or all of pathways for: Lys, Trp, Arg, Thr, Val, Ile, Leu (no His pathway) Quiz on readings • What were the two habitats studied in the two readings – Venter et al. – Tyson et al. • Which was more diverse? 11 GW Tyson et al. 2004 Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature. • Sequencing of DNA cloned from acidic biofilm at mining site in California • Simple community – library of 384 16S rRNA sequences – 3 bacterial and 3 archaeal lineages – minor variants within each group • Dominated by Leptospirillum Group II (Bacteria) and Ferroplasma (Archaea) • Designed rRNA probes for in situ to visualize organisms within biofilm Simple community Acid mine drainage Pink biofilm Dominated by Leptospirillum (Bacteria, most) & Ferroplasma (Archaea fewer) 12 Assembly of acid mine sequences Tyson et al 2004 • • • They allowed alignment discrepancies above sequencing error, if consistent with end-pairing Why? Assigning scaffolds to organism types: – Separated scaffolds by GC content: >43.5% GC v <43.5% GC High GC scaffold bin (~10X coverage). – single 16S rRNA corresponding to Leptospirillum species, – total length similar to sequenced Leptospirillum genome – implied that most of the genome was recovered • Low GC scaffolds (~10X coverage). – – – – single 16S rRNA corresponding to a Ferroplasma species, similar overall length as another sequenced Ferroplasma genome average sequence divergence of 22% As for Leptospirillum, implied that essentially whole genome was recovered Metagenomic sequencing can reveal complex histories of recombination among member genomes Polymorphism & recombination in Ferroplasma Type I strains from acid mine metagenome Tyson et al 2004 13 3 ancestral strains have undergone ~400 recombination events No recombination with Ferroplasma type II strains Implications for species definitions in bacteria Tyson et al 2004 Metabolic inferences e.g., Infer N fixation In Leptospirillum Gr III, N fixed by Lepto. Gr III Then used by Ferroplasma 14 Venter et al. 2004 study on the Sargasso Sea (Science 304:66) • DNA extracted from ~1500 liters of surface seawater from the subtropical North Atlantic Ocean off Bermuda • ~2 million cloned DNA fragments, 2 to 6 kb in size. • 1.2 million new genes • 1164 different 16S ribosomal DNA (rDNA) genes sampled • able to reconstruct only two, almost-complete genomes • 9 large plasmids Do different genes capture the same distribution of phylogenetic diverstiy? 15 Prochlorococcus scaffolds showing synteny to fully sequenced genome of one isolate that was previously sequenced. Previous sequence scaffolds heterogeneity of Prochlorococcus scaffolds not derived from a single strain, but instead represent a conglomerate assembled from distinct but closely related Prochlorococcus biotypes. Example--> Individual reads 16 Venter et al. 2004 Fig. 4. Circular diagrams of 9 complete megaplasmids from the Sargasso Sea. Layers represent forwardand reverse-coding genes. Colors indicate functions inferred by sequence homology. Including putative genes for arsenate, mercury, copper, & cadmium resistance Sargasso Sea Sample: Gene count breakdown by TIGR role category. TIGR role category Amino acid biosynthesis Biosynthesis of cofactors, prosthetic groups, and carriers Cell envelope Cellular processes Central intermediary metabolism DNA metabolism Energy metabolism Fatty acid and phospholipid metabolism Mobile and extrachromosomal element functions Protein fate Protein synthesis Purines, pyrimidines, nucleosides, and nucleotides Regulatory functions Signal transduction Transcription Transport and binding proteins Unknown function Miscellaneous Conserved hypothetical Total number of roles assigned Total number of genes Total genes 37,118 25,905 27,883 17,260 13,639 25,346 69,718 18,558 1,061 28,768 48,012 19,912 8,392 4,817 12,756 49,185 38,067 1,864 794,061 1,242,230 17 Environmental samples can reveal diversity of particular gene families Eg Proteorhodopsin Is hyperdiverse in Sargasso sample Venter et al. 2004 Nov 9 2006 Expects to Build UCSD Lab to Develop Drugs from Ocean Microbes J. Craig Venter Institute Community Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis $24.5M in support from the Bay Area-located Gordon and Betty Moore Foundation. Craig Venter on the Sorceror II http://www.sorcerer2expedition.org 18 Question: Can useful information be obtained without worrying about assembling into chromosomes and organisms? • SG Tringe et al 2005 Science “Comparative Metagenomics of Microbial Communities”: – “The sequence data have posed challenges to genome assembly, which