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Research protocol on stable isotope probing to elucidate the role of soil
microorganisms in nutrient cycling and soil quality
A
A
B
non-labeled
DNA
newly grown
labeled DNA
Figure 4. Isopycnic centrifugation of DNA
extracted from mixed conifer soil incubated with
H218O (tubes A) or H216O (tube B) for 10 days.
Dr. Egbert Schwartz
Northern Arizona University
Requisition 12750
(IAEA staff: Joseph Adu-Gyamfi)
Table of Contents
I.
Introduction to centrifugation and stable isotope probing.
II.
Stable Isotope Probing experimental design
a. Cost of isotopes limits size of experiments
b. Most SIP experiments to date involve a single labeled substrate
c. Type of stable isotope impacts separation of labeled DNA from nonlabeled DNA.
i.
ii.
iii.
13Carbon
15Nitrogen
18Oxygen
d. In SIP experiments a majority of atoms in “labeled” DNA must be the
heavy isotope
e. Adding high concentrations of a labeled substrate may introduce a
fertilizer artifact into the experiment
f. The labeled substrates, once added to soil, will be incorporated into a
variety of compounds, making interpretation of experimental results more
difficult with extended incubations.
III.
Stable Isotope Probing Experimental Protocol
a. Incubation of soil with labeled substrate
b. DNA extraction
c. DNA quantification
d. Centrifugation
2
i. Rotor Choice
1. Vertical Rotor
ii. Fixed Angle Rotor
iii. The compromise between speed and length of centrifuge spin.
iv. Guanine/cytosine content affects density of DNA
v. DNA binding dyes may be used to exaggerate the impact of GC
content
e. Preparing the ultracentrifuge tubes for isopycnic centrifugation
f. Photographing DNA Bands Within the Centrifuge Tubes
g. Fractionation of centrifuge tube contents.
h. Purifying DNA from CsCl Solution
i. The SIP experiment is successful if significantly more heavy DNA is
present in the labeled substrate treatment than the non-labeled substrate
treatment.
j. Analysis of DNA fractions
i. Quantitative PCR
ii. Sequencing of fractions
IV.
Use of SIP in elucidating the role of microorganisms in nutrient
cycling and soil quality
a. Carbon Cycle
b. Nitrogen Cycle
i. Nitrogen Fixation
ii. Ammonia oxidation
c. Food web analysis
3
d. Characterization of microorganisms in the plants rhizosphere
V.
List of Published SIP studies
4
Introduction
Stable isotope probing is a technique that is used to identify the microorganisms
in environmental samples that use a particular growth substrate. The method relies on
the incorporation of a substrate that is highly enriched in a stable isotope, such as
15N
or
18O
13C,
and allows identification of active microorganisms by the selective recovery
and analysis of isotope-enriched cellular components. DNA and rRNA are the most
informative taxonomic biomarkers and 13C-labelled molecules can be purified from nonlabeled nucleic acid by density-gradient centrifugation. The future holds great promise
for SIP, particularly when combined with other emerging technologies such as
metagenomics.
The report is divided into five separate sections: 1. An introduction to
centrifugation and stable isotope probing, 2. Experimental design of stable isotope
probing experiments, 3. A detailed experimental protocol for stable isotope probing
experiments 4. A discussion of possible applications of stable isotope probing to
nutrient cycling and soil quality is given, and 5. A list of published SIP studies is given in
the Reference section. Stable Isotope Probing sometimes refers to the study of
incorporation of stable isotopes into any biomolecule including RNA, DNA,
phospholipid fatty acids, and proteins. This report is limited to the application of stable
isotope probing to the study of DNA and RNA.
5
VI.
Introduction to centrifugation and stable isotope probing.
Centrifugation has played an extremely important role in biological research. It
has been used to separate cell types, organelles within a cell and a variety of biological
macromolecules.
Among the most famous researchers was the Swedish chemist
Theodore Svedberg who received the Nobel Prize in 1926. The Svedberg unit, for
sedimentation rate is named after him, and he was the first to show that pure proteins
could be separated from each other through ultra-centrifugation.
There are several types of centrifugation including differential centrifugation,
rate zonal centrifugation and isopycnic centrifugation. In differential centrifugation
particles of various densities are sedimented at different rates thereby separating them.
