Microbial Diversity and Microbial Diversity and Assessment (III)Assessment (III)
Guangyi Wang, Ph.D.POST103B
Spring, 2007
http://www.soest.hawaii.edu/marinefungi/OCN403webpage.htm
Overview of Last Lecture
• Taxonomy (three components)– Classical
• physiological and metabolic characteristics
– Molecular taxonomy• Phylogenetic tree
• Molecular clock and 16S rRNA gene
– Why care about taxonomy
• Major groups of bacteria and archea
• Three domains of life
Terminal Restriction Fragment Length Polymorphism (T-RFLP)
T-RFLP: Measurement of the size polymorphism of terminal restriction fragments from a PCR amplified marker.
T-RFLP
RFLPPCR Nucleic acid electrophoresis
Amplification of a signal from a high background of unrelated markers
Judiciously selected restriction enzymes produce terminal fragments appropriate for sizing on high resolution (sequencing gel)
Automated systems such the DNA sequencer that provides digital output. (each peak is a terminal fragment with a size calculated by the software on the basis of internal size markers).
Fluo
resc
ence
Size (bp)
Electropherogram
Terminal restriction fragment (T-RFP) Internal DNA standard
Principle of T-RFLP (cont.)
Web-based phylogenetic assignment tool for analysis of T-RFLP profiles
Prediction of T-RFs from 16S rRNA gene sequences presently in the database based on the user input of PCR primers and restriction enzymeshttp://rdp.cme.msu.edu (Ribosomal database project)http://mica.ibest.uidaho.edu/ (University of Idaho)http://trflp.limnology.wisc.edu/index.jsp
1. Compare fragments obtained from T-RFLP analysis to the fragment sized predicted from known 16S rRANgene sequences.
2. More difficult in complex communities when each individual peak from each digest has the potential to represent multiple species.
Advantages of T-RFLP1. Not limited to makers such as rRNA2. It can identify 60-80 unique terminal fragments
(ribotype)3. Sensitive and rapid technique for assessing
amplication product diversity within a community as well as comparative distribution across communities.
Disadvantages of T-RFLP1. Difficulty to select restriction enzyme when
sequences are unknown. 2. Many species share the same length of fragment
even when an optimal enzyme is elected.
Summary
Principles of fluorescence in situ hybridization (FISH)
What does FISH do?visualize and map the genetic material in an individual's cells,including specifc genes or portions of genes
prepare short sequences of single-stranded DNA that match a portion of the gene the researcher is looking for (probe).
label these probes by attaching one of a number of colors of fluorescent dye.
single-stranded binds to the complementary strand of DNA
tRNA 23S rRNA gene16S rRNA gene 5S
1500 bp 3000 bp 120 bp
• found in all living organisms
rRNA are the main target molecules for FISH for several reasons:
• are relatively stable and occur in high copy numbers (usually several thousand per cell)• include both variable and highly conserved sequence domain
Principles of fluorescence in situ hybridization (FISH)
rRNA Secondary Structure & FISH Probe Design
Increased variability
Van de Peer et al. 1996, J. Mol. Evol. 42:201-210. Nucleic Acids Res. 24: 3381-3391
Procedures of fluorescence in situ hybridization (FISH)
1. Bacterial separation and sample treatment
2. Treatment of bacterial cells with appropriate chemical fixatives
3. Selecting probe (12-25 bp) and probe labeling
5. Hybridization under stringent conditions on a glass slide or in solution with oligonucleotide proble
6. Detection via epifluorescence microscopy or flow cytometry (confocal laser scanning microscopy (CLSM)
4. Prehibridization (optional)
1. Bacterial separation and sample treatment
Fresh samples???Cell structural integrity???Break cells and filtrations???
2. Treatment of bacterial cells with appropriate chemical fixatives
Fixation of materials on slidesPrecipitation (e.g. ethanol)Crosslinkage (e.g. formaldehyde)
Slide preparationsGelation or poly-lysingeSiliconization of coverslides
Technical Considerations for FISH
3. Selecting probe (12-25 bp) and probe labeling•Loy A, Horn M, Wagner M. Probe database: an online resource for rRNA-targeted oligonucleotideprobes. Nucleic Acids Research 2003, 31:514-516
•http://www.mikro.biologie.tu-muenchen.de (the most comprehensive tool for phylogenetic analysis and probe design).
