microbial ecology: basic concepts and methods

Microbial ecology: basicconcepts and methods

Dr. András Táncsics

Regional University Center of Excellence

[email protected]

Part III.

Molecular methods to reveal microbialdiversity

Taxonomic profiling of microbial communities

• basic problem 1.

– Most of the bacterial species cannot be cultivated, or we have

not cultivated them yet

Habitat Reference

Marine water Ferguson et al. 1984

Lake water Staley & Konopka 1985

Estuary Ferguson et al. 1984

Activated sludge Wagner et al. 1993

Sediment Jones 1977

Soil

Cultivation efficiency [%]

0.001 to 0.1

0.1 to 1

0.1 to 3

1 to 15

0.25

0.3 Torsvik et al. 1990

Hugenholtz et al., J. Bac. 1998

Aquificales

Thermodesu

lfobacte

rium

ThermotogalesCoprothermobacter

DictyoglomusOP9

OP5

Green non-sulfur

Actinobacteria

WS1

OP10

Pla

nctomycete

sO

P3

Cla

mydia

Verru

com

icro

bia

OS

-KN

itro

spira

Acid

obacte

rium

Term

ite g

roup I

OP8 S

yner

gist

es

Flex

istip

es

Cya

noba

cter

ia

Low G+C g

ram

posit

ive

Fibrobacter

Marine group A

Green sulfur

Cytophagales

Thermus/DeinococcusSpirochetesTM 6

WS6TM

7

OP11

Fusob

acteria

Pro

teobacte

ria

Archaea

0.10

Yet uncultured

microbial diversity

• Basic problem 2.

– morphology of microbes is limited, conventional microscopic

observations do not allow identification

Electron microscopic images DAPI stained cells

Taxonomic profiling of microbial communities

What is Fluorescence in situ Hybridization (FISH)?

sample

fixed cells,

permeabilized

fixation

hybridization ribosomes

fluorescently labelled

oligonucleotides (probes)

hybridized cells

washing

detection

probe fluorescent dye

target (rRNA)

epifluorescence microscopy

Bloom of Archaea

in the North-Sea

DAPI – general DNA

staining

FISH – eubacterial staining

30% compared to DAPI

Localisation: seawater microbial consortium -

anaerobic oxidation of methane

Methanosarcinales(ANME-2; ~100 cells)

Sulphate-reducing

bacterium

Desulfosarcina/Desulfococcus

(~200 cells/aggregate)

5 µm 7 x 107 aggregate/ml seawater

• The solution – molecular „clock” - nucleotide sequence of the16S ribosomal RNA coding gene (16S rDNA)

• Why the a 16S rDNA?

– Can be found in every bacterium (universal among prokaryotes)

– Its evolution is slow, changes become to be specific for the giventaxon (bacterial species)

– the rRNA genes cannot be transferred by horizontal gene transfer(not like antibiotic resistance genes), because they are essentiallynecessary for cells

– Can be isolated easily from pure cultures, or from anyenvironmental sample, appropriately informative macromolecule(~1500 bp lon gene)

Identification of microbes with molecular

methods

Fingerprint of bacterial species

Structure of the 16S rDNA

• The 16S rDNA is a ~1500 bp long DNA stratch

which can be found in every bacterium, and some

parts are similar in each of them – constant parts –

and there are variable parts as well

• In bacterial genetics the 16S rDNA sequence is

routinely used for identification

Grey rectangulars show the constant parts which are almost the same in every bacterium!!

• Secondary structure

of 16S rRNA

Identification of microbes with molecular methods

Escherichia coli16S rRNA

Primary and Secondary Structure

16S rRNA

• How do we analyse the sequence of the 16S rDNA?

– first step is the isolation of DNA from the strain, or from the

environmental sample

– The second step is the amplification of the 16S rDNA fragments from

the DNA pool by polymerase chain reaction – PCR

• Story of the PCR and its mechanism

– Basic steps of PCR were already known in the 1970’s – DNA

replication, steps of elongation and enzymes catalyzing these steps

– In 1983 Kary Mullis – Cetus Corp. California – one of the first

biotechnology companies – aimed to develop a new method to detect

DNA mutations, but instead he invented the PCR


methods

The DNA isolation

There are two main steps: lysis of the cells,

and pusirifcation of DNA from cell debris

and proteins

Methods:

• DNA Isolation Kits – bead beating –

mechanical lysis

• ultrasonication

• Cell wall lysis with enzymes e.g..:

lysozym, proteinase K,

• detergents e.g.: SDS, Triton X100, which

solve the lipids in membranes

• Mechanism of the PCR


methods

denaturation – at 95 °C for 3-4 min –

the DNA double helix denaturated and

become single stranded

anellation – at ~50 °C for 30-60 sec –

hybridization of primers to the target

sequences

extension – at 72 °C for 30-60 sec ––

copy of the DNA strand

Steps of one cycle

By repeating this cycle for 30-32x the initial DNA

strand is getting amplificated exponentially.

