By: Jitesh

Jalthuria

MBT (Prev)

Roll No.1299

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 metagenome (genomic content of entire microbial

community), genetic material recovered directly

from environmental samples.

Also referred as Environmental genomics, Ecogenomics, or

community genomics.

The term "metagenomics" was first used by

Jo Handelsmann, 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

METAGENOMICS AND SYMBIOSIS

E.g. 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 and does not conduct many of the life functions.

• The deep-sea tube worm, Riftia pachyptila, and a bacterium (Boetius,

2005). o These creatures live in harsh environments near thermal vents 2600m

below the ocean surface.

o The tube worm provides the bacterium with carbon dioxide, hydrogen

sulfide and oxygen, which it accumulates from the seawater.

o 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

Desert Deep 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 e.g. 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.

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 groups

Venter, 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 hybridization probes; 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 hostTherefore, the two approaches are complementary and should be

pursued in parallel.

GENERAL METHODOLOGY

• Nucleic acid extraction and enrichment technologies

• Genome and gene enrichment

• Metagenomic libraries

• Transcriptome libraries

• Metagenome sequencing

TECHNIQUE

• Genome enrichment: o 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.

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

o Suppressive subtractive hybridization (SSH)

o Phage display

o DNA microarray

• Nucleic Acid Extraction:

o Cell Extraction and Direct Lysis

- Cell lysis (chemical, enzymatic or mechanical) followed by

removal of cell fragments and nucleic acid precipitation and

purification.

Metagenome sequencing:• Complete metagenome 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

biodegradative capabilities

•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

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 micro biome 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

• 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.

Thank You