Microbial Community Analysis
With 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



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

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 T G
1 40
A A G G C C G C C G
1 50
T C T C G
T G C C G
1 60
G
T C G T
T C T G G C G
1 70
G G
T G C C C G
1 80
A C C C G
T G
C G C G T T G
1 90
A T G T A G T C G
2 00
A T G
T C C T C G
2 10
G G G
G
C
What can molecules tell us ?
The Central Dogma of Molecular Biology

DNA





RNA



Who is there ?
Who is not there ?
What functional genes are there ?
BUT can not tell you who is active
Who is active ?
Who is expressing genes?
Protein


Enzyme activity
Rate measurement (e.g. primary
production by 14C carbon fixation)
Traditional vs. culture independent methods
Dunbar et al., AEM No. 4, Vol 65, 1999, pp. 1662-69
known
novel
Microbial Life, BOX 17.5
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)
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 PCR
Conserved
sequence shared
by all species
* * *
*
*
* Ambiguities in the sequence
5’-TWCGTSGARCTGCACGGVACCGGYAC-3’
IUPAC degeneracies: W = A or T
V = C or G or A
S = G or C
Y = C or T
R = A or G
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 ?
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
(Woese, Giovannoni, Ward, Stahl, Pace and
others – late 1980s and early 1990s)
Microbial Life-’02
Perry et al.
How do we estimate bacterial community composition ?
99 Streptomyces nodosus AAK73514 I
99
35
Streptomyces nodosus AAK73514 V
Streptomyces noursei AF263912 IV
99 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
4
Streptomyces sp. FR-008 AY310323 I
6
Streptomyces sp. FR-008 AY310323 VI
32
Streptomyces sp. FR-008 AY310323 V
Streptomyces natalensis AJ278573 II
99 Streptomyces nodosus AAK73514 III
43
66
Streptomyces nodosus AAK73514 VI
92
Streptomyces nodosus AAK73514 II
Streptomyces nodosus AAK73514 IV
99
1
Streptomyces noursei AF263912 III
56
Streptomyces noursei AF263912 II
64
Streptomyces noursei AF263912 V
99
Streptomyces noursei AF263912 VI
99
Streptomyces nanchangensis AF521085 II
54
Streptomyces cinnamonensis AF440781 II
11
Streptomyces natalensis AJ132222 I
Streptomyces natalensis AJ132222 II
25
90
Streptomyces caelestis AF016585 II
99
Streptomyces caelestis AF016585 III
Streptomyces hygroscopicus AAF86396 V
15
Streptomyces hygroscopicus AAF86396 IV
53
Streptomyces hygroscopicus AAF86396 II
87
Streptomyces hygroscopicus AAF86396 III
99
Streptomyces venezuelae T17409 II
20
Streptomyces avermitilis BAB69303 II
Streptomyces sp. HK803 AAQ84157 II
90
Streptomyces sp. HK803 AAQ84157 I
19
Streptomyces sp. FR-008 AY310323 II
87
98
Streptomyces sp. FR-008 AY310323 III
73
Streptomyces sp. FR-008 AY310323 IV
44
Streptomyces natalensis AJ132222 III
Streptomyces natalensis AJ132222 IV
99
Streptomyces avermitilis BAB69303 III
97
Streptomyces halstedii BAD08359 III
57
Streptomyces halstedii BAD08359 II
46
Streptomyces noursei AF263912 VII
85
Streptomyces fradiae AAB66504 II
77
Streptomyces antibioticus AF220951 II
94
Streptomyces antibioticus AF220951 III
99
Streptomyces venezuelae T17409 III
Streptomyces venezuelae T17409 I
Streptomyces coelicolor A3 NP 733695 I
Streptomyces antibioticus AF220951 I
39
99
35
Streptomyces nanchangensis AF521085 I
23
Streptomyces cinnamonensis AF440781 I
35
Streptomyces griseoruber AY196994 I
99 Micromonospora griseorubida AB017641 I
53
Micromonospora griseorubida AB089954 I
16
51
Streptomyces fradiae AAB66504 I
Streptomyces caelestis AF016585 I
Saccharopolyspora erythraea AY330485 I
9
Saccharopolyspora spinosa AY466441 I
7
Streptomyces avermitilis AB070949 I
11
Streptomyces avermitilis AB070949
99 Streptomyces avermitilis BAB69303 I
22
56
99
2
96
C G C C T T G A A G G C C G C C G T C T C G T G C C G G T C G T T C T G G C G G G T G C C C G AC C C G T G C G C G T T G AT G T A G T C G A T G T C C T C G G G G G C
140
150
160
170
180
190
200
210
DNA extraction
Primer design
and PCR
Sequencing
Phylogenetic
analysis
0.05
Library
screening
TA cloning
Comparison to other
samples – hypothesis
testing
Examples of what you can do with 16S PCR technology
DGGE
TRFLP
SIP
FISH
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
20%
80%

You obtain a ‘barcode’ of the
community
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)
cut with 4bp RE
FU
fragment size

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
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