In rate zonal centrifugation the sample is not homogenously distributed throughout the
centrifuge tube but rather is layered on top of the density gradient. Differences in
sedimentation rates are still exploited to separate particles from each other. It is less
likely that particles will cross contaminate in Rate zonal centrifugation than in
differential centrifugation. In isopycnic centrifugation the particles do not sediment.
Instead the density gradient is designed in such a way that the particles will migrate
within the density gradient to the location where the particles density is equal to the
density of the gradient. Isopycnic centrifugation is used in SIP so that both labeled and
non-labeled DNA hang in the middle of the tube.
In isopycnic centrifugation a variety of gradient media are used to separate
biomolecules. In SIP cesium chloride (CsCl) is most commonly used to separate labeled
DNA from non-labeled DNA while cesium trifluoroacetate (CsTFA) is used to separate
labeled RNA from non-labeled RNA. However in the 1960’s and 1970’s other media,
such as sodium iodide, were also employed to separate labeled DNA molecules.
6
Representation of isopycnic centrifugation.
At time = 0 (A) all compounds, such as
labeled and non-labeled nucleic acids are
homogenously mixed. After centrifugation
(B) the compounds hang in the density
gradient according to their buoyant density.
Labeled nucleic acids are more dense and
would position lower in the centrifuge tube.
None of the compounds pellet in isopycnic
centrifugation
In SIP a highly labeled substrate, as much as 99 atom% but rarely less than 50
atom%, is added to an environmental sample.
Microorganisms in the sample will
assimilate the substrate thereby incorporating heavy isotopes from the substrate into
their nucleic acids. These nucleic acids will have higher buoyant densities than those of
organisms that did not assimilate the labeled substrate, either because they were not
active or because they assimilated other non-labeled substrates present in the
environmental sample. The difference in buoyant density is subsequently exploited to
separate the labeled nucleic acids from non-labeled nucleic acids along a density
gradient of CsCl or CsTFA, generated in an ultracentrifuge. The labeled nucleic acids
are subsequently recovered from the tube and can be analyzed in a variety of ways
including quantitative PCR or next generation sequencing such as pyrosequencing or
sequencing on the illumina platform.
7
Stable Isotope Probing has it origins in the 1950’s and 60’s when a large number
of researchers focused on how DNA in cells replicated. The classic studies of Messelson
and Stahl (1958) showed a new strand DNA formed by using an old strand as a template
also known as semi-conservative replication. In these experiments DNA was labeled
with 15N, the heavier stable isotope of nitrogen. The researchers were able to analyze the
DNA in their experiments after one generation through isopycnic centrifugation; 2
distinct DNA bands formed in their ultra-centrifuge tubes, one containing
while the other had incorporated
15N
14N
atoms
atoms. When labeled DNA from subsequent
generations where included in the comparisons a third DNA band appeared. The only
reasonable interpretation of these results was that, after one generation, DNA with one
14N
strand and one
appeared
in
15N
strand formed and DNA in which both strands contained
subsequent
generations
thereby
establishing
the
15N
principle
of
semiconservative DNA replication. During this important period of innovation in the
field of centrifugation, and biology in general, studies were limited to pure cultures and
labeled substrates were not administered to environmental samples.
These were
experiments in the fields if cell and molecular biology and not microbial ecology.
The first report of a Stable Isotope Probing study was published in the journal
Nature in 2000 and was authored by Radajewski, Ineson, Parkeh and J.C. Murrell. The
report was entitled “stable-isotope probing as a tool in microbial ecology” and involved
the use of 13C-methane to label DNA of methane oxidizers in environmental samples.
Subsequently numerous SIP studies appeared not only using 13C labeled substrates but
also compounds labeled with 15N or 18O. Theoretically any atom present in nucleic
acids including, Carbon, Nitrogen, Oxygen, Hydrogen and Phosphorus, can be used in
SIP to label nucleic acids. Phosphorus has only radioactive isotopes and to date no SIP
8
Overview of Stable Isotope Probing. (Taken from Marc G. Dumont & J.
Colin Murrell Nature Reviews Microbiology 3, 499-504 (June 2005)
doi:10.1038/nrmicro1162
9
studies have employed this element.