suggests that complex communities will demand enormous sequencing expenditure for the assembly of even the most predominant members (Venter et al. 2004)” • • Can specific communities be characterized by gene content, without assembling the individual genomes? Compared three microbial assemblages: – Minnesota farm soil – Deep sea “whale falls” (community on whale carcasses in the ocean) – Sargasso Sea samples Fig. 4. Enrichments of gene functional groups in specific environments. Three-way comparisons of soil, whale fall, and Sargasso Sea environments in terms of COGs, operons, KEGG processes, and COG functional categories. Each dot shows relative abundance of an item in the 3 environmental samples, such that proximity to a vertex is proportional to the level of enrichment in the respective sample. 1, COG5524 bacteriorhodopsin; 5, COG3459 cellobiose phosphorylase; 7, ABC-type proline/glycine betaine transport system; 10, Na+-transporting NADH:ubiquinone reductase; 14, osmosensitive, active K+transport system; 18, photosynthesis; and 19, type I polyketide biosynthesis (antibiotics). Tringe et al 2005 19 KEGG (Kyoto Encyclopedia of Genes and Genomes) 1.1 Carbohydrate Metabolism Glycolysis / Gluconeogenesis Citrate cycle (TCA cycle) Pentose phosphate pathway Pentose and glucuronate interconversions Fructose and mannose metabolism Galactose metabolism Ascorbate and aldarate metabolism … 1.2 Energy Metabolism Oxidative phosphorylation Photosynthesis Revised! Photosynthesis - antenna proteins Carbon fixation … 20 EF DeLong et al 2005 Science “Community genomics among stratified microbial assemblages in the ocean's interior.” KEGG categories at different depths, from photic zone to deep water More photosynthesis genes in the photic zone! Implications of metagenomic studies for bacterial community ecology • • Metabolic interdependence of closely associated microbes may underlie inability to establish many of them in pure culture Examples: – Acid mine Leptospirillum and Ferroplasma (N fixation and supply) – Oral cavity bacteria in gum disease: later-colonizing species (Porphyromonas gingivalis) depend on adherence to surface molecules of earlier-colonizing species (Streptococcus spp. & Actinomyces spp.) • • Understanding of these relationships could provide ways of directing community succession, so as to prevent disease, improve remediation of wastes, etc. Many recent efforts to establish prokaryotes in culture by exposing them to products of other organisms. – Pure culture still the best way to show causation of microbial process – Pure culture allows collection of DNA sample for shotgun sequencing 21 Culturing prokaryotes dependent on products of other organisms Membrane with pores smaller than bacterial width Cage in natural environment Human gut: In a person, bacterial cells exceed human cells by >10 fold Human intestinal microbiota: 500-1,000 different species, Total biomass of ~ 1.5 kg/person Number of genes in the human ‘microbiome’ may exceed number of human genes by 100-fold Most are noncultivable Major effects on development of intestine and immune system, nutrition, disease resistance JI Gordon lab 22 Metagenomics of viruses • bacteriophage = largest pool of uncharacterized diversity • Major sampling efforts from oceans, soil, human gut – Requires methods to remove DNA from cellular sources – Average bacterial genome size is 5 Mb, average viral genome size is about 50 kb, (100 fold difference). • Represent a huge mobile reservoir of genes that is sampled by a wide array of bacterial lineages • Can deposit genes permanently into bacterial genomes – major source of lateral gene transfer • Ecologically important – – – – Disease-causing genes (Shiga toxin and others) Genes underlying photosynthesis in marine cyanobacteria Many toxin gene involved in biological defenses Most phage genes are uncharacterized Edwards RA, Rohwer F. Viral metagenomics. Nat Rev Microbiol. 2005 23 Reading for TUESDAY Nov 21 Bejerano, G. et al 2004. Ultraconserved elements in the human genome Science 304:1321 AND/OR Derti, A. et al. 2006. Mammalian ultraconserved elements are strongly depleted among segmental duplications and copy number variants. Nature Genetics 38:1216 Be ready to summarize at least one of these papers! Cicadellinae (=“sharpshooters”) Major plant pests - disease vectors Diet = xylem sap --very poor in all organic compounds sugar, vitamins & most amino acids low or absent Contains very dilute amino acids: asparginine, glutamine, ammonia, ureides, organic acids, minerals Photos D. Takiya & Regents, UCalifornia 24