molecular methods in microbial ecology

Molecular Methods in Microbial Ecology

Contact Info: Julie HuberLillie [email protected]

Schedule: 21 October: Introductory Lecture, DNA extraction23 October: Run DNA products on gel

Lecture on PCR Prepare PCR reactions

28 October: Analyze gels from PCR Lecture on other molecular methods

Readings: Head et al. 1998. Microbial Ecology 35: 1-21.

Day 1

• Introduction to molecular methods in microbial ecology

• Extract DNA from Winogradsky Columns

Habitat Culturability (%)Seawater 0.001-0.1

Freshwater 0.25Sediments 0.25

Soil 0.3

From Amann et al. 1995 Microbiological Reviews

The Challenge for Microbial Ecology

How do you study something you can’t grow in the lab?

DNA

mRNA

Transcription

The Solution: Molecular Biology

Protein

TranslationRibosome

•Are present in all cells- Bacteria, Archaea and Eukaryotes

•Are documents of evolutionary history

•Are the basis of all molecular biological techniques

Head et al. 1998

DNA extraction from Winogradsky Columns

DNA Extraction1. Lyse cell membrane

a. Chemically detergentb. Physically bead beating

2. Pellet cell membrane, proteins and other cell parts while DNA stays in solution

3. Remove other inhibitors from DNA

4. Mix DNA with acid and salt stick to filter

5. Wash filter-bound DNA several times with alcohol

6. Elute DNA off membrane with pH 8, low-salt buffer

Day 2

• Run an electrophoresis gel of the DNA products extracted from your columns

• Learn about PCR

• Set up PCR reactions using the DNA from your extractions and an assortment of primers

Head et al. 1998

Basics of Gel Electrophoresis

• The gel is a matrix (like jello with holes)

• DNA is negatively charged- will run to positive

• Smaller fragments run faster than larger ones

• Gel contains Ethidium Bromide, which binds to DNA and fluoresces when hit with UV light (WEAR GLOVES!!!)

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What to do

• Mix 10 µl of your DNA with 2 µl loading buffer

• Load in well on gel

• I’ll load the ladder

• Run it

• Take a picture of it

Molecular Biology

DNA

mRNA

Transcription

Protein

TranslationRibosome

•Are present in all cells- Bacteria, Archaea and Eukaryotes

•Are documents of evolutionary history

•Are the basis of all molecular biological techniques

Ribosomes

• Make proteins

• rRNA is transcribed from rDNA genes

70S Ribosome

50S subunit

30S subunit

21 different proteins

16S rRNA

31 different proteins

23S rRNA 5S rRNA

The Star of the Show: SSU rRNA•Everybody has it

•Contains both highly conserved and variable regions

-allows making comparisons between different organisms

over long periods of time (evolutionary history)

•Not laterally transferred between organisms

•HUGE and growing database

SSU rRNA

Universal Tree of Life

BACTERIA

EUKARYA

ARCHAEA

Modified from Norman Pace

BACTERIA

EUKARYA

You Are Here

ARCHAEA

Polymerase Chain Reaction (PCR)

• Rapid, inexpensive and simple way of making millions of copies of a gene starting with very few copies

• Does not require the use of isotopes or toxic chemicals

• It involves preparing the sample DNA and a master mix with primers, followed by detecting reaction products

Every PCR contains:

• A DNA Polymerase (most common, Taq)

• Deoxynucleotide Triphosphates (A, C, T, G)

• Buffer (salt, MgCl2, etc)

• A set of primers, one Forward, one Reverse

• Template DNA

Typical PCR Profile

Temperature Time Action

95ºC 5 minutes DNA Taq polymerase activation

35 cycles of:95ºC54ºC72ºC

1 minute1 minute1 minute

DNA denaturizationPrimer annealingExtension creation

72ºC 10 minutes Final extension created

Slide courtesy of Byron Crump

Things you can optimize

• Temperature and time to activate Taq polymerase

• Temperature and time to allow primer annealing

• Temperature and time for extension

• Concentration of reagents, especially primers, dNTPs, and MgCl2

• Concentration of template DNA

• Number of replication cycles

• Etc…

Primers we are using

• 16S Bacteria

• 16S Archaea

• mcr Methanogens (Methyl coenzyme M reductase)

• dsr Sulfate reducers (Dissimilatory bisulfite reductase

Reagent Volume (µl) per reaction # of reactions final volume Sterile H20 22.7 5X PCR buffer 10 dNTPs (8mM) 5 Taq polymerase (5 Units/µl) 0.3 Tube Master mix Target Template Vol F primer Vol R primer Vol

