SURENDER RAWAT

Msc. Microbial Biotechnology

Roll No. 1784

SIGNIFICANCE OF MICROORGANISMS

INTRODUCTION

• Total number of prokaryotic cells on earth 4–6 × 1030

• Less than 0.1% are culturable

• Yet to discover the correct culture conditions for culturing the rest 99.9%

• Metagenomics presently offers a way to access unculturable microorganisms

because it is a culture-independent way to study them.

• It involves extracting DNA directly from an environmental sample –e.g. seawater, soil, the human gut – and then studying the DNA sample.

Metagenomics

• Study of metagenomes, genetic material recovered directly from environmental samples.

• Also reffered as Environmental genomics, ecogenomics, or community genomics.

• The term "metagenomics" was first used by Jo Handelsman,

Jon Clardy, Robert M. Goodman, and others,

and first appeared in publication in 1998

“The application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”

- Kevin Chen and Lior Pachter

HISTORY• Late 17th century, Anton van Leeuwenhoek :

• First metagenomicist who directly studied organisms from pond water and his own teeth.

• 1920’s:

• Cell culture evolved, 16 S rRNA sequencing of culturable microbes

• If an organism could not be cultured, it could not be classified.

• 1980’s:

• Discrepancies observed:

• (1) Number of organisms under microscope in conflict with amount on plates.

• Ex: Aquatic culture differed by 4-6 orders of magnitude from direct observation.

• (2) Cellular activities in situ conflicted with activities in culture.

• Ex: Sulfolobus acidocaldarius in hot springs grew at lower temperatures than required for culture.

• (3) Cells are viable but unculturable.

• Norman Pace proposed the idea of cloning DNA directly from environmental samples in 1985

• The first report was published by Pace and colleagues in 1991 which reported non fuctional genes.

In 2002, Mya Breitbart & Forest Rohwer, and colleagues used shotgun sequencing to show that

200 liters of seawater contains over 5000 different viruses.

In 2003, Craig Venter led the Global Ocean Sampling Expedition (GOS), circumnavigating the globe and collecting metagenomic samples throughout the journey. All of these samples are sequenced using shotgun sequencing, in hopes that new genomes (and therefore new organisms) would be identified.

The pilot project, conducted in the Sargasso Sea, found DNA from nearly 2000 different species, including 148 types of bacteria never before seen.

After leaving the Pace laboratory, Edward DeLong continued in the field and has

published work that has largely laid the groundwork for environmental phylogenies based

on signature 16S sequences, beginning with his group's construction of libraries from marine samples

Healy reported the metagenomic isolation of functional genes from "zoolibraries" constructed from a complex culture of environmental organisms grown in the laboratory on dried grasses in 1995

In 2005 Stephan C. Schuster at Penn State University and colleagues published the first sequences of an environmental sample generated with high-throughput sequencing, in this case massively parallel pyrosequencing developed by 454 Life Sciences

In 2004, Gene Tyson, Jill Banfield, and colleagues at the University of California, Berkeley and the Joint Genome Institute sequenced DNA extracted from an acid mine drainage system

Venter has circumnavigated the globe and thoroughly explored the West Coast of the United States, and completed a two-year expedition to explore the Baltic, Mediterranean and Black Seas. Analysis of the metagenomic data collected during this journey revealed two groups of organisms, one composed of taxa adapted to environmental conditions of 'feast or famine', and a second composed of relatively fewer but more abundantly and widely distributed taxa primarily composed of plankton

METAGENOMICS AND SYMBIOSIS

• Eg. the Aphid and Buchnera, • First example of genomics on an uncultured microorganism.

• lost almost 2000 genes since it entered the symbiotic relationship 200–250 million years ago.

• It contains only 564 genes

• Does not conduct many of the life functions

The deep-sea tube worm, Riftia pachyptila, and a bacterium (Boetius, 2005). • These creatures live in harsh environments near thermal vents 2600m below the

ocean surface. • The tube worm provides the bacterium with carbon dioxide, hydrogen sulfide and

oxygen, which it accumulates from the seawater. • The bacterium, converts the carbon dioxide to amino acids and sugars needed by

the tube worm, using the hydrogen sulfide for energy

Many microorganisms with symbiotic relationships with their hosts are difficult to culture away from the host are prime candidates for metagenomics.

Extreme environments

DesertDeep sea

GlacialHalophilic environments

METAGENOME OF EXTREME HABITATS

• Metagenomic analyses of seawater revealed some interesting aspects of ocean-dwelling microorganisms.

• More than one million genes were sequenced and deposited in the public databases.

• Groups of bacteria that were not previously known to transduce light energy appear to contain genes for such a function eg. Rhodopsin.

• Metagenomic analysis of the biofilm led to the computer-based reconstruction of the genomes of some of the community members.

• A model for the cycling of carbon, nitrogen and metals in the acid mine drainage environment was developed.

GUT METAGENOMICS

• The human intestinal microbiota is composed of 1013 to 1014 microorganisms

• Collective genome (‘‘microbiome’’) contains at least 100 times as many genes as our own genome.

• About 10 to 100 trillion microbes inhabit our gastrointestinal tract.

• The greatest number residing in the distal gut. • They synthesize essential amino acids and

vitamins and process components of otherwise indigestible contributions to our diet

• 70 divisions of Bacteria and 13 divisions of Archaea described to date

• The distal gut and fecal microbiota was dominated by just two bacterial divisions, the Bacteroidetes and the Firmicutes, which made up 999% of the identified phylogenetic types, and by one prominent methanogenic archaeon, Methanobrevibacter smithii.

