Microbial ecology: basicconcepts and methods
Dr. András Táncsics
Regional University Center of Excellence
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
Identification of microbes with molecular
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
Identification of microbes with molecular
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
Identification of microbes with molecular
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.
Identification of microbes with molecular
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 the DNA sequence
Analysis of the DNA sequence
• 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
Taxonomic profiling of microbial communities -
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)
Taxonomic profiling of microbial communities -
methods
• Terminal Restriction Fragment Length Polymorphism – T-
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
Taxonomic profiling of microbial communities -
methods
• Terminal Restriction Fragment Length Polymorphism – T-
RFLP
Taxonomic profiling of microbial communities -
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)
Taxonomic profiling of microbial communities -
methods
• Denaturing Gradient Gel-electrophoresis – DGGE
– 16S rDNA is amplificated from the environmental DNA sample
– 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
Taxonomic profiling of microbial communities -
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
Taxonomic profiling of microbial communities -
methods
Process of DGGE
• DGGE in practice
Taxonomic profiling of microbial communities -
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!
Taxonomic profiling of microbial communities -
methods
• Using molecular cloning to separate different 16S amplicons
– 16S rDNA is amplificated from the environmental DNA sample
– Ligation of the 16S rDNA amplicons into a vector – which is a plasmid
in this case
Taxonomic profiling of microbial communities -
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
Taxonomic profiling of microbial communities -
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
Taxonomic profiling of microbial communities -
methods
• The blue-white selection
Taxonomic profiling of microbial communities -
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
Quantitative PCR – qPCR or Real-Time PCR
• 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)
Quantitative PCR – qPCR or Real-Time PCR
• 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
Quantitative PCR – qPCR or Real-Time PCR
• 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
Quantitative PCR – qPCR or Real-Time PCR
• 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
Quantitative PCR – qPCR or Real-Time PCR
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
Next Generation Sequencing
Next Generation Sequencing
• 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
Next Generation Sequencing
• 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!
Next Generation Sequencing
Next Generation Sequencing
• Illumina Genome Analyzer
– Preparation of DNA library, than „bridge” amplification
• Illumina Genome Analyzer
– Generation of fluorescent signal and sequence information
Next Generation Sequencing
Next Generation Sequencing
• 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
Next Generation Sequencing