Microbial Community Analysis

Boris Wawrik, Ph.D.

Molecular methods
Advantages
Disadvantages
Open questions
Outline

z What can molecules tell us ?

z DNA extraction methods sample preservation and results
z Describing a microbial community with molecular tools
PCR and primer design
TA-cloning and clone libraries
Screening for diversity in clone libraries by ARDRA
z Hypothesis testing in microbial ecology
DGGE
TRFLP
FISH
SIP
Real-time PCR
Remote sensing
Microbial ecology -

Long term goals:

To gain a better understanding of the ecology of important

microorganisms in environmental samples

Questions we ask:

Which bacterial are present in a sample?

How many different types?
Which bacteria are active and growing?
Whats the ecology of microorganisms in the context of their
environment ?
How can we apply this knowledge (e.g. bioremediation,
fermentation)?
Traditional vs. culture independent methods

Dunbar et al., AEM No. 4, Vol 65, 1999, pp. 1662-69

known
novel
Microbial Life, BOX 17.5
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)
Sample preservation: Crucial depending on target
DNA

Preferred by most people, because easiest to work with

DNA is usually stable as long as sample is frozen in TE buffer
Soil can be frozen directly
Water samples are best filtered onto 0.22 or 0.45 m filters and filters stored in TE

RNA

RNA degrades rapidly because RNAse is EVERYWHERE (!!)

DEPC (diethylpyrocarbonate) treat all reagents (inactivates enzymes)
Bake glassware at 450C overnight
Store samples in specialized buffers to prevent RNA degradation

Protein

Assays are usually are preformed immediately with source material and not stored
DNA is the preferred target for most people

Easy to work with

There are many extraction methodologies
DNA is fairly stable can be frozen for years

Extraction methods
Old methods
Organic phase extractions with phenol and chloroform
Precipitation with ethanol or isopropanol

Newer methods
Binding to a column that contains charged silica or activated cellulose
Salt and pH are used to wash away impurities and to elude DNA
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)
 Examples of different extraction methods

1.800 1800.00

1.600 1600.00

1.400 1400.00

1.200 1200.00

ng total recovery
absorbance

1.000 1000.00
Paul et al.
0.800 Kerkhoff 800.00
Qiagen
0.600 600.00
MO BIO
0.400 400.00

0.200 200.00

0.000 0.00
200 250 300 350 Paul et al. Kerkhoff Qiagen MO BIO

lamda M ethod

Humic acid contamination DNA yield

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
(Woese, Giovannoni, Ward, Stahl, Pace and Perry et al.
others late 1980s and early 1990s)
 How do we estimate bacterial community composition ?
99 Streptomyces nodosus AAK73514 I
99 Streptomyces nodosus AAK73514 V
Streptomyces noursei AF263912 IV
35
99 Streptomyces noursei AF263912
Streptomyces natalensis AJ278573 V
22 Streptomyces natalensis AJ278573 III
56
99 Streptomyces natalensis AJ278573 IV
2 Streptomyces natalensis AJ278573 I
96 Streptomyces noursei AF263912 I
Streptomyces natalensis AJ278573 VI
4
6
Streptomyces sp. FR-008 AY310323 I
32 Streptomyces sp. FR-008 AY310323 VI
Streptomyces sp. FR-008 AY310323 V
Streptomyces natalensis AJ278573 II
43 99 Streptomyces nodosus AAK73514 III
66 Streptomyces nodosus AAK73514 VI
92
Streptomyces nodosus AAK73514 II
99 Streptomyces nodosus AAK73514 IV
1
Streptomyces noursei AF263912 III
56 Streptomyces noursei AF263912 II
64
99
Streptomyces noursei AF263912 V
99 Streptomyces noursei AF263912 VI
54 Streptomyces nanchangensis AF521085 II
11 Streptomyces cinnamonensis AF440781 II
Streptomyces natalensis AJ132222 I
25 Streptomyces natalensis AJ132222 II
90 99 Streptomyces caelestis AF016585 II
Streptomyces caelestis AF016585 III
15 Streptomyces hygroscopicus AAF86396 V
Streptomyces hygroscopicus AAF86396 IV
53
Streptomyces hygroscopicus AAF86396 II
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 87
99 Streptomyces hygroscopicus AAF86396 III
20 Streptomyces venezuelae T17409 II
140 150 160 170 180 190 200 210 Streptomyces avermitilis BAB69303 II
90 Streptomyces sp. HK803 AAQ84157 II
Streptomyces sp. HK803 AAQ84157 I
19 87 Streptomyces sp. FR-008 AY310323 II
98 Streptomyces sp. FR-008 AY310323 III
44 73 Streptomyces sp. FR-008 AY310323 IV
Streptomyces natalensis AJ132222 III
99 Streptomyces natalensis AJ132222 IV
97 Streptomyces avermitilis BAB69303 III
57 Streptomyces halstedii BAD08359 III
Streptomyces halstedii BAD08359 II