Furthermore the element is relatively heavy
compared to Carbon, Nitrogen and Oxygen and a one or two neutron difference between
the isotopes would only increase buoyant density by a small fraction. Deuterium can be
used in SIP. DNA from E.coli grown in D2O based media can be separated along a CsCl
gradient from DNA extracted from E.coli grown in media with H2O, but to date no
environmental studies employing deuterium have been published. Hydrogen atoms on
molecules may exchange more readily with water molecules, which may complicate
interpretation of SIP experiments that use deuterium. Most of the SIP studies to date
are in the field of bioremediation and focus on the degradation of pollutants in
environmental samples.
II Stable Isotope Probing experimental design
Before designing a SIP experiment, it is important that the researcher is aware of
several limitations imposed on SIP experiments and she must adjust her experimental
design to accommodate these limitations.
Cost of isotopes limits size of experiments
SIP experiments require that nucleic acids are heavily labeled, in excess of 50
atom%, and therefore the substrate added to an environmental sample must also be
heavily labeled. Highly labeled substrates are expensive. 95 atom % H218O for instance
can cost upwards of $200 a mL. Also it is important that the label of the added
substrate is not diluted with non-labeled substrates already present in the soil. If for
instance, the object is to study degradation of labeled litter in soil the carbon already
present in soil may dilute the added labeled litter. A small amount of labeled litter in a
large amount of non-labeled soil will not produce nucleic acids sufficiently labeled for
10
SIP experiments.
Consequently SIP experiments are often conducted on small
quantities of soil (1-5 grams) and often relatively large quantities of substrate are added
to produce enough labeled nucleic acids.
As discussed below these experimental
limitations may lead to fertilizer artifacts.
Most SIP experiments to date involve a single labeled substrate
To perform SIP a highly labeled substrate must be added to an environmental
sample. Usually this substrate is purchased from a chemical company and is delivered
to the environmental sample in pure form. For some substrates, including 13CO2, 13CH4
and H218O, that is representative of environmental conditions. But in other studies,
especially those focusing on pollution degradation or carbon metabolisms in soil, the
addition of a single substrate does not conform to realistic circumstances. For instance,
it is highly unlikely that pure benzene or glucose is ever added to an environmental
sample.
More likely, benzene pollutes soil in combination with other compounds
including toluene, ethylbenzene and xylene. Thus SIP experiments in which multiple
labeled substrates are added to a soil remain rare. Some scientists would argue that
adding multiple substrates at once defeats the main purpose of SIP experiments which
is to identify microbial populations that assimilate specific compounds. However, in
studies of soil quality or nutrient cycling we are especially keen to understand the role of
microorganisms in degrading complex cocktails of substrates such as litter or organic
nitrogen.
Type of stable isotope impacts separation of labeled DNA from non-labeled
DNA
Stable isotopes are more dense because the nuclei of the atom incorporates extra
neutrons. An extra neutron can shift the density of a small element substantially but
11
may have less impact, on a relative basis, on larger elements.
Furthermore the
concentrations of different elements in nucleic acids are not equivalent. Thus while 15N
is on a percentage basis, relative to 14N, only slightly less heavy than 13C is to 12C, there is
substantially less N in nucleic acids than C, so that it is more difficult to label nucleic
acids sufficiently for SIP with 15N than 13C.
13Carbon
There is a long and rich history of the use of carbon isotopes in biology dating
back to the classical experiments on photosynthesis of Kammen, Benson and Calvin
employing
14C.
The first experiments that demonstrated stable isotope probing was
feasible in environmental samples used 13C labeled methane. The methane was the sole
carbon source for methane oxidizers and as the communities were incubated with
13C
methane substantial quantities of 13C (DNA) formed. Methane was a particularly good
choice for the first SIP experiments because it is relatively easy to remove methane from
soil samples so that in an incubation with newly added 13C-methane almost all the added
methane is labeled with
13C.
In studies of litter decomposition this is not feasible
because soil organic matter cannot be removed from soil without severely disturbing the
sample. Therefore the added labeled litter will be diluted by the 12C in the soil organic
matter. Microorganisms in soil are often limited by the availability of labile carbon.
Therefore SIP studies that employ labile carbon are particularly susceptible to
fertilization artifacts. Most SIP experiments to date involved
13C
substrates, partly
because there is great interest in metabolism of organic compounds in our environment,
but also because 13C labeled substrates are readily available. Specifically, most studies to
date have focused on degradation of pollutants in the environment and assimilation of
methane or simple compounds such as acetate.