• Basic problem – denaturation of DNA takes place at 95 °C

– Normal proteins denatirate at this temp., in early PCRs the polymerase enzyme

was replenished in every cycle

– 1969 – isolation of Thermus aquaticus termophilic bacterium (can grow at 80

°C) from hot spring of Yellowstone National Park

– 1976 – expression of DNA-polymerase of Thermus aquaticus (Taq polymerase)

its halflife at 98 °C is 9 min, temp. opt. 72 °C

– However, its first application in the PCR took place in the– 1989 – Science –

„Molecule of the year”

– 1993 – Kary Mullis Nobel-prise in chemistry


methods

Thermus aquaticus

Purification of the PCR product and quality check

• To get rid of enzymes and unincorporated nucleotides

• Quality check with agarose gel-electrophoresis

Agarse gel-electrophoresis

Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores through which biomolecules can pass

The gel contains ethyidium-bromide, which binds to the DNA and illuminates under UV-light.

DNA, so the PCR has a negative charge at neutral pH, under directcurrent DNA starts to migrate from the cathode to the anode. Short DNA fragments migrate faster than longer ones.

Agarose gel-electrophoresis

• Sequence analysis of the 16S rDNA

– Sequencing reaction – a modified PCR

– the Sanger dideoxy sequencing method is used generally

– Basic principles of the reaction:

• A part of the nucleotides (A, T, G, C) are present in dideoxy form

• When a dideoxy nucleitode is getting incorporated to the DNA the

synhesis stops (in the lack of free 3’ OH-group) – incorpotation is

random, thus DNA fragments with different length are produced

with a dideoxy-nucleotide at their ends

• dideoxy-nucleotides are fluorescently labeled (with different

color!), the incorporated nucleotide can be detected with a laser

• With capillary gel-electrophoresis the fragments can be precisely

separated.


methods

The sequencing reaction

• process of the capillary gel-electrophoresis

Structure of the „Genetic analyser”

ABI 310

• Result of the capillary gel-electrophoresis – the sequence

electropherogram

Analysis of the DNA sequence

• Last step – identification of the bacterial strain based on its „fingerprint”– GenBank database – exists since 1983 (NCBI – National Center for Biotechnology Information) –

Bethesda, Maryland, USA

– Alignment of the sequence online by using BLAST (Basic Local Alignment Searching Tool)

– anybody can upload sequences – has to be used with due caution!

– A type strain database: EzTaxon server at Ezbiocloud – bacterial strains can be compared to validly described species

– Databases for next generation sequencing: Silva, Greengenes


• Analysis of community 16S rDNA fragments

• But! – new problem – environmental sample– communities

contain thousands of species

Taxonomic profiling of microbial communities -

methods

The PCR product will be mixed – we amplify

the 16S rDNA of all species present in the

sample

A mixed amplicon cannot be uesed directly in a

sequencing reaction, since a mixed

electropherogram would be the result


methods

„clear” sequence, one type of

template DNA sequence in the

template

Mixed sequence, mixed

amplicon template

• How can we analyse a mixed PCR product?

– Molecular ecology helps to do that

• Terminal Restriction Fragmentlength Polymorphism – T-RFLP

• Denaturing Gradient Gel-Electrophoresis - DGGE

• Molecular cloning

• Terminal Restriction Fragment Length Polymorphism – T-

RFLP

– 16S rDNA is amplificated from the environmental DNA sample

– trick – during PCR, the amplicons are getting labeled at their 5’ end

with a fluorescent tag, which makes possible the detection – we use a

labeled forward primer (the solution is usually colorful)


methods


RFLP

– trick II.: enzymatic digestion of the PCR product

– How restriction enzymes work?