Phylogenetic
46
85 Streptomyces noursei AF263912 VII
Streptomyces fradiae AAB66504 II
77
94
Streptomyces antibioticus AF220951 II
99 Streptomyces antibioticus AF220951 III
Streptomyces venezuelae T17409 III
Streptomyces venezuelae T17409 I
Streptomyces coelicolor A3 NP 733695 I
39 Streptomyces antibioticus AF220951 I
99 35 Streptomyces nanchangensis AF521085 I
23 Streptomyces cinnamonensis AF440781 I
35
Streptomyces griseoruber AY196994 I

DNA extraction
99 Micromonospora griseorubida AB017641 I

Sequencing analysis
53 Micromonospora griseorubida AB089954 I
16 51
Streptomyces fradiae AAB66504 I
Streptomyces caelestis AF016585 I
9 Saccharopolyspora erythraea AY330485 I
Saccharopolyspora spinosa AY466441 I
7 Streptomyces avermitilis AB070949 I
11 Streptomyces avermitilis AB070949
99 Streptomyces avermitilis BAB69303 I

0.05

Library
Primer design
screening
and PCR

Comparison to other
samples hypothesis
TA cloning testing
 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

 TA cloning

5
PCR products generated with TAQ
3
3 5 polymerase contain an A-overhang at
the 3 end of each strand

Plasmid Plasmid We can generate a

plasmid with a T-overhang
at the 3 ends

Using a specialized topoisomerase we

can ligate the two together
TA-cloning (cont.)
Ligate the mixed PCR
product from a mixed natural
+ population in to a TA-vector

mixed PCR
TA-vector
we get a mixed population of
product
clones (different sequences
in different clones)

Problem :

Sequencing is expensive

We need to prescreen to
select clones for
sequencing
 How to screen clones: ARDRA
A B C A B C
Individual clones are PCR
amplified using the gene
specific primers
4bp RE

The PCR products are then cut

with a 4 bp cutting restriction
endonuclease

The cut products are run on 3%

low melting point agarose gel

Banding patterns are compared

either visually or by means of
special (and very expensive)
software

Unique clones are selected for

sequencing
 99 Streptomyces nodosus AAK73514 I
99 Streptomyces nodosus AAK73514 V
Streptomyces noursei AF263912 IV
35
99 Streptomyces noursei AF263912
Streptomyces natalensis AJ278573 V
22 Streptomyces natalensis AJ278573 III
56
99 Streptomyces natalensis AJ278573 IV
2 Streptomyces natalensis AJ278573 I
96 Streptomyces noursei AF263912 I
Streptomyces natalensis AJ278573 VI
4 Streptomyces sp. FR-008 AY310323 I
6
32 Streptomyces sp. FR-008 AY310323 VI
Streptomyces sp. FR-008 AY310323 V
Streptomyces natalensis AJ278573 II
43 99 Streptomyces nodosus AAK73514 III
66 Streptomyces nodosus AAK73514 VI
92
Streptomyces nodosus AAK73514 II
99 Streptomyces nodosus AAK73514 IV
ACACAGATCTAAGGAAGAGGGAGTCTAGAATGGCTAGCAAAGGAGAAGAACT 1
56
64
Streptomyces noursei AF263912 III
Streptomyces noursei AF263912 II
99 Streptomyces noursei AF263912 V
TTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGC 11
54
99 Streptomyces noursei AF263912 VI
Streptomyces nanchangensis AF521085 II
Streptomyces cinnamonensis AF440781 II

ACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCTACATACGGAAAGCT 90
25
99
Streptomyces natalensis AJ132222 I
Streptomyces natalensis AJ132222 II
Streptomyces caelestis AF016585 II

TACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACAC
TTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATA
15
Streptomyces caelestis AF016585 III
Streptomyces hygroscopicus AAF86396 V
Streptomyces hygroscopicus AAF86396 IV
53
20
87
99
Streptomyces hygroscopicus AAF86396 II
Streptomyces hygroscopicus AAF86396 III
Streptomyces venezuelae T17409 II

TGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGA 90
Streptomyces avermitilis BAB69303 II
Streptomyces sp. HK803 AAQ84157 II
Streptomyces sp. HK803 AAQ84157 I
19
Streptomyces sp. FR-008 AY310323 II
ACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTC 44 73
98
87
Streptomyces sp. FR-008 AY310323 III
Streptomyces sp. FR-008 AY310323 IV
Streptomyces natalensis AJ132222 III
AAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTT 97
99 Streptomyces natalensis AJ132222 IV
Streptomyces avermitilis BAB69303 III
57 Streptomyces halstedii BAD08359 III
AAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACA 46
85
Streptomyces halstedii BAD08359 II
Streptomyces noursei AF263912 VII
Streptomyces fradiae AAB66504 II
CAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCA 77
94
99
Streptomyces antibioticus AF220951 II
Streptomyces antibioticus AF220951 III
Streptomyces venezuelae T17409 III
AAATTCGCCACAACATTGAAGA Streptomyces venezuelae T17409 I
Streptomyces coelicolor A3 NP 733695 I
39 Streptomyces antibioticus AF220951 I
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
9 Saccharopolyspora erythraea AY330485 I
Saccharopolyspora spinosa AY466441 I
7 Streptomyces avermitilis AB070949 I
11 Streptomyces avermitilis AB070949
99 Streptomyces avermitilis BAB69303 I

0.05
Hypothesis testing in microbial ecology

Are my sample and my control different ? How different ?

How does a microbial community respond to a particular treatment ?

How many individuals of each species are there ?

Which are the active species ?

How does it relate to their ecology ?

DGGE (Denaturing Gradient Gel Electrophoresis)
simple complex PCR products of mixed
communities are loaded on a gel
with a gradient of denaturant
20%
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
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 (Transcribed Restriction Fragment Length
Polymorphism)

Mixed population is amplified

using a 16S primer with a
fluorescent tag

cut with 4bp RE

PCR product is cut with a 4bp
cutting restriction endonuclease

Different sequences will give

different length fragments

FU Sample is injected into a

capillary sequencer to sort
fragment size 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

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)?
Quantitative PCR (real-time PCR)

fluorochrome/quencher
labeled probe in addition of
PCR primers

5-3 exonuclease activity of

the polymerase degrades
the probe during
amplifications releasing the
flurochrome

Laser + detector read

fluorescence in reaction
tube, which is proportional to
amplification
 40
A 40 B

35
35

30 30

CT
CT

25 25

20 20

15 15
0.001 0.01 0.1 1 10 100 1000 0.001 0.01 0.1 1 10 100 1000

pg plasmid DNA pg rbcL mRNA

 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
grow on will incorporate it into their DNA.
labeled Dormant cells will not
substrate

