Download Stable-isotope probing

Advanced Environmental
Biotechnology II
Week 09 - Stable-isotope
probing
Based on Chapter 7
Stable-isotope probing
Stefan Radajewski and J.Colin Murrell
in Molecular Microbial Ecology BIOS
Advanced Methods. (2005) Osborn, A.
Mark.; Smith, Cindy J. Eds. Taylor &
Francis Routledge
7.1 Introduction
Finding the metabolic function of groups in
microbial communities is difficult.
One way to do this has involved isolating,
identifying and characterizing
microorganisms which have a particular
function.
A functional group can sometimes be found
by small subunit rRNA gene similarities,
then molecular biological techniques are
used to investigate these closely related
populations in situ.
Another method looks at the similarities of
genes that encode key enzymes in
metabolic pathways, ‘functional genes’.
Many microorganisms will share metabolic
functions, and therefore some new
organisms will be found using these
molecular approaches.
However these methods will not work for all
uncultivated taxa.
A different way to link metabolic function
with taxonomic identity is first to find the
function of uncultivated microbial
populations and then find their identity
using molecular biological techniques.
Several techniques involve the use of
substrates labeled with radioisotopes or
stable-isotopes. These methods can link
identity, activity and function under
conditions similar to in situ.
Microautoradiography, developed
for microscopic observation of
microorganisms involved in
uptake of radiolabeled substrates,
has recently been combined with
molecular identification using 16S
rRNA probes and fluorescent in
situ hybridization.
Figure 1 | Cultivation-independent
identification of microorganisms using
radioisotopes.
a FISH (fluorescence in situ
hybridization)–microautoradiography.
An environmental sample is incubated with
labelled substrates such as 3H-acetate, 14Cpyruvate, 14C-butyrate or 14C-bicarbonate, and
then fixed onto a glass slide.
Fluorescently labelled oligonucleotide probes
specific for various 16S rRNA sequences are
hybridized to the sample.
The slides are treated to show radioactive cells.
FISH plus radioactivity can find microorganisms
that are present and metabolizing the
radiolabelled substrate.
Figure 1 | Cultivation-independent
identification of microorganisms using
radioisotopes.
b Isotope array.
The environmental sample is incubated with a
14C-labelled substrate, then RNA is extracted
from the sample.
RNA is then labelled with a fluorescent dye and
hybridized to an oligonucleotide array
containing DNA probe sequences specific for
the 16S rRNA genes of the bacteria of
interest.
Fluorescence can show which microorganisms
are in the sample, and radioactivity shows
which of the microbes metabolized the
labelled substrate and incorporated the
isotope into their RNA.
Substrates labeled with 14C or 13C have
also been added to environmental
samples and found as labeled lipids that
can be compared with the lipids of
cultivated strains.
The technique of stable-isotope probing (SIP)
used substrates highly enriched with 13C to
recover the DNA of functional groups of
microorganisms, which were identified
using molecular biological techniques.
This lecture will introduce the basis of SIP,
outline technical considerations for the use
of stable-isotopes, and provide examples
of its application.
7.2 Stable-isotope labeling of DNA
There is a low natural abundance of certain
stable-isotopes: the stable carbon isotopes
are 12C (98.9%) and 13C (1.1%), and
stable nitrogen isotopes are 14N (99.63%)
and 15N (0.37%).
Substrates that are highly enriched in the
rare stable-isotopes (e.g. >99%, 13C or 15N)
are added to complex environments, and
the labeled isotopes tracked by finding the
mass increase due to the single additional
neutron.
Idealized structure of the two most common stable isotopes of carbon,
12C (left) and 13C (right).
+ = protons, which are positively charged,
O = neutrons (neutral), and
the path taken by the six negatively charged electrons as they orbit the
nucleus, balancing the charge of the protons.
SIP relies on the fact that DNA
synthesized during microbial
growth on a substrate enriched
with a ‘heavy’ stable-isotope
becomes sufficiently heavy to be
separated from unlabeled DNA by
equilibrium centrifugation in a
CsCl density-gradient.
Escherichia coli grown on
15NH +.