• endonucleases, which can be only found in microbes

• Their biological role is the recognition and cleavage of alien DNA

• Recognize DNA motifs precisely and cleave the DNA at these sites

• Different enzymes have different recignition motifs – e.g. AluI – AG CT,

MspI – C CGG

– The PCR product is digested with one of these enzymes – the

16S rDNA of different bacterial species will be differently

cleaved due to the different 16S sequence – this will generate

terminal fragments with different length and labeled at their 5’

end


methods


RFLP


methods

Process of T-RFLP

• T-RFLP in applied science –comparative analysis of groundater microbialcommunities at a BTEX-contaminated site (SKV – pristine groundwater, ST5 – low contamination, ST2 – high contamination)


methods

• Denaturing Gradient Gel-electrophoresis – DGGE


– trick I.: during PCR we put a GC-clamp on either the 5’ or the 3’ end of

the amplicons – guanine and citosine rich

– trick II. gel-electrophoresis on acrylamide gel with a denaturing

gradient – denaturing agent - urea

– Different 16S rDNA amplicons will be denaturated at different

concentrations of urea

– When double stranded DNA is denaturated its migration slows down or

simply getting stopped – „fork” like structure due to the GC-clamp

which stays in double helix form and keeps together the two strands of

DNA


methods

The 16S rDNA of different bacteria will denaturate at different

„places” in the gel, and by theory they can be separated – they will

appear as distinct bands from which DNA can be isolated


methods

Process of DGGE

• DGGE in practice


methods

DGGE gel casting system

Typical DGGE image

• The pros and cons of DGGE

– highly complicated methodology, the akrylamide in monomer form is carcinogenic, excision of bands from the gel requires constant UV-light usage – damage of skin and the eyes!

– Technical drawback: both preapartion of the gel, and formation of the denaturing gradient requires high preciseness; the electrophoresis takes12-15 h – if something went wrong during any step of the process, can be seen after the ethidium-bromide staining

– In practice it can occurr that different 16S rDNA fragments denaturate at the same concentration of urea, and cannot be separated – the back isolated DNA can be still a mixture

– high amount of labwork – high uncertainty of success – not a popular method any more

– Nikolausz et al. (2005) Observation of bias associated with re-amplification of DNA from denaturing gradient gels. FEMS Microbiology Letters 244: 385-390. (ELTE Dept. of Microbiology, Budapest, Hungary) – highly cited paper!


methods

• Using molecular cloning to separate different 16S amplicons


– Ligation of the 16S rDNA amplicons into a vector – which is a plasmid

in this case


methods

The vector contains:

ampicillin resistance gene,

lacZ gene

the PCR product is getting

incorporated into the a lacZ gene,

thus it is not working any more

• Role of lacZ gene

– Encodes the small subunit of β-galactosidase, which takes part in the degradation of lactose, but can also degrade the chromogenicsubstrate X-GAL– the product has a blue color

• Plasmides are transferred into Escherichia coli (E. coli) cells

– one E. coli cell takes one plasmid, with either a 16S fragment or asempty

– the plasmid provides ampicillin resistance to the cells, so only those E. coli cells will grow on the ampicillin agar plates, which took upplasmid

– The plasmid may be empty– not all vector take up 16S fragment –some of them are closed without it, thus the lacZ gene can operate in these plasmids


methods

• The blue-white selection

– E. coli cells are spreaded on ampicillin containing agar plate

– Those which contain plasmid start to grow

– In case of empty plasmid the lacZ gene operates, and degrades the X-

Gal, due to this the colony will have a blue color

– If the plasmi contains insert, the lacZ is not working properly, so the

colony will be white – we need these colonies

– Isolation of plasmids from the white colonies– every colony contains

one type of 16S rDNA insert - with a simple 16S PCR we can

reamplify it – now we have a clone library


methods

• The blue-white selection


methods

• Process of molecular cloning

Taxonomic profiling of microbial

communities - methods

Quantitative PCR – qPCR or Real-Time PCR• Basic problem

– the standard PCR gives only +/- result

– Independently from the initial amount of DNA template after a given number of PCR cycles the amount of amlicons won’t be increased anymore

– reason: the high concentration of DNA (the PCR product) inhibits the reaction, enzyme degradation, lack of nucleotides

– By applying high cycle numbers (32-40) the final amoutn of the PCR product is independent from the initial concentration of the templat –similar band will appear in the gel

Maximum amount of

PCR product

• qPCR

– what was the initial copy number?