•Based on signature sequences unique to a chosen group of microbes, probes can be designed for bacteria ranging from whole phyla to individual species.
•Probes can be covalently linked at the 5’-end to a single fluorescent dye moleculeCommon fluorophors: fluorescein, tetramethylrhodamine, Taxas red, and carbocyaninedyes (Cy3 and Cy5) (more sensitive!)
•Nearly full-length 16S and/or 23S rRNA genes can be used to detect low abundant bacteria in natural habitat (AEM, 65:5554-5563)
4. Prehibridization (optional)Just like Southern or Northern blots (hybridization buffer without probe)
5. Hybridization under stringent conditions on a glass slide or in solution with oligonucleotide proble
Denhart’s solution (BSA, Samon sperm DNA, tRNA, etc) SSPE buffer, Formamide
6. Detection via epifluorescence microscopy or flow cytometry (confocal laser scanning microscopy (CLSM)
•one sample can be hybridized with different probes •one sample can be hybridized with one probe labeled with different fluorescent dyes •Sorting different groups of bacteria
1)Marine environments2)Limnology3)Wastewater treatment4)Symbioses5)Biofilms6)Soil Bacteria7)biomedical research, etc.
General Application and Advantage of FISH
1) Overcome bias of PCR-based techniques2) Can be used to detect uncultured bacteria
without DNA extraction3) Spatial distribution in samples
Advantages
Application
Bacteria and Poribacteria Poribacteria
Poribacteria Poribacteria and Chloroflexi
Poribacteria and Planctomycetes Planctomycetes
Results
16S rDNA Gene Library
Insert DNA fragment into a carrier DNA molecular, to produce recombinant DNA.
Transformation & amplification.
Selection & identification of clones.
Validation of clones
Positive recombinant DNA
1. General cloning procedures
1) Genomic DNA extraction from environmental samples.
2) Cloning 16S rDNA genes into plasmid to make the 16S rDNA gene library.
3) Miniprep plasmid DNA for sequencing.
4) Blast and phylogenetic analysis.
2. General cloning procedures
16S rDNA Gene Library (cont.)
DGGE analysis of microbial communities
1. DGGE-PCR
3. Examples
2. DGGE electrophoresis
DGGE – Denaturing Gradient Gel Electrophoresis
General PCRMix DNA sample with primers, polymerase, and dNTP. Transfer to thermycycler
Step 1: Separation of DNA strands at 94˚C
Step 2: Annealing of primers at abt. 55˚C
Step 3: Elongation step at 72˚C
DGGE-PCR: PCR with GC-clamp
Supply PCR oligonucleotide primers with a GC-tail:
5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’3’-NNNNNNNNNGGATGCCCTCCGTCGTCNNNNNN….-5’
GC tail
elongation
Example 1: The microbial community in a gypsum crust
Several bands of brightly colored phototrophic organisms
40% FA
70% FA
Example 1: The microbial community in a gypsum crust
Band patterns highly variable:
Different communities in each depth
Example 2: Surface sediment from 4 stations at Coral Island
Band pattern highly reproducible between samples:
Little difference in community between samples
Information gained from DGGE analysis:
-How diverse is the microbial community in the sample, i.e. how many bands show up
-Detect differences and similarities between microbial communities
-Often used for preliminary screening of samples prior to detailed (expensive) analysis
Technique Strong points Weak Points
16S rDNA gene library High resolution Tedious, expensive
RFLP/ARDRA Straightforward; no expensive equipment
Number of bands not directly related to number of community members
T-RFLP High resolution; intra-lane makers; direct quantification of fragments
No phylogeneticinformation obtained; expensive equipment
DGGE Identification of community members possible
No phylogeneticinformation obtained; expensive equipment; reproducibility
FISH Identification of community members possible, quantification, monitoring subgroup
Noise/no specific hybridization (hybridize to unknown microbes, lack of automation
Advantages and Weakness of Different Methods
Applicability of various fingerprinting and DNA techniques at different levels of taxonomic resolution
Family Genus Species Subspecies strain16S rDNA library
sequencingARDRA
RFLP
T-RFLP/ARISA
DGGE
FISH