microbial community analysis with thanks to: boris wawrik, ph.d. jerome kukor, ph.d. lee kerkhof,...
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Microbial Community AnalysisWith thanks to:
Boris Wawrik, Ph.D.Jerome Kukor, Ph.D.Lee Kerkhof, Ph.D.
Microbial ecology -
Long term goals:
To gain a better understanding of the ecology of important microorganisms in environmental samples
Questions we ask:
Which bacteria are present in a sample? How many different types? Which bacteria are active and growing? What’s the ecology of microorganisms in the context of their
environment ? How can we apply this knowledge (e.g. bioremediation,
fermentation)?
Traditional Approach
Culture Organisms Isolate Pure Cultures Study Metabolism of Cultures
Direct plating of seawater Electron microscopy
One ml of seawater typically yields 102-103 colonies
This is a low number => bacteria were not
considered important in the marine environment
Electron microscopy suggested much higher bacterial abundance in the marine environment (1920s)
Here are some reasons why
1) Most cells are dead.
2) Many bacteria can not grow on the media.
3) In principle the media components are fine, the concentration is off.
4) The cells grow too slowly for you to assay.
5) Many cells become inactivated by fast growing colonies producing inhibitors.
6) Changes in conditions inhibit growth (e.g. temperature, pressure, placement on agar)
7) Cells clump.
8) Cells stick to pipettes, dilution tubes, sampling gear.
The Revolution : Polymerase Chain Reaction (PCR)
C G C C T T140
G A A G G C C G C C150
G T C T C G T G C C160
G G T C G T T C T G170
G C G G G T G C C C180
G A C C C G T G C G190
C G T T G A T G T A200
G T C G A T G T C C210
T C G G G G G C
What can molecules tell us ?
DNA
Who is there ? Who is not there ? What functional genes are there ? BUT can not tell you who is active
RNA
Who is active ? Who is expressing genes?
Protein
Enzyme activity Rate measurement (e.g. primary
production by 14C carbon fixation)
The Central Dogma of Molecular Biology
Traditional vs. culture independent methods
Dunbar et al., AEM No. 4, Vol 65, 1999, pp. 1662-69 knownnovel
Microbial Life, BOX 17.5
Why use Ribosomal RNA Genes?
(Pauling and Zuckerkandl-1965; Woese, 1987)
1. Everybody’s got ‘em
2. All perform the same function--protein synthesis
3. High homology--good for probing or PCR.
4. Good for telling us big picture lineages.
5. Many new rapid molecular biological methods to detect
Why is the small subunit rRNA gene so useful ?
Conserved in parts – highly variable in other parts. Thus it a very good phylogenetic marker
VERY large database of sequences
Cell have many ribosomes which can be targeted with probes (e.g. FISH, &TRFLP) for community analysis
16S rRNA gene sequencing is now the gold standard for community analysis
Primer design: degenerate PCRConserved
sequence shared by all species
* * * * *
* Ambiguities in the sequence
5’-TWCGTSGARCTGCACGGVACCGGYAC-3’
W = A or T S = G or C R = A or G V = C or G or A Y = C or T
IUPAC degeneracies:
2*2*2*3*2 = 48 different primers sequences
Some caveats:
Not all methods yield the same results
Different samples require different extraction methods
It is best to try several methods and determine effort, yield, and purity
Most people nowadays opt for extractions kits, because they are simple, rapid, reproducible and reasonable cheap
Biggest problems:
PCR inhibitors that co-extract Low DNA yields (e.g. clay)
Who are all these uncultivated bacteria ?
Microbial Life-’02 Perry et al.
(Woese, Giovannoni, Ward, Stahl, Pace and others – late 1980s and early 1990s)
There are regions that are highly similar among all bacteria
These regions can be used to design universal 16S PCR primers
Using these primers we can amplify the 16S sequences from a natural population
This mixture of PCR products can be cloned and the inserts from individual colonies sequenced
How do we estimate bacterial community composition ?
