Microbial Diversity and Assessment (III) Spring, 2007 Guangyi Wang, Ph.D. POST103B guangyi@hawaii.edu 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 PCR Amplification of a signal from a high background of unrelated markers RFLP Nucleic acid electrophoresis 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).
Principle of T-RFLP U-F1 U-R1 Restriction enzyme The ideal situation is one ban per species as
Principle of T-RFLP (cont.) Terminal restriction fragment (T-RFP) Internal DNA standard Fluorescence Size (bp) Electropherogram
Case Study
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 enzymes http://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 rran gene sequences. 2. More difficult in complex communities when each individual peak from each digest has the potential to represent multiple species.
Summary Advantages of T-RFLP 1. Not limited to makers such as rrna 2. 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-RFLP 1. 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.
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
Principles of fluorescence in situ hybridization (FISH) 16S rrna gene trna 23S rrna gene 5S 1500 bp 3000 bp 120 bp rrna are the main target molecules for FISH for several reasons: found in all living organisms are relatively stable and occur in high copy numbers (usually several thousand per cell) include both variable and highly conserved sequence domain
rrna Secondary Structure & FISH Probe Design Van de Peer et al. 1996, J. Mol. Evol. 42:201-210. Nucleic Acids Res. 24: 3381-3391 Increased variability
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 4. Prehibridization (optional) 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)
Technical Considerations for FISH 1. Bacterial separation and sample treatment Fresh samples??? Cell structural integrity??? Break cells and filtrations??? 2. Treatment of bacterial cells with appropriate chemical fixatives Slide preparations Gelation or poly-lysinge Siliconization of coverslides Fixation of materials on slides Precipitation (e.g. ethanol) Crosslinkage (e.g. formaldehyde)
3. Selecting probe (12-25 bp) and probe labeling Loy A, Horn M, Wagner M. Probe database: an online resource for rrna-targeted oligonucleotide probes. 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 molecule Common fluorophors: fluorescein, tetramethylrhodamine, Taxas red, and carbocyanine dyes (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
General Application and Advantage Application of FISH 1)Marine environments 2)Limnology 3)Wastewater treatment 4)Symbioses 5)Biofilms 6)Soil Bacteria 7)biomedical research, etc. Advantages 1) Overcome bias of PCR-based techniques 2) Can be used to detect uncultured bacteria without DNA extraction 3) Spatial distribution in samples
Detection of Different Microbial Groups in Sponges Appl. Environ. Microbiol. 70 (6) : 3724-3732
Results Bacteria and Poribacteria Poribacteria Poribacteria Poribacteria and Chloroflexi Poribacteria and Planctomycetes Planctomycetes
16S rdna Gene Library 1. General cloning procedures Insert DNA fragment into a carrier DNA molecular, to produce recombinant DNA. Transformation & amplification. Selection & identification of clones. Validation of clones Positive recombinant DNA
16S rdna Gene Library (cont.) 2. 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.
DGGE analysis of microbial communities DGGE Denaturing Gradient Gel Electrophoresis 1. DGGE-PCR 2. DGGE electrophoresis 3. Examples
General PCR Mix 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
General PCR
DGGE-PCR: PCR with GCclamp Supply PCR oligonucleotide primers with a GC-tail: 5 -CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3 3 -NNNNNNNNNGGATGCCCTCCGTCGTCNNNNNN.- 5 elongation GC tail
Denaturing Gradient Gel Electrophoresis (DGGE) Low GC High GC 30% FA 70% FA +
Example 1: The microbial community in a gypsum crust Solar salterns in Eilat, Israel
Example 1: The microbial community in a gypsum crust Several bands of brightly colored phototrophic organisms
Example 1: The microbial community in a gypsum crust Band patterns highly variable: 40% FA Different communities in each depth 70% FA
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
Application of 16S rdna library, DGGE, and FISH in Environmental Microbiology
Advantages and Weakness of Different Methods Technique Strong points Weak Points 16S rdna gene library High resolution RFLP/ARDRA T-RFLP DGGE FISH Straightforward; no expensive equipment High resolution; intra-lane makers; direct quantification of fragments Identification of community members possible Identification of community members possible, quantification, monitoring subgroup Tedious, expensive Number of bands not directly related to number of community members No phylogenetic information obtained; expensive equipment No phylogenetic information obtained; expensive equipment; reproducibility Noise/no specific hybridization (hybridize to unknown microbes, lack of automation
Applicability of various fingerprinting and DNA techniques at different levels of taxonomic resolution Family Genus Species Subspecies strain 16S rdna library sequencing ARDRA RFLP T-RFLP/ARISA DGGE FISH
Summary T-FRLP, FISH, 16S rdna library, and DGGE Principles of each method Strength and weakness General application