– In which case is this important? – quantifying exact amount of

microbes, or quantifying copy number of functional genes in a

microbial community

– Qualitative analysis of gene expression – how actively described

a gene under given conditions – gives information on the amoutn

of mRNA

• The basis of quantification

– Monitoring the formation of amplicons in every PCR cycle

– Two types of chemistry – SYBR Green, TaqMan probe

Quantitative PCR – qPCR or Real-Time PCR

• SYBR Green

– Fluorescent dye – emits light only when biding to DNA

– disadvantage – detects primer dimers and aspecific PCR

products as well


• TaqMan method

– Oligonucleotide probe with a fluorescetn tag

– can hybridize to an intermediate part of the DNA template to be

amplified

– During extension the Taq polymerase cleaves the fluorescent

molecule – moves away from the quencher molecule

specific signal – primer dimers,

aspecific amplicons do not give signal

– more reliable than the SYBR Green

method (and much more expensive)


• Method of quantification

– „treshold cycle” – CT – the first PCR cycle, where the

fluorescent signal appears

– ez alapján határozzuk meg, hogy mennyi volt a kiindulási

kópiaszám


• Way of quantification – absolute vs. relative quantification

– absolute – to tell exactly how many 16S rDNA (or any other

target DNA) parts can be found in a given sample (e.g. soil,

groundwater) – amount of microbes can be estimated

– Requires a calibration curve – (i) ligation of a 16S rDNA

amplicon into a plasmid, (ii) preparation of a serial dilution –

from the DNA concentration the number of plasmids can be

calculated


• Way of quantification – absolute vs. relative quantification

– relative – there is no exact (absolute) quantification,

simplier method from this viewpoint

– Can be only used to compare samples

• example – analysis of the activity of

functional genes

– Expression of key genes of toluene

degradation under different dissolved

oxygen levels

– Oxygen is not just a cosubstrate of

dioxygenase enzymes but also

modulates their expression


What is the function of a given bacterium in the

community?

Stable Isotope Probing (SIP), Radioisotope Probing

(RIP)

• „macro nature” – coral-reef

– The role of the species can

be easily resolved

• „micro nature” – groundwater

microbial community

– Tricky techniques are

needed to determine the role

of species

Stable Isotope Probing (SIP)

• Basics of the method

– question – which microorganisms can use a given organic

compound as sole source of carbon and energy – e.g.

aromatic hydrocarbons (benzene, toluene)

– Substrate containing 13C isotope is added to the

environmental sample, short incubation (few days)

– The heavy isotope is getting incorporated into the DNA of

the microbe which consumed the substrate

– Isolation of environmental DNA, fractionation by isopycnic

centrifugation – CsCl solution (1.72 g/mL) – 72 hours, 180

000 g – ultracentrifuge is required

Radio Isotope Probing (RIP)

• Basics of the method

– Substrate containing 14C isotope is added to the

environmental sample, short incubation (few days)

– The radioactive isotope is getting incorporated into the DNA

of the microbe which consumed the substrate – two

opportunities for further analysis

• Basic requirement – high throughput

• Different companies – different chemistries and sequencing strategies

• Roche 454 pyrosequencing

• Illumina Genome Analyzer

• ABI/Life Technologies – Ion Torrent

Next Generation Sequencing

• Roche 454 pyrosequencing – the first NGS method

– Preparation of DNA library

– emPCR – emulsion PCR



• Roche 454 pyrosequencing – further steps

– Fixation of the beads into the micropockets – 1 pocket – 1

bead – 1 kind of template

• Roche 454 pyrosequencing – further steps

– Detection of the fluorescent signal


• washing unique nucleotides on the chip

• during binding a pyrophosphate is getting

released, followed by ATP formation

•ATP is getting used by a luciferase enzyme

– light emission

• Roche 454 pyrosequencing – pro and contra

– Long sequence reads – ~1000 bases

– 1 million read / reaction

– One run takes 8-10 hours

– But! If one base is repeated more than 6x, the detection can

be ambiguous

– the preparation of the emulsiun PCR requires high

precisity, the weakest point of the method – number of

reads highly depends on the quality of this step

– Highly expensive reagents!




– Preparation of DNA library, than „bridge” amplification


– Generation of fluorescent signal and sequence information



• Illumina Genome Analyzer – pro and contra

– High specificity, repetitive bases do not cause problems

– The most commonly used platform,

– Short sequence reads, 250 – 300 basis max.

– Requires high coverage, but generates high amount of

sequence information

– One reaction takes days!

• IonTorrent - ABI/Life Science Technologies

– incorporation of a nucleotide – this generates a free H+ –

changes the pH


microbial ecology: basic concepts and methods

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