µl µl µl µl

1 38 Sulfate reducers Column DNA 2 dsr1F 5 dsr4R 5

2 38 Methanogens Column DNA 2 ME1 5 ME2 5

3 38 Bacteria Column DNA 2 8F 5 1492R 5

4 38 Archaea Column DNA 2 20F 5 958R 5

5 38 Archaea + control M. jannaschii

2 20F 5 958R 5

6 38 Nothing - control (water) 2 20F 5 958R 5

Day 3

• Examine gels from PCR

• Learn about more molecular methods in microbial ecology

Andrea

Paliza

Jessica

Sarah

Rob

Amy

Class DNA10 kb

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Methane Concentration (uM)

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Col 1

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Col 8

Rob

Sarah

Paliza

Jessica

Andrea

Amy

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Hydrogen Sulfide Concentration (mM)

Dep

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m)

Col 1

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Col 8

Rob

Sarah

Paliza

Jessica

Andrea

Amy

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Rob

Paliza

Amy

Andrea

Sarah

Jessica

3 kb 1 kb 3 kb 1 kb

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So you have a positive PCR product: Now what?

• Clone and sequence clones

• Go straight into sequencing (454)

• Get “community fingerprint” via DGGE and sequence bands

• Get “community fingerprint” via T-RFLP

Schematic courtesy of B. Crump


The 454 Approach

The 454 Approach

400,000 reads 250 bp in length

Average ~ 250 bp

454• Avoids laborious/expensive cloning procedures

• Sample diversity deeply and quickly

• Find “rare” or low abundance organisms

• Limited to short reads (<250bp)

• 454 has revolutionized sequencing- many new technologies every day!

What if we wanted to compare the general community composition of

each column rather than getting detailed taxonomic information?

FINGERPRINTING METHODS

Now we know who is there:What next?

• Let’s say we have identified an interesting sequence that we want to know more about in the column and quantify it

–FISH

–Dot Blot

–Q-PCR

Head et al. 1998

Fluorescent In-Situ Hybridization (FISH)



Fluorescent In-Situ Hybridization (FISH)

Schleper et al. 2005

Quantitative (Real Time) PCR

Real time PCR monitors the fluorescence emitted during the reactions as an indicator of

amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

• Detection of “amplification-associated fluorescence” at each cycle during PCR

• No gel-based analysis

• Computer-based analysis

• Compare to internal standards

• Must insure specific binding of probes/dye

Quantitative (Real Time) PCR

Quantitative PCR

Some Problems with PCR

• Inhibitors in template DNA

• Amplification bias

• Gene copy number

• Limited by primer design

• Differential denaturation efficiency

• Chimeric PCR products may form

• Contamination w/ non-target DNA

• Potentially low sensitivity and resolution

Metagenomics a.k.a., Community Genomics, Environmental Genomics

Does not rely on Primers or Probes (apriori knowledge)!

Image courtesy of John Heidelberg

Metagenomics

Access genomes of uncultured microbes:Functional PotentialMetabolic Pathways

Horizontal Gene Transfer…

Metagenomics

From the Most “Simple” Microbial Communities…

•Acid Mine Drainage (pH ~0!)

•Jillian Banfield (UC Berkeley)

•Well-studied, defined environment with ~4 dominant members

•Were able to reconstruct almost entire community “metagenome”

•Tyson et al. 2004

… to the potentially most diverse!

•The Sorcerer II Global Ocean Sampling Expedition

•J. Craig Venter Institute “Sequence now, ask questions later”

•Very few genomes reconstructed

•Sequenced 6.3 billion DNA base pairs (Human genome is ~3.2) from top 5 m of ocean

•Discovered more than 6 million genes… and they are only halfway done!

Venter et al. 2004

Most of these methods are “who is there” not “who is active”

• Use RNA

• Link FISH with activity/uptake

DNA

mRNA

Transcription

Protein

TranslationRibosome

Reverse Transcription PCR (RT-PCR)

• Looks at what genes are being expressed in the environment

• Isolate mRNA

• Reverse transcribe mRNA to produce complementary DNA (cDNA)

• Amplify cDNA by PCR

• Analzye genes from environment

RT-PCR

• RNA + Reverse Transcriptase + dNTPs= cDNA

• cDNA + Primers + Taq + dNTPs = gene of interest

• Who is active? What genes are active?

Metatranscriptomics

Access expressed genes of uncultured microbes

(Some) Problems with Molecular Methods

DNA extraction Incomplete sampling

Resistance to cell lysis

Storage Enzymatic degradation

PCR Inhibitors in template DNA

Amplification bias

Gene copy number

Fidelity of PCR

Differential denaturation efficiency

Chimeric PCR products

Anytime Contamination w/ non-target DNA

The “best approach?”

• A little bit of everything!

And the list goes on…

• Optical tweezers• Single cell genomics• Meta-proteomics• Microarrays• Flow Cytometry• Nano-SIMS FISH• In-situ PCR and FISH• …

molecular methods in microbial ecology

Documents

dna products

dnamix dna

dna stays

dna polymerase

alcoholelute dna

sample dna

dna extraction23

microbial ecologyextract