1. Microbial Community Analysis
With thanks to:
Boris Wawrik, Ph.D.
Jerome Kukor, Ph.D.
Lee Kerkhof, Ph.D.

2. Microbial ecology - Long term goals: Questions we ask:
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)?

3. Traditional Approach Culture Organisms Isolate Pure Cultures
Study Metabolism of Cultures

4. Direct plating of seawater Electron microscopy
Electron microscopy suggested much higher bacterial abundance in the marine environment (1920s)
One ml of seawater typically yields colonies
This is a low number
=> bacteria were not considered important in the marine environment

5. Here are some reasons why
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. 16

6. The Revolution : Polymerase Chain Reaction (PCR)

7. What can molecules tell us ?
The Central Dogma of Molecular Biology
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)

8. Traditional vs. culture independent methods
known
novel
Microbial Life, BOX 17.5
Dunbar et al., AEM No. 4, Vol 65, 1999, pp

9. Why use Ribosomal RNA Genes?
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
(Pauling and Zuckerkandl-1965; Woese, 1987)

10. 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

11. Primer design: degenerate PCR
Conserved sequence shared by all species * * * * *
* Ambiguities in the sequence
5’-TWCGTSGARCTGCACGGVACCGGYAC-3’
IUPAC degeneracies:
W = A or T S = G or C R = A or G
V = C or G or A Y = C or T
2*2*2*3*2 = 48 different primers sequences

12. 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)

13. Who are all these uncultivated bacteria ?
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
Microbial Life-’02 Perry et al.
(Woese, Giovannoni, Ward, Stahl, Pace and others – late 1980s and early 1990s)

14. How do we estimate bacterial community composition ?
Phylogenetic
analysis
DNA extraction
Sequencing
Library
screening
Primer design
and PCR
Comparison to other samples – hypothesis testing
TA cloning

15. Examples of what you can do with 16S PCR technology
DGGE
TRFLP
FISH
SIP

16. DGGE (Denaturing Gradient Gel Electrophoresis)
simple complex
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
20%
80%

17. DGGE (cont.) Advantages Disadvantages
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

18. 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 size
cut with 4bp RE FU
fragment size

19. TRFLP (cont.) Advantages Disadvantages 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

20. 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

21. 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)?

22. Stable isotope probing
Bacterial population
13C apple pie
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 +
grow on
labeled
substrate
12C DNA
CsCl gradient
extract
DNA/RNA
13C DNA
centrifugation

23. SIP (cont.)
Who is there ?
Who is eating
apple pie ?

24. 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

25. FISH (cont.) Advantages Disadvantages
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)

26. Microautoradiography of labeled substrate
and fluorescent in situ hybridization F F
Allows for co-localization of radiolabel
and phylogenetic probe 16

27. DAPI and Flo-probed cells exposed to 3H amino acids
Cottrell and Kirchman 2000, AEM 66: 1692–1697

28. 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