Molecular Methods in Microbial Ecology Contact Info: Julie Huber Lillie 305 x7291 jhuber@mbl.edu Schedule: 21 October: Introductory Lecture, DNA extraction 23 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

The Challenge for Microbial Ecology Habitat Culturability (%) Seawater 0.001-0.1 Freshwater 0.25 Sediments Soil 0.3 How do you study something you can’t grow in the lab? From Amann et al. 1995 Microbiological Reviews

The Solution: Molecular Biology DNA Transcription mRNA Translation Ribosome Protein 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

Head et al. 1998 7

DNA extraction from Winogradsky Columns

DNA Extraction Lyse cell membrane Chemically  detergent Physically  bead beating Pellet cell membrane, proteins and other cell parts while DNA stays in solution Remove other inhibitors from DNA Mix DNA with acid and salt  stick to filter Wash filter-bound DNA several times with alcohol 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

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Head et al. 1998 13

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 Protein Transcription Protein Translation Ribosome 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 16S rRNA 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 BACTERIA ARCHAEA ARCHAEA EUKARYA You Are Here EUKARYA EUKARYA Modified from Norman Pace

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: 95ºC 54ºC 72ºC 1 minute DNA denaturization Primer annealing Extension creation 72ºC 10 minutes Final extension created

Slide courtesy of Byron Crump PCR. This graphic is meant to explain how PCR amplifies or makes many copies of a target gene. Here is the target gene on a long strand of genomic DNA. Short sequences of 10 to 30 bases on both ends of the gene, called primer sites, are figured out in advance and are used to direct the reaction to that gene. The reaction begins when the double stranded DNA is denatured into two single strands. Those short pieces of single-stranded DNA called primers match up with the DNA sequences of the primer sites and bind to them. An enzyme called DNA polymerase then takes free nucleotides that are included in the reaction mixture and builds complimentary strands of DNA starting at the primers. Now we have two copies of the original gene. The steps are repeated and we have 4 copies. So after 30 cycles of PCR there is a tremendous number of copies of the target gene. 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

Day 3 Examine gels from PCR Learn about more molecular methods in microbial ecology

Class DNA 10 kb Andrea Paliza Jessica Sarah Rob Amy

Rob Sarah Paliza Jessica Andrea Amy

Rob Sarah Paliza Jessica Andrea Amy

Rob Andrea Paliza Sarah Amy Jessica 1 4 3 2 5 6 1 4 3 2 5 6 1 4 3 2 5 3 kb 1 kb 3 kb 1 kb 1 4 3 2 5 6 1 4 3 2 5 6 Rob Andrea 1 4 3 2 5 6 1 4 3 2 5 6 Paliza Sarah 1 4 3 2 5 6 1 4 3 2 5 6 Amy Jessica

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 Clone Library sequencing: The first method is the method used in some of the original papers on the diversity of natural bacterial communities, and it is called clone library sequencing. DNA is extracted from environmental samples, including DNA from bacteria, flagellates, diatom chains, everything. PCR is used with primers designed to amplify 16S rRNA genes from all the bacteria in the sample. The rest of the method separates these genes so they can be analyzed individually. The 16S rRNA genes are put into circular DNA called plasmids and inserted into E. coli cells such that there is one plasmid per cell. The E. coli cells are then grown up on plates such that each colony contains many copies of one of these 16S rRNA genes. The plasmids are then extracted from these colonies and sequenced. Schematic courtesy of B. Crump

The 454 Approach Schematic courtesy of B. Crump Clone Library sequencing: The first method is the method used in some of the original papers on the diversity of natural bacterial communities, and it is called clone library sequencing. DNA is extracted from environmental samples, including DNA from bacteria, flagellates, diatom chains, everything. PCR is used with primers designed to amplify 16S rRNA genes from all the bacteria in the sample. The rest of the method separates these genes so they can be analyzed individually. The 16S rRNA genes are put into circular DNA called plasmids and inserted into E. coli cells such that there is one plasmid per cell. The E. coli cells are then grown up on plates such that each colony contains many copies of one of these 16S rRNA genes. The plasmids are then extracted from these colonies and sequenced. Schematic courtesy of B. Crump