12
15Nitrogen
As previously mentioned
replication in E.Coli.
15N
15N
was used by Messelson and Stahl to study DNA
has also been used in SIP to study nitrogen fixation and to
identify pollutant degraders in environmental samples. The table below lists the limited
number of studies that have used 15N SIP. It is the most difficult of the elements to use
in SIP because 15N labeled nucleic acids are only slightly more dense than
14N
labeled
nucleic acids. The maximum shift in buoyant density that can be achieved in CsCl
gradients for
15N
labeled nucleic acids is approximately 0.016 g ml-1 DNA, relative to
0.036 g ml- for 13C labeled isotopes. GC content of microbial genomes can result in
DNA samples that vary in buoyant density by as much as 0.05 g ml-1. Some researchers
employ a DNA binding dye, bis-benzimide and further discussed later in the report, to
help separate 15N labeled DNA from non-labeled DNA.
18Oxygen
Paul Boyer and coworkers studied DNA replication in E. coli using H218O to label
DNA showing that branch oxygen atoms of E. coli DNA are almost entirely derived from
water and that
18O
labeling of DNA is not due to formation of hydration shells around
DNA. When analyzing environmental samples of microorganisms by stable isotope
probing (SIP), labeling the DNA with H218O, instead of organic or nitrogenous
compounds, offers important advantages because water cannot be used as an energy,
carbon, or nitrogen source. As a result, addition of the label is unlikely to influence
microbial growth rates in soil directly and microbial communities can be exposed to the
label for long periods of time because they are not exposed to abnormally high substrate
13
concentrations. Because all organisms incorporate water into their DNA, performing
SIP with H218O is a method for identifying microorganisms that have grown during
incubation with H218O, as well as, microorganisms that have not grown (i.e., did not
incorporate the label) but survived the incubation. Though there are fewer oxygen atoms
in DNA than carbon (Radajewski et al., 2003), more neutrons can be added to DNA
through
18O
than any other isotope.
There are, on average, 11 oxygen atoms per
nucleotide unit in DNA whereas on average 3.75 nitrogen and 14.75 carbon atoms are
present per unit DNA. The carbon and nitrogen isotopes
neutron relative
12C
or
14N
while
18O
used in SIP contain one extra
has 2 more neutrons than the more prevalent
oxygen isotope 16O. Thus by labeling DNA with
18O
it is possible to introduce 22 extra
neutrons per unit DNA while only 3.75 or 14.75 extra neutrons can be added with
15N
and 13C respectively.
To test if soil DNA could be labeled sufficiently to use H218O in SIP of soil
microbial communities, 0.2 ml of 95 atom% H218O was added to one gram of Ponderosa
Pine soil, with a moisture content of 8%. DNA was extracted from soil and isopycnic
centrifugation produced 2 or 3 DNA bands after 7 or 21 days of incubation, respectively
(Schwartz, 2007). The third band appears to form after microorganisms feed on
labeled organic matter while in H218O.
18O-
DNA extracted from Ponderosa pine soil
incubated with H216O for 6 or 21 days did not produce multiple bands after isopycnic
centrifugation indicating that changes in % GC of bacterial genomes in soil did not cause
the formation of multiple DNA bands.
14
A
A
B
B
C
C
D
D
E
E
18
Timecourse of O labeling of DNA in Ponderosa Pine soil. A = DNA
18
extracted from soil incubated with H2 O for 0 days, B = DNA
18
extracted from soil incubated with H2 O for 6 days, C= DNA
18
extracted from soil incubated with H2 O for 21 days, D = DNA
16
extracted from soil incubated with H2 O for 6 days, E = DNA
16
extracted from soil incubated with H2 O for 21 days.
There are important advantages in using H218O over other types of labels, such as NH4+,
in order to measure growth in soil. These include:
1. More neutrons can be added to DNA when H218O is used than when 15N is added
as a label in SIP experiments. As a result labeled DNA separates from nonlabeled DNA along a CsCl gradient whereas in experiments with 15N a continuous
smear of DNA forms. Migration of DNA within this smear is controlled by both
DNA label incorporation and GC content of the DNA whereas in 18O-SIP changes
in % GC are not sufficient to cause DNA to migrate to the lower labeled band. In
18O-SIP,
but not
15N-SIP,
only two DNA fractions are retrieved (labeled and
unlabeled DNA) making subsequent analysis much more simple and cost
effective.
