Microbial Diversity and Assessment (III) · • Molecular clock and 16S rRNA gene ......

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Microbial Diversity and Microbial Diversity and Assessment (III) Assessment (III) Guangyi Wang, Ph.D. POST103B [email protected] Spring, 2007 http://www.soest.hawaii.edu/marinefungi/OCN403webpage.htm

Transcript of Microbial Diversity and Assessment (III) · • Molecular clock and 16S rRNA gene ......

Microbial Diversity and Microbial Diversity and Assessment (III)Assessment (III)

Guangyi Wang, Ph.D.POST103B

[email protected]

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).

Principle of T-RFLP

U-F1 U-R1

Restriction enzyme

The ideal situation is one ban per species as

Fluo

resc

ence

Size (bp)

Electropherogram

Terminal restriction fragment (T-RFP) Internal DNA standard

Principle of T-RFLP (cont.)

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 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

Detection of Different Microbial Groups in Sponges

Appl. Environ. Microbiol. 70 (6) : 3724-3732

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

General PCR

DGGE-PCR: PCR with GC-clamp

Supply PCR oligonucleotide primers with a GC-tail:

5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’3’-NNNNNNNNNGGATGCCCTCCGTCGTCNNNNNN….-5’

GC tail

elongation

30% FA

70% FA

Low GC High GC

÷

+

Denaturing Gradient Gel Electrophoresis (DGGE)

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

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

Application of 16S rDNA library, DGGE, and FISH in Environmental Microbiology

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

Summary

• T-FRLP, FISH, 16S rDNA library, and DGGE• Principles of each method• Strength and weakness• General application