400,000 reads 250 bp in length Average ~ 250 bp The 454 Approach 400,000 reads 250 bp in length Average ~ 250 bp

454 Sample diversity deeply and quickly 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!

FINGERPRINTING METHODS What if we wanted to compare the general community composition of each column rather than getting detailed taxonomic information? FINGERPRINTING METHODS

Schematic courtesy of B. Crump DGGE: The method I have been working with is called denaturing gradient gel electrophoresis. 16S rRNA genes are amplified using PCR just like the clone libraries. These pieces of DNA have different sequences because they are from different organisms, and the differences in the sequences means that they will denature from double-stranded to single-stranded DNA at different concentrations of chemical denaturant. So the mixture of different genes are run on an acrylamide gel containing a gradient of denaturing chemicals, and the different genes denature and stop moving down the gel when they reach their critical denaturant concentration. The result is that each band on the gel represents a different organism or group of organisms from your original sample. Schematic courtesy of B. Crump

Schematic courtesy of B. Crump

Schematic courtesy of B. Crump TRFLP: The first one is called Terminal Restriction Fragment Length Polymorphism or TRFLP. The first part of the method is the same as Clone Library sequencing, but one of the primers is labeled with a fluorescent molecule or with P-32. The mix of PCR products are then cut up with a restriction enzyme. Restriction enzymes seek out very specific short sequences on a piece of DNA and they cut the DNA only at those sequences. In this example I am using a restriction enzyme that cuts at the sequence “ggcc”. It cuts these pieces of DNA in different places, because there are differences in the sequences. Only the terminal piece of each different gene is labeled, and they are all different lengths. You then separate these pieces of DNA using electrophoresis. This is done in a gel matrix of agarose or acrylamide. An electric field is set up across the gel and DNA migrates towards the positive pole. Smaller pieces of DNA move faster, and so the fragments are separated by length. The banding pattern is used as a community fingerprint, with each band representing some subset of the organisms in the original sample. Schematic courtesy of B. Crump

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

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Fluorescent In-Situ Hybridization (FISH) FISH: Another use of probes is quite different. It is called fluorescent in situ hybridization, which unfortunately abbreviates to FISH. With this method you actually use probes to label whole organisms. In one approach cells are immobilized on a microscope slide. The Cell walls of the organisms are made permeable and probes are introduced. The slides are then looked at under a microscope and the fluorescent cells are counted. Another approach is to count the fluorescently labeled cells with a flow cytometer. Schematic courtesy of B. Crump

Fluorescent In-Situ Hybridization (FISH) FISH: Another use of probes is quite different. It is called fluorescent in situ hybridization, which unfortunately abbreviates to FISH. With this method you actually use probes to label whole organisms. In one approach cells are immobilized on a microscope slide. The Cell walls of the organisms are made permeable and probes are introduced. The slides are then looked at under a microscope and the fluorescent cells are counted. Another approach is to count the fluorescently labeled cells with a flow cytometer. Schleper et al. 2005 Schematic courtesy of B. Crump

Schematic courtesy of B. Crump Dot Blots: One of the most common use of probes is called Dot Blot Hybridization. DNA is extracted from a natural sample and is serially diluted. The DNA is then attached to a membrane in dots. Probes are then introduced to these dilution series dots. The first row of dots was exposed to a general bacteria probe, and the second row of dots was exposed to a probe specific for the proteobacteria. We used factor of ten dilutions so proteobacteria compose approximately 10% of the bacteria in the original sample. Sulfate reducers are approximately 1% of the bacteria in the original sample. Schematic courtesy of B. Crump

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

Quantitative (Real Time) PCR 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 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)! Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS Image courtesy of John Heidelberg

Metagenomics Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS

Metagenomics Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS

Access genomes of uncultured microbes: Metagenomics Access genomes of uncultured microbes: Functional Potential Metabolic Pathways Horizontal Gene Transfer … Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS

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! Venter et al. 2004 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!

Most of these methods are “who is there” not “who is active” Use RNA Link FISH with activity/uptake DNA mRNA Transcription Protein Translation Ribosome

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?

Access expressed genes of uncultured microbes Metatranscriptomics Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS Access expressed genes of uncultured microbes

Schematic courtesy of B. Crump

(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 …