DNA extracted and heavy (13C

containing) DNA is separated from
12C DNA light (only 12C containing) DNA by

CsCl gradient
CsCl density gradient centrifugation
extract 13C
DNA/RNA DNA
The heavy band is isolated and the
community analyzed by PCR TA
cloning approach

centrifugation
 69
WS01ST2CH16
WS01ST3CH19
70
WS01ST3CH4
WS01ST2CH36

SIP (cont.)
96
WS01ST5CH4
WS01ST7CH32
WS01ST7CH38
WS01ST6CH15
WS01ST6CH37
52 WS01ST6CH17
62
WS01ST6CH2
WS01ST7CH7
WS01ST6CH19
WS01ST7CH4
WS01ST5CH1
WS01ST6CH6
WS01ST2SY14
77 WS01ST2SY18
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

Who is there ?
WS01ST7CH19
Skeletonema costatum
WS01ST4CH14
54 WS01ST6CH1
50 Phaeodactylum tricornutum
Bollidomonas pacifica
WS01ST7CH3 Bollidophytes
Bollidomonas mediterranea
WS01ST4CH16 Dictyochophyceae
76 Pseudopedinella elastica
100 WS01ST4CH4
67 WS01ST6CH33 Xanthophyceae
Heterococcus caespitosus
74 WS01ST1CH14
99 WS01ST3CH27
74
WS01ST8CH5 Eustigmatophytes
Nannochloropsis CCMP533
WS01ST1CH4
100 WS01ST1CH9
WS01ST3CH8
98
WS01ST1CH1
WS01ST3CH3
WS01ST1CH8
WS01ST3CH36
WS01ST8CH16
WS01ST1CH33
WS01ST8CH3
97 WS01ST8CH4
P994AH1
52 WS01ST8CH2
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
73 WS01ST8CH15

0.05

WS01ST3SY29
WS01ST7SY24
A13
WS01ST3SY2
P99SY12
S13
GG3L
P99SY5
B13
N5D
P99SY1 Marine Synechococcus
WS01ST2SY27
WS01ST6SY9
70 J15
WS01ST6SY3
WS01ST8SY9
WS01ST8SY18
70 WS01ST2SY30
WS01ST2SY26
95
WS01ST2SY4
98 P99SY22
Prochlorococcus marinus PAC1
WS01ST2SY19
WS01ST8SY4
WS01ST8SY26
77

Who is eating
Prochlorococcus marinus SB
91 Prochlorococcus marinus GP2
WS01ST1SY15 Prochlorococcus
83
WS01ST3SY1
WS01ST3SY5
WS01ST2SY24
WS01ST2SY33
WS01ST2SY35

apple pie ? 65
97
98 WS01ST4SY12
WS01ST8SY15
Hydrogenovibrio marinus

Trichodesmium thiebautii
Prochlorothrix hollandica
66 WS01ST4SY3
Trichodesmium

78
81 WS01ST6SY8
81 WS01ST5SY21
Pycnococcus provasolii
WS01ST3SY25
WS01ST1SY3
99 WS01ST8SY13
WS01ST8SY25
96 WS01ST8SY3
Spniach
WS01ST1SY10
WS01ST8SY7
70 WS01ST5SY4
99 WS01ST7SY6
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)
Disadvantages

Cross-hybridization

Different groups often do not add

up to 100% of the population

Relatively expensive and time

(chromosome mapping) consuming
Gulf of Mexico
 60

40

Dissimilarity
20

0
1 3 8 4 5 2 6 7

N on Syn D iatom
plume plume plume

ST6 ST5 ST4 ST3 ST2 ST7 ST8 ST1 6A 5A 4A 3A 2A 7A 8A 1A

2.5e+5 5000
60
Prochlorococcus
% chl a due to diatoms Synechococcus
50 2.0e+5 Picoeukaryotes 4000
Pro & Syn cells ml-1

% chl a due to Syn

-1
40

Picoeuk cells ml
1.5e+5 3000
30
%

20 1.0e+5 2000

10
5.0e+4 1000
0
0.0 0
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

International Cooperative Biodiversity Group

United States Department of Agriculture

National Institute of Health

National Science Foundation

Rutgers University

University of Illinois