DNA extraction
Primer design and PCR
TA cloning
Library screening
Streptomyces nodosus AAK73514 I Streptomyces nodosus AAK73514 V Streptomyces noursei AF263912 IV Streptomyces noursei AF263912 Streptomyces natalensis AJ278573 V
Streptomyces natalensis AJ278573 III Streptomyces natalensis AJ278573 IV Streptomyces natalensis AJ278573 I
Streptomyces noursei AF263912 I Streptomyces natalensis AJ278573 VI
Streptomyces sp. FR-008 AY310323 I Streptomyces sp. FR-008 AY310323 VI Streptomyces sp. FR-008 AY310323 V Streptomyces natalensis AJ278573 II Streptomyces nodosus AAK73514 III Streptomyces nodosus AAK73514 VI Streptomyces nodosus AAK73514 II
Streptomyces nodosus AAK73514 IV Streptomyces noursei AF263912 III
Streptomyces noursei AF263912 II Streptomyces noursei AF263912 V Streptomyces noursei AF263912 VI Streptomyces nanchangensis AF521085 II
Streptomyces cinnamonensis AF440781 II Streptomyces natalensis AJ132222 I
Streptomyces natalensis AJ132222 II Streptomyces caelestis AF016585 II
Streptomyces caelestis AF016585 III Streptomyces hygroscopicus AAF86396 V
Streptomyces hygroscopicus AAF86396 IV Streptomyces hygroscopicus AAF86396 II
Streptomyces hygroscopicus AAF86396 III Streptomyces venezuelae T17409 II
Streptomyces avermitilis BAB69303 II Streptomyces sp. HK803 AAQ84157 II
Streptomyces sp. HK803 AAQ84157 I Streptomyces sp. FR-008 AY310323 II Streptomyces sp. FR-008 AY310323 III
Streptomyces sp. FR-008 AY310323 IV Streptomyces natalensis AJ132222 III Streptomyces natalensis AJ132222 IV Streptomyces avermitilis BAB69303 III
Streptomyces halstedii BAD08359 III Streptomyces halstedii BAD08359 II
Streptomyces noursei AF263912 VII Streptomyces fradiae AAB66504 II
Streptomyces antibioticus AF220951 II Streptomyces antibioticus AF220951 III
Streptomyces venezuelae T17409 III Streptomyces venezuelae T17409 I
Streptomyces coelicolor A3 NP 733695 I Streptomyces antibioticus AF220951 I
Streptomyces nanchangensis AF521085 I Streptomyces cinnamonensis AF440781 I
Streptomyces griseoruber AY196994 I Micromonospora griseorubida AB017641 I Micromonospora griseorubida AB089954 I
Streptomyces fradiae AAB66504 I Streptomyces caelestis AF016585 I Saccharopolyspora erythraea AY330485 I
Saccharopolyspora spinosa AY466441 I Streptomyces avermitilis AB070949 I Streptomyces avermitilis AB070949 Streptomyces avermitilis BAB69303 I99
9953
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C G C C T T140
G A A G G C C G C C150
G T C T C G T G C C160
G G T C G T T C T G170
G C G G G T G C C C180
G A C C C G T G C G190
C G T T G A T G T A200
G T C G A T G T C C210
T C G G G G G C
SequencingPhylogeneticanalysis
Comparison to other samples – hypothesis testing
Examples of what you can do with 16S PCR technology
TRFLP
FISHSIP
DGGE
DGGE (Denaturing Gradient Gel
Electrophoresis) PCR products of mixed
communities are loaded on a gel with a gradient of denaturant
Typically 20-80% formamide
double stranded DNA will run down the gel until it melts
Melting determined by sequence and GC content
Different sequences migrate different distances
You obtain a ‘barcode’ of the community
simple complex
20%
80%
DGGE (cont.)
