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 natalens is AJ278573 IV Streptomyces natalensis AJ278573 I

Streptomyces noursei AF263912 I Streptomyces natalens is AJ278573 VI

Streptomyces sp. FR-008 AY310323 I Streptomyces sp. FR-008 AY310323 VI Streptomyces sp. FR-008 AY310323 V Streptomyces natalens is 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 natalens is 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 natalens is 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

39

51

35

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99

99

99

99

87

99

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94

99

98

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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

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77

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96

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9787

81

78

69

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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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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