15
2. In contrast to an organic or nitrogenous molecule, water by itself does not induce
growth because it is not used as an energy, carbon or nitrogen source. As a result
fertilization artifacts by adding large quantities of labeled substrate to soil are
avoided.
3. Water can easily be distributed homogenously throughout soil because it is a
small molecule that does not interact with the cation exchange capacity of soil. As
a result microorganisms in soil are uniformly exposed to the label. In SIP with
15NH4+,
microorganisms will incorporate unlabeled ammonium, formed through
N mineralization, as well as labeled ammonium into their DNA. Because some
organisms may grow in N-mineralization hot spots not all microorganisms are
exposed to the same amount of 15NH4+.
4. During precipitation events water is normally added to soil while semiarid soils
in northern Arizona never receive pure ammonium solutions.
5. H218O SIP allows study of environmental manipulations that do not involve
substrate assimilation including the impact of temperature, moisture, soil bulk
density and pH on ammonia oxidizing microorganisms. These environmental
parameters could be very important in determining which genotype grows fastest
in soils.
In SIP experiments a majority of atoms in “labeled” DNA must be the
heavy isotope
As previously mentioned the majority of atoms of an element in nucleic acids
must be the heavy isotope in order to have sufficient difference in buoyant density to
separate labeled and non-labeled nucleic acids along a CsCl or CsTFA density gradient.
Consequently SIP experiments should only be designed for labeled substrates that may
16
be obtained with 50 atom% heavy isotope or higher. Furthermore the researcher needs
to consider to what extent the added labeled substrate will be diluted by non-labeled
substrates already present in the environmental sample. It may, for instance be difficult
to do SIP with 13C-methane in an environmental sample that produces large quantities
of 12C-methane. Similarly when using 15N2, it may be prudent to replace the atmosphere
of the sample thereby removing the 14N2 from the incubation.
Adding high concentrations of a labeled substrate may introduce a
fertilizer artifact into the experiment
Because high concentrations of substrate are required in small environmental
samples, SIP experiments are susceptible to fertilizer artifacts. When studying glucose
assimilators in soil, for instance, it is important to recognize that addition of glucose will
alter the active microbial community in soil. Therefore results may not reflect the
community that would assimilate low concentrations of substrate.
In soils
microorganisms are often limited by labile carbon and adding a large amount of labile
carbon to a soil may not reflect average conditions in the environment.
Often in
bioremediation experiments microorganisms are exposed to high concentrations of
carbon substrates but SIP studies of nutrient cycling or soil quality will need to be
designed in such a way that the fertilization artifact is avoided.
The labeled substrates, once added to soil, will be incorporated into a
variety of compounds, making interpretation of experimental results more
difficult with extended incubations.
Once microorganisms assimilate the added labeled substrate and produce new
biomolecules, a wide range of labeled organic compounds are formed in soil including
proteins, lipids, nucleic acids, carbohydrates, cell wall components and smaller
17
metabolites. These compounds also serve as excellent substrates for microbial growth
so that the longer the incubation proceeds the more types of microorganisms will
become labeled. While it is reasonable to presume that the first labeled nucleic acids are
derived from organisms that assimilated the original added labeled substrate, once the
incubation proceeds further it will become increasingly difficult to ascribe labeled
nucleic acids to specific substrate assimilators. By conducting a SIP time series analysis
it is feasible to track an element such as carbon through different microbial populations
as the incubation proceeds. This approach may be highly suitable for studies of nutrient
cycling and soil quality.
III Stable Isotope Probing Experimental Protocol
Incubation of soil with labeled substrate
1. Combine 1 g of soil with 200 μL of H218O (95 atom%) or other labeled substrate and
place in a 15-mL Falcon tube. Stir the soil with a small spatula so that it becomes
homogenously moist. The moisture content of the soil matters because unlabeled H2O
present in the soil, prior to addition of labeled H2O, will dilute the label present during
the incubation. If the soil is too wet, so that addition of 200 μL of H218O results in a soil
completely saturated with H2O, it may be necessary to air dry the soil before adding the
H218O. If 13C or 15N labeled substrates are used it is important to add a sufficient quantity
of substrate to cause growth of the microbial population. It is likely that at least 50µg
substrate/g soil is required. The more substrate is added the more likely there will be a
fertilization artifact.