Advantages
Can cut individual bands and clone or sequence them
Can detect very small differences in DNA sequences
Disadvantages
High complexity samples give smears
Requires specialized gel rig
Acryl-amide is highly toxic
TRFLP (Terminal Restriction Fragment Length Polymorphism)
Mixed population is amplified using a 16S primer with a fluorescent tag
PCR product is cut with a 4bp cutting restriction endonuclease
Different sequences will give different length fragments
Sample is injected into a capillary sequencer to sort fragments by sizefragment size
FU
cut with 4bp RE
TRFLP (cont.)
Advantages
Very sensitive
Fast, easy and cheap
Disadvantages
Can NOT cut bands to get sequence data
Requires capillary sequencer
Hard to distinguish noise from little peaks sometimes
PCR is inherently NOT quantitative
Amplification of some sequences maybe be sub-optimal
Primer binding Secondary structure of template
Reaction kinetics
Amplification tends to lead to a 1:1 product ratio regardless of the starting DNA ratios
Amplification of low abundance templates in a mixed template experiment will often be suppressed
PCR can produce erroneous sequences
Mis-incorporation of nucleotides by TAQ polymerase Formation of chimeric sequences
LIBRARY CONTENT CANOT BE USED TO CALCULTE DIVERSITY INDICES
Many questions in ecology involve determining the active portion of a community
Many species may be present but only a few might be active
If you are looking for a functional gene, only some of the bacteria that contain this gene may be involved in actual substrate transformation
Among the active ones, who is most dominant/active?
Which bacteria are stimulated by a treatment (treatments may not kill other bacteria and 16S can detect them, although they are no longer active)?
Stable isotope probing
A population is grown on a substrate that contains 13C carbon
Cells that eat the 13C labeled substrate will incorporate it into their DNA. Dormant cells will not
DNA extracted and heavy (13C containing) DNA is separated from light (only 12C containing) DNA by CsCl density gradient centrifugation
The heavy band is isolated and the community analyzed by PCR – TA cloning approach
13C apple pie
+
Bacterial population
12C DNA
13C DNA
grow on labeled substrate
extract DNA/RNA
CsC
l gradient
centrifugation
SIP (cont.)
WS01ST3SY29 WS01ST7SY24
A13 WS01ST3SY2 P99SY12 S13 GG3L P99SY5 B13 N5D P99SY1 WS01ST2SY27 WS01ST6SY9 J15
WS01ST6SY3 WS01ST8SY9
WS01ST8SY18 WS01ST2SY30
WS01ST2SY26 WS01ST2SY4
P99SY22
Marine Synechococcus
Prochlorococcus marinus PAC1 WS01ST2SY19 WS01ST8SY4 WS01ST8SY26 Prochlorococcus marinus SB Prochlorococcus marinus GP2
WS01ST1SY15 WS01ST3SY1
WS01ST3SY5 WS01ST2SY24
WS01ST2SY33 WS01ST2SY35
Prochlorococcus
Hydrogenovibrio marinus WS01ST4SY12 WS01ST8SY15
Trichodesmium thiebautiiTrichodesmium
Prochlorothrix hollandica WS01ST4SY3 WS01ST6SY8 WS01ST5SY21
Pycnococcus provasolii WS01ST3SY25
WS01ST1SY3 WS01ST8SY13
WS01ST8SY25 WS01ST8SY3
Spniach WS01ST1SY10
WS01ST8SY7 WS01ST5SY4
WS01ST7SY6 Chlamydomonas reinhardtii
WS01ST2SY2 WS01ST4SY17 WS01ST3SY26 WS01ST7SY29
WS01ST3SY4 WS01ST4SY39 WS01ST4SY7
Chlorella Chlorella ellipsoidea
Bathycoccus prasinos WS01ST1SY35
Platydorina caudata Yamagishiella unicocca