4
The buoyant density of DNA varies with
its guanine-cytosine content, but a high
proportion of a naturally rare stableisotope (2H, 15N or 13C) into DNA
increases the density difference
between labeled and unlabeled DNA
fractions.
Eg. bacterial cultures grown on 13CH3OH
and 13CO2 as a carbon source, and soil
microorganisms that actively used
methanol and methane.
To see if SIP is a suitable
technique we must see whether
each DNA molecule in the target
microorganisms will contain
enough stable-isotope (13C) to
collect a 13C-labeled ‘heavy’ DNA
fraction ([13C]DNA).
There might be:
13C
dilution because of naturally occurring
carbon substrates (i.e. 12C-labeled),
13C turnover due to substrate co-oxidation or
eating of the target microorganisms, or
13C assimilation without DNA replication.
All might change the proportion of a
microbial genome that will become
labeled.
13C-
SIP has been used to target
metabolically restricted groups
[methylotrophs and ammoniaoxidizing bacteria (AOB)] that grow
in the presence of high
concentrations of labeled substrate.
Figure 7.1
CsCl/ethidium bromide density gradients of DNA fractions
extracted from Methylobacterium extorquens grown on
either [12C]- or [13C]methanol as the carbon source.
ultracentrifugation at (A) 265 000 g for 16 h at 20°C and
(B) 140 000 g for 60 h at 20°C. Bar=1 cm.
Figure 2 | DNA-based stable isotope probing
(SIP) and 13C-phospholipid fatty acids (PLFA)
analyses.
a A 13C-labelled substrate is added to an
environmental sample, and incubated so that the
labelled carbon from the substrate can be
incorporated into the biomass of the active
microorganisms in the sample.
b Total DNA that has been purified from the
incubated sample should represent those
microorganisms that grew using the 13Clabelled substrate. This genomic DNA —
enriched with the 13C isotope — can be
separated from the community DNA (12C-DNA)
by CsCl gradient centrifugation.
Figure 2 | DNA-based stable isotope probing (SIP) and
13C-phospholipid fatty acids (PLFA) analyses.
Phylogenetic analyses of sequence data produced by
PCR amplification of the isolated 13C-labelled DNA
using selected primers sets (chosen by the
researcher based on their knowledge of probable
community members) such as 16S rRNA, pmoA
(particulate methane monooxygenase), mmoX
(soluble methane monooxygenase), cmuA
(chloromethane utilization) and mxaF (methanol
dehydrogenase) can help to identify organisms that
are active in the soil sample
Microarrays can also be used to find which of the
amplified genes are the most numerous.
c PLFA can also be purified, and PLFA profiles can
reveal which microorganisms incorporated the 13C
isotope.
7.3 Application of stable-isotope probing
PCR primers that are universal for the smallsubunit rRNA genes of Bacteria, Archaea
and Eukarya are needed to use SIP for
identification of microorganisms involved
in a specific function.
More selective PCR primers, such as those
targeting functional genes, can also be
applied to study populations that are
known to be involved in specific processes.
7.3.1 Methylotroph populations
Methylotrophs are microorganisms that can
use reduced one-carbon compounds as a
sole source of carbon and energy.
Although the known methylotrophs include a
variety of Bacteria, Archaea and Eukarya,
most aerobic strains are Bacteria
belonging to the class Proteobacteria.
A specialized subgroup of methylotrophs is
the methane oxidizing bacteria
(methanotrophs).
7.3.1 Methylotroph populations
Proteobacterial methylotrophs have
common features of their biochemistry
So we can design PCR primers that target
key functional genes of methylotrophs
and methanotrophs; those encoding the
active-site subunits of methanol
dehydrogenase (MDH) and methane
monooxygenase (MMO).
7.3.1.1 Methanol assimilation
Stable-isotope probing shows the active
methanol using microorganisms in an
acidic forest soil.
Soil in a microcosm (small container) was
exposed to 13CH3OH (0.5% v/w) for 44
days.
Then a [13C]DNA fraction was found from
total community DNA using a CsCl
density-gradient.
7.3.1.1 Methanol assimilation
Domain level PCR primers only found
bacterial sequences in the
[13C]DNA fraction.