• The human distal gut microbiome is estimated to contain ˃100 times as many genes as our 2.85–billion base pair (bp) human genome.

• Oral metagenome is also done

GUT METAGENOMICS

Metagenomic studies have revealed that each person carries a unique microbial community in his or her gastrointestinal tract; in fact these communities have been called a ‘second fingerprint’ because they provide a personal signature for each of us.

ACID MINE DRAINAGE METAGENOME

Low Diversity6 species identified with 16 S rRNA

10X coverage of dominant species Leptospirillum

Ferroplasma

Identified genes ion transport iron-oxidation• carbon fixation

N2-fixation genes found only in a minor community memberLeptospirillum

Metagenomics

• Scope of diversity: Sargasso Sea– Oligotrophic environment– More diverse than expected

• Sequenced 1x109 bases• Found 1.2 million new genes• 794,061 open reading frames with no known function• 69,718 open reading frames for energy transduction– 782 rhodopsin-like photoreceptors

• 1412 rRNA genes, 148 previously unknown phylotypes(97% similarity cut off)

– α- and γ- Proteobacteria dominant groupsVenter, J.C. 2004. Science 304:66

METHODOLOGY

• Data Storage:

– Metagenomic Library – 2 Approaches

• Function-Driven: Focuses on activity of target protein and clones that express a given trait.

• Sequence-Driven: Relies on conserved DNA to design PCR primers and hybrdizationprobes; gives functional information about the organism.

•rRNA: –“Evolutionary Chronometer:” Very slow mutation rate.–Universal and functionally similar–16S rRNA sequences used.

•Data Collection Methods:–Initially, direct sequencing of RNA and sequencing reverse transcription generated DNA.–Progressed to PCR

TWO APPROACHES FOR METAGENOMIC STUDY

TWO APPROACHES FOR METAGENOMICS• In the first approach, known as

‘sequence-driven metagenomics’, DNA from the environment of interest is sequenced and subjected to computational analysis.

• The metagenomic sequences are compared to sequences deposited in publicly available databases such as GENBANK.

• The genes are then collected into groups of similar predicted function, and the distribution of various functions and types of proteins that conduct those functions can be assessed.

• In the second approach, ‘function-driven metagenomics’, the DNA extracted from the environment is also captured and stored in a surrogate host, but instead of sequencing it, scientists screen the captured fragments of DNA, or ‘clones’, for a certain function.

• The function must be absent in the surrogate host so that acquisition of the function can be attributed to the metagenomic DNA.

LIMITATIONS OF TWO APPROACHES

• The sequence driven approach• limited existing knowledge: if a metagenomic gene does not look like a gene

of known function deposited in the databases, then little can be learned about the gene or its product from sequence alone.

• The function driven approach • most genes from organisms in wild communities cannot be expressed easily

by a given surrogate host

Therefore, the two approaches are complementary and should be pursued in parallel.

TECHNIQUE

• Genome enrichment: • Sample enrichment enhances the screening of metagenomic libraries for a

particular gene of interest, the proportion of which is generally smaller than the total nucleic acid content.

• Stable isotope probing (SIP) and 5-Bromo-2-deoxyuridine labeling of DNA or RNA, followed by density-gradient centrifugal separation.

• Suppressive subtractive hybridization (SSH)• Phage display• DNA microarray

Nucleic Acid Extraction: Cell Extraction and Direct Lysis

Cell lysis (chemical, enzymatic or mechanical) followed by removal of cell fragments and nucleic acid precipitation and purification.

GENERAL METHODOLOGY

• Nucleic acid extraction and enrichment technologies

• Genome and gene enrichment

• Metagenomic libraries

• Transcriptome libraries

• Metagenome sequencing

• Metagenome sequencing:• Complete metagenomes sequencing using large fragments of genomic DNA

from uncultured microorganisms.

• The objectives have been to sequence and identify the thousands of viral and prokaryotic genomes as well as lower eukaryotic species present in small environmental samples such as a gram of soil or liter of seawater.

Gene Targeting:PCR is used to probe genomes for specific metabolic or biodegradativecapabilities•Primer design based on known sequence information•Amplification limited mainly to gene fragments rather than full-length genes, requiring additional procedures to attain the full-length genes•RT-PCR has been used to recover genes from environmental samples since RNA is a more sensitive biomarker than DNA

Shotgun sequencing

Metagenomics and applications

• Successful products

• • Antibiotics

• • Antibiotic resistance pathways

• • Anti-cancer drugs

• • Degradation pathways• Lipases, amylases, nucleases, hemolytic

• • Transport proteins

LIMITATIONS

• – Too much data?

• • Most genes are not identifiable

• – Contamination, chimeric clone sequences

• – Extraction problems

• – Requires proteomics or expression studies to demonstrate phenotypic characteristics

• – Need a standard method for annotating genomes

• – Requires high throughput instrumentation – not readily available to most institutions

• Can only progress as library technology progresses, including sequencing technology

FUTURE OF METAGENOMICS

• To identify new enzymes & antibiotics

• To assess the effects of age, diet, and pathologic states (e.g., inflammatory bowel diseases, obesity, and cancer) on the distal gut microbiome of humans living in different environments

• Study of more exotic habitats

• Study antibiotic resistance in soil microbes

• Improved bioinformatics will quicken analysis for library profiling

• Investigating ancient DNA remnants

• Discoveries such as phylogenic tags (rRNA genes, etc) will give momentum to the growing field

• Learning novel pathways will lead to knowledge about the current nonculturable bacteria to then culture these systems