WS01ST5SY7 WS01ST4SY23 WS01ST6SY14 WS01ST5SY20 WS01ST6SY26
Chlorophytes
70
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77
98
91
9897
65
6678
8181
99
96
99
74
9787
81
78
69
67
92
70
0.05
WS01ST2CH16 WS01ST3CH19 WS01ST3CH4
WS01ST2CH36 WS01ST5CH4
WS01ST7CH32 WS01ST7CH38
WS01ST6CH15 WS01ST6CH37 WS01ST6CH17 WS01ST6CH2
WS01ST7CH7 WS01ST6CH19 WS01ST7CH4 WS01ST5CH1
WS01ST6CH6 WS01ST2SY14
WS01ST2SY18 WS01ST6CH21
WS01ST5CH11 WS01ST6CH22
Cylindrotheca sp WS01ST4CH12
WS01ST7CH1 WS01ST5CH14
WS01ST2SY17 Detonula confervacea
Odontella sinensis WS01ST4CH15
WS01ST2CH2 WS01ST4CH20 WS01ST7CH27 WS01ST4CH36 WS01ST7CH18 WS01ST6CH25
WS01ST2CH4 WS01ST4CH31
WS01ST7CH19 Skeletonema costatum
WS01ST4CH14 WS01ST6CH1
Phaeodactylum tricornutum
Diatoms
Bollidomonas pacifica WS01ST7CH3
Bollidomonas mediterraneaBollidophytes
WS01ST4CH16 Pseudopedinella elastica Dictyochophyceae
WS01ST4CH4 WS01ST6CH33
Heterococcus caespitosusXanthophyceae
WS01ST1CH14 WS01ST3CH27 WS01ST8CH5
Nannochloropsis CCMP533 WS01ST1CH4 WS01ST1CH9
Eustigmatophytes
WS01ST3CH8 WS01ST1CH1 WS01ST3CH3 WS01ST1CH8
WS01ST3CH36 WS01ST8CH16
WS01ST1CH33 WS01ST8CH3 WS01ST8CH4 P994AH1
WS01ST8CH2 WS01ST8CH9
P994BH5 WS01ST1CH3
WS01ST6CH16 WS01ST5CH18
WS01ST1CH5 WS01ST2CH10 Chrysochromulina hirta
WS01ST1CH27 WS01ST3CH24
WS01ST4CH34 WS01ST7CH21 WS01ST8CH14 WS01ST8CH23
WS01ST8CH26 Emiliania huxleyi
Umbilicospaera sibogae WS01ST5CH10
Chrysochromulina parva WS01ST3CH23
Calcidiscus leptoporus Platychrysis sp
Prymnesium parvum
Prymnesiophytes
P994AH12 WS01ST5CH2
WS01ST8CH12 WS01ST8CH15
Unknown deeply rooted chromophytes
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100
98
97
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9799
96
67
73
99
0.05
Who is there ?
Who is eatingapple pie ?
FISH (Fluorescent In Situ Hybridization)
A cell population is fixed with formaldehyde
The cell membranes are permeablized
DNA or RNA probe is hybridized to cells In-Situ i.e. while the cells are still mostly intact
The oligonucleotide contains a fluorescent label, which can be visualized by epifluorescence microscopy
FISH (cont.) Advantages
Allows visualization of a particular population of cells (e.g. a species of interest)
Gives quantitative information about a microbial population
Can probe for DNA, mRNA and ribosomal RNA
Disadvantages
Cross-hybridization
Different groups often do not add up to 100% of the population
Relatively expensive and time consuming
(bacterial population)
(chromosome mapping)
Microautoradiography of labeled substrate and fluorescent in situ hybridization
Allows for co-localization of radiolabel
and phylogenetic probe
F
F
DAPI and Flo-probed cells exposed to 3H amino acids
Cottrell and Kirchman 2000, AEM 66: 1692–1697
Take home messages:
Molecular methods
Most people prefer to work with DNA, because it is easiest and there are now many standard methods, reagents, and kits
PCR based techniques have important limitations/biases DNA based methods can not determine who is active Phylogenetic analysis can not be used to calculate diversity
indices (like the Shannon index) Molecular methods should be put into context of the biology
and ecology of a system
THE BETTER OUR METHODS THE MORE WE LEARN