16S rDNA sequences showed that
three closely related genera within
the α-Proteobacteria had taken up
the [13C] methanol. This was also
seen in genes coding MDH.
Other 16S rDNA sequences from the
[13C]DNA fraction seemed to be from the
Acidobacterium division.
Acidobacterium don’t often grow in culture.
Finding these bacteria take up methanol or
the by-products of methylotrophic carbon
metabolism tells us about the metabolic
function of a diverse, poorly studied and
maybe important group of bacteria.
7.3.1.2 Methane assimilation
The active methanotrophs in a peat soil
with 13CH4 (8% v/v) were studied
using a ‘heavy’ [13C]DNA fraction.
The [13C]DNA fraction was a ‘smear’,
possibly intermediate density, from:
Pieces of DNA from the growth of
methanotrophs and
bacteria using 13C-labeled intermediates
or by-products of methanotroph
metabolism.
7.3.1.2 Methane assimilation
PCR of 16S rRNA genes and functional genes for
MDH and MMO had many methanotroph
sequences, showing the activity of these
bacteria in situ.
The [13C]DNA fraction also had a large proportion
of 16S rDNA sequences of bacteria not known
as methanotrophs or methylotrophs.
Maybe these other groups of bacteria are also
involved in cycling the carbon derived from CH4
(possibly in the form of 13C-labeled metabolites
or biomass).
So SIP can identify the microbial population
involved in the cycling of a specific compound,
even though their function is unclear.
7.3.2 Ammonia-oxidizing populations
Autotrophic ammonia-oxidizing bacteria
(AOB) are slow-growing and difficult to
study in culture, but are important in the
global cycling of nitrogen.
Phylogenetic analysis of rRNA gene
sequences places nearly all AOB in a
group within the β-Proteobacteria.
Now there is wide use of selective 16S
rDNA PCR primers to study their ecology.
7.3.2 Ammonia-oxidizing populations
In enrichment cultures inoculated with a freshwater sediment, SIP identified AOB species
which used 13CO2.
Although several types of AOB were detected in
total DNA extracted from the enrichment cultures,
only some subgroups of AOB were present in
the [13C]DNA fraction.
This suggests that certain subgroups of AOB are
out competed in laboratory culture.
It also shows that ‘heavy’ isotope labeled DNA can
be used to identify which members of a
metabolically defined population are active
under set conditions.
7.4 Future
SIP is able to enrich and isolate the
combined ‘genome’ of a microbial
population that is involved in a
specific function.
So SIP helps us to investigate the
ecology of these microorganisms
in situ using a variety of molecular
biological techniques.
7.4 Future
We could use cloning and
hybridization techniques to find
entire gene clusters from
uncultivated bacteria whose
function has been defined.
Maybe then biases in the use of
selective PCR primers may be
avoided and improved PCR
primers could be designed.
7.4 Future
Also in the future, we could look for
molecules that do not need replication
of the chromosome for 13C-labeling
(e.g. rRNA).
Ribosomes are naturally amplified in
active cells, so this would improve the
sensitivity of SIP.
We could use less label but still be able
to link identity with function.
DNA-SIP has a long incubation time for
DNA replication and incorporation of
the 13C-label into newly synthesized
DNA.
Because there is much more RNA
synthesis than DNA synthesis, it is
possible to get 13C-RNA more quickly
than 13C-DNA.
RNA-SIP has helped to identify bacteria that
degrade phenol in an aerobic industrial
bioreactor.
A pulse of 13C-phenol was added to a
bioreactor sludge sample and RNA was
collected for analysis 8 hours later.
The 13C-labelled RNA was separated from
12C-RNA by density gradient centrifugation.
Reverse transcription (RT-) PCR
amplification of 13C-labelled 16S rRNA
showed that a Thauera species was
important in phenol degradation in this
bioreactor.
13C-labelled
RNA with a specific buoyant
density is found in several fractions in
density gradients.
We must analyse each gradient fraction by
RT-PCR and denaturing gradient gel
electrophoresis (DGGE).
Shifts in band intensities that occurred
during the pulse of 13C-phenol in the
bioreactor showed which bacteria were
being labelled.