MICROBIAL GENETICS

Metagenomics

Viable, but non-cultivable microorganisms

VBNC= viable but not culturable

Cases & de Lorenzo (2001) Environ Microbiol 4: 623-627

Number of species versus number of genes

Individual genomes

Metagenome= > 10.000 genes

Methods to detect non-viable Bacteria (or Archaea)

Extraction of DNA from soil or water

Amplification of 16s DNA by PCR Cloning of fragments in plasmid vectors

Sequence determination
Comparison with data banks

Soil bacteria stained with DAPI

Torsvik & Ovreas (2002) Curr Opin Microbiol 5: 240-245

Denatured Gradient Gel Electrophoresis

The DGGE/TGGE technique

DNA PCR with GC clamp 16srDNA fragments

Soil sample

Phylogenetic tree DGGE

Gl obal (G apcost: 0% ) (Jukes&Cant or)

(denaturation gradient gel electrophoresis)

4 3 2 1 0

opcL

LMG 13010 B. m ult. (II)

63 100 52

LMG 16660 B. m ult. (II) LMG 16668 B. m ult. (II) R 2900 B. cc. (VI) LMG 13544 B. am b. (VIII)

31 11 11

LMG 16656 B. c. (III) LMG 16671 B. c. (III) R 11794 B.pyrr. (IX)

99

Re-amplification
Sequence analysis

R 12631 B. pyrr. (IX)

15 91

LMG 14086 B. s tab. (IV) LMG 6997 B. stab. (IV)

50

LMG 1325 B. viet. (V)

97 95

58

LMG 6032 B. cep. (I) R 1464 B. cep. (I)

47 96

R 12633 B. c . (III) R 2879 B. cc. (VI) R 13514 B. am b. (VIII)

89 98

LMG 9927 B. anth. (VII)

69

R 696 B. anth. (VII) LMG 17828 B. anth. (VII)

85 53 79

LMG 17829 B. anth. (VII) R 4183 B. am b. (VIII) LMG 10824 B. viet. (V)

Metagenomics: Craig Venter

Direct cloning of the metagenome

DNA Large fragments

Soil sample

(20-100kb)

Cloning in BACs or fosmids (Bacterial Artificial Chromosomes)

Transformation of E. coli

Amylase activity

Metagenomics/Environmental genomics
Traditional Microbial Genomics Environmental Genomics

prepare DNA, sequence randomly isolate individuals

cultivation

prepare DNA, sequence randomly

assemble (partial) genomes assemble genome

Direct cloning of the metagenome

Direct cloning of the metagenome

http://www.asmusa.org/memonly/asmnews/jul00/feature3.html

Direct cloning of the metagenome

E. coli clones producing a new pigment

http://www.asmusa.org/memonly/asmnews/jul00/feature3.html

Direct cloning of the metagenome

Discovery of new signal molecules

Direct cloning of the metagenome

Discovery of new Phyla

http://www.asmusa.org/memonly/asmnews/jul00/feature3.html

Metagenomics 2004-now: 7mio ORFs and counting

#ORFs
Environmental metagenome Eukaryotic genome Microbial genome Global ocean sampling

Sargasso Sea

1,000,000
Minnesota Soil Whale fall community Anaerobic methane oxidation Acid mine drainage Human Arabidopsis Rice

Mouse gut

North Pacific depth series Marine phages SAR, Arctic

Human gut EBPR sludge (AU) EBPR sludge (US) Marine phages GO M, BBC

Worm
Fly

10,000
H.influenza

Yeast

Mycoplasma

100

1995

2000

2005

2007

Raes et al (2007) Curr Opin Struct Biol

Goals in metagenomics
Medical: understanding human-microbial interplay Biotech: discovering novel enzymes, streamlining industrial processes

Fundamental sciences: ecology, evolution, biogeography, microbiology Understanding ecosystem functioning and complexity

Understanding earths biogeochemical cycles Discovery: novel species, novel genes

Understanding microbial communities from their DNA: Bioinformatics

Not for the faint-hearted

1000s of species, present in various degrees of

ecological dominance Sequencing is not exhaustive! Incomplete genomes, half- or totally not assembled Short reads (no assembly) with incomplete genes Huge datasets computing power (or smart programming) needed! Sampling issues: biases, contaminations Before you can understand microbial communities from their genome data, you first need to determine which influencing factors/properties of the organisms/environments might be influencing computational analyses!

>100 ongoing metagenomics projects

Comparative Metagenomics
What is different between communities? What is the same? Can we learn something about how organisms adapt to their environment?

Relationship between genomic properties and habitat

Influence of the environment on genome composition

Sargasso sea: 34% GC Minnesota soil: 61% GC Whale falls: 48 % GC Acid mine: 47 % GC

Update with GOS survey data confirms early observations

avg GC%
GC content

Foerstner et al. (2005) EMBO reports

Influence of the environment on genome size For some samples, clear

difference between general and bact-spec EGS Clear difference between environments: Soil is bigger than all others, sargasso is smallest One aberrant Sargasso, all others are remarkably similar

Raes et al. (2007) Genome Biology

Differences in EGS reflect differences in ecological complexity

1.6 Mb

3.2 Mb

3.6 Mb

4.5 Mb

Organism density (competition)

Environment stability (T, weather)

Nutrient diversity

Influence of the environment on gene functional repertoire

Protein content profile of environments functional fingerprints

Tringe, Von Mering et al. (2005) Science

Preferred habitats

currently known genomes not very representative clear habitat preferences of microbial clades changes in habitat rather rare ?

Long tail of rare novelty

Many small, unknown protein families

Harrington et al. (2007) PNAS

Data integration: Reconciling species diversity with functional diversity and habitat (geochemical) data
(collab. P.Hughenholz JGI / N.Pace Boulder CO)

Link between habitat and evolutionary rate

Soil organisms are slow-evolving (+high GC, +large genomes), Surface water organisms evolve rapidly (+low GC, +small genomes).

Von Mering et al. (2007) Science

Metabolic adaptation along global physico-chemical gradients

R ight: world map showing combined oxygen levels and ocean temp erature for selected Global Ocea n Survey samples. B elow: K EGG metabolic map showing processes linked to environmental constraints. Processes were color-coded according to a significant contribution (p< 0.05) of an environmental factor to the variation of that map (using a stepwise m ultiple reg ression analysis) accross samples.
Color le ge nds:

silica te tem pe ra ture nitra te /phos pha te salinity oxygen sam pling de pth multiple fa ctors

temperature

Data integration: Determining metabolic adaptation to environmental constraints

(collab. M.Gerstein Yale)

oxygen

photosynthesis gene a bundance

oxygen

carbon fixa tion gene abunda nce

combined oxygen + temperature model

Data integration: comprehensive approaches to avoid pitfalls

Raes and Bork, Nat Rev Microbiol, in press

Systems biology from proteins to ecosystems

Now metagenomics, soon metatranscriptomics, meta-metabolomics Computational integration will be main challenge

Raes and Bork, Nat Rev Microbiol, in press

Future: the human ecosystem

Eco-systems biology in health and disease