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Reaction kinetics


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Primer binding
Secondary structure of template
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
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
DNA
13C
DNA
centrifugation
CsCl gradient
extract
DNA/RNA
12C
WS01ST2CH16
69
WS01ST3CH19
WS01ST3CH4
WS01ST2CH36
WS01ST5CH4
WS01ST7CH32
WS01ST7CH38
WS01ST6CH15
WS01ST6CH37
52
WS01ST6CH17
62 WS01ST6CH2
WS01ST7CH7
WS01ST6CH19
WS01ST7CH4
WS01ST5CH1
WS01ST6CH6
WS01ST2SY14
WS01ST2SY18
77
WS01ST6CH21
WS01ST5CH11
WS01ST6CH22
Diatoms
Cylindrotheca sp
WS01ST4CH12
WS01ST7CH1
WS01ST5CH14
WS01ST2SY17
Detonula confervacea
Odontella sinensis
WS01ST4CH15
WS01ST2CH2
WS01ST4CH20
95 WS01ST7CH27
WS01ST4CH36
55 WS01ST7CH18
WS01ST6CH25
WS01ST2CH4
WS01ST4CH31
WS01ST7CH19
Skeletonema costatum
WS01ST4CH14
WS01ST6CH1
54
Phaeodactylum tricornutum
50
Bollidomonas pacifica
WS01ST7CH3
Bollidophytes
Bollidomonas mediterranea
WS01ST4CH16
Dictyochophyceae
Pseudopedinella elastica
76
100 WS01ST4CH4
67
WS01ST6CH33
Xanthophyceae
Heterococcus caespitosus
74 WS01ST1CH14
99
WS01ST3CH27
74
WS01ST8CH5
Eustigmatophytes
Nannochloropsis CCMP533
WS01ST1CH4
100 WS01ST1CH9
WS01ST3CH8
WS01ST1CH1
98
WS01ST3CH3
WS01ST1CH8
WS01ST3CH36
WS01ST8CH16
WS01ST1CH33
WS01ST8CH3
97 WS01ST8CH4
P994AH1
WS01ST8CH2
52
97 WS01ST8CH9
P994BH5
WS01ST1CH3
WS01ST6CH16
WS01ST5CH18
WS01ST1CH5
WS01ST2CH10
Prymnesiophytes
Chrysochromulina hirta
WS01ST1CH27
96
WS01ST3CH24
WS01ST4CH34
97 WS01ST7CH21
99 WS01ST8CH14
WS01ST8CH23
WS01ST8CH26
Emiliania huxleyi
Umbilicospaera sibogae
WS01ST5CH10
Chrysochromulina parva
WS01ST3CH23
Calcidiscus leptoporus
Platychrysis sp
Prymnesium parvum
P994AH12
WS01ST5CH2
Unk nown deeply rooted chromophytes
99
WS01ST8CH12
WS01ST8CH15
73
70
SIP (cont.)
96
Who is there ?
0.05
Who is eating
apple pie ?
65
WS01ST3SY29
WS01ST7SY24
A13
WS01ST3SY2
P99SY12
S13
GG3L
P99SY5
B13
N5D
P99SY1
Marine Synechococcus
WS01ST2SY27
WS01ST6SY9
70
J15
WS01ST6SY3
WS01ST8SY9
WS01ST8SY18
WS01ST2SY30
70
WS01ST2SY26
95
WS01ST2SY4
P99SY22
98
Prochlorococcus marinus PAC1
WS01ST2SY19
WS01ST8SY4
WS01ST8SY26
77
Prochlorococcus marinus SB
Prochlorococcus marinus GP2
91
Prochlorococcus
WS01ST1SY15
83
WS01ST3SY1
WS01ST3SY5
WS01ST2SY24
WS01ST2SY33
WS01ST2SY35
Hydrogenovibrio marinus
98 WS01ST4SY12
97
WS01ST8SY15
Trichodesmium
Trichodesmium thiebautii
Prochlorothrix hollandica
66 WS01ST4SY3
78 WS01ST6SY8
81
WS01ST5SY21
81
Pycnococcus provasolii
WS01ST3SY25
WS01ST1SY3
99 WS01ST8SY13
WS01ST8SY25
96 WS01ST8SY3
Spniach
WS01ST1SY10
WS01ST8SY7
70
WS01ST5SY4
WS01ST7SY6
99
Chlamydomonas reinhardtii
74
WS01ST2SY2
WS01ST4SY17
Chlorophytes
97 WS01ST3SY26
87
WS01ST7SY29
78
WS01ST3SY4
WS01ST4SY39
81 WS01ST4SY7
Chlorella
Chlorella ellipsoidea
69
Bathycoccus prasinos
WS01ST1SY35
Platydorina caudata
Yamagishiella unicocca
WS01ST5SY7
WS01ST4SY23
92 WS01ST6SY14
67 WS01ST5SY20
WS01ST6SY26
0.05
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
(bacterial population)

(chromosome mapping)
Disadvantages

Cross-hybridization

Different groups often do not add
up to 100% of the population

Relatively expensive and time
consuming
Microautoradiography of labeled substrate
and fluorescent in situ hybridization
Allows for co-localization of radiolabel
and phylogenetic probe
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