2. Incubate the soil with the labeled H2O or labeled substrate for ~1 week in a Falcon
tube at room temperature (approximately 20 °C). Keep the tube closed to avoid
18
evaporation of H2O and drying of the soil sample.
A preliminary experiment to
determine the optimum incubation time to allow formation of labeled DNA which can
be detected along the CsCl gradient is highly recommended. More than one week may be
required to produce sufficient labeled DNA. If the sample is incubated too long the label
will turnover and DNA of organisms that did not originally assimilate the substrate will
become labeled. For many 13C labeled substrates incubation times are shorter than one
week; 24 to 72 hours may be sufficient to label a large fraction of the microbial
population.
3. After incubation, the soil may be frozen at -20°C or -80°C until time is available for
DNA extraction. If the soil is frozen at -20°C, DNA should be extracted within a month,
whereas soil may be stored at -80°C for at least a year.
4. A non-labeled substrate control must be included in the experiment to ascertain that
nucleic acids in the labeled treatment did become labeled during the incubation. There
must be significantly greater quantities of heavy nucleic acids (for DNA usually greater
than 1.72 g/mL) in the labeled treatment than in the non-labeled substrate control.
DNA extraction
Extract the DNA from soil using a commercially available soil DNA extraction kit
according to the manufacturer’s instructions. The frozen soil should not be thawed prior
to extraction. Extract only half of the incubated sample at one time, leaving the other
half frozen, in case the first attempt at SIP analysis is unsuccessful.
During the
extraction procedure it is important to maximize yield, not purity, of the DNA because
subsequent centrifugation of the DNA on a CsCl gradient will further purify the DNA.
The yield can be improved by eluting the DNA, in the final step of purification, with
larger amounts of elution buffer, because the DNA does not need to be concentrated for
19
centrifugation. Some researchers include a phenol/chloroform step in their protocol to
improve DNA yield. One microgram of DNA is sufficient to perform SIP.
DNA quantification
Nucleic acids should be quantified before they are loaded onto the cesium
chloride gradient. Quantification can be done via fluorescent methods such as the use of
picogreen a fluorescent DNA binding dye and the QUBIT system. Alternatively a
spectrophotometer can be used to quantify the DNA. If using a spectrophotometer it is
important to measure the absorbance at 230, 260 and 280nm. The absorbance at 260
nm will be used to calculate the concentration of DNA and the ratio of absorbance at
260 nm over 230 nm or 260 nm over 280 nm are indicative of the cleanliness of the
Change in position of DNA bands as the samples are spun in an
ultracentrifuge
DNA. If the A260/A280 or the A260/A230 are not over 1.5 the DNA is not very clean
and the A260 is not a reliable measurement of DNA concentration.
20
Centrifugation
Rotor Choice
Both fixed angle and nearly vertical rotors may be used in SIP experiments. Vertical
rotors appear to set up the density gradient faster and therefore require shorter spin
times. However, it appears that greater separation is feasible with a fixed angle rotor.
Furthermore contaminants in DNA such as humic acids may be pelleted in a fixed angle
rotor.
Vertical Rotor
An example of a vertical rotor used in SIP analysis is the TLN-100 from Beckman. It
positions the tubes at a 9 degree angle, so it is referred to as a nearly vertical rotor.
In a nearly vertical rotor, such as the Beckman TLN-100 the
tubes are nearly upright in the rotor
21
If the samples are spun too fast CsCl will precipitate in the
centrifuge tubes. This graph shows how fast the TLN-100 rotor
can be spun without having CsCl precipitate
22
In isopycnic centrifugation the gradient media needs to be of a density similar to the
molecules the researcher is attempting to hang in the middle of the tube. DNA has a
density of approximately 1.7 g/mL. The number is not exact since it varies due to
differences in GC content. As a result Cesium Chloride is a good salt to use for DNASIP. RNA has a higher buoyant density and will pellet when centrifuged in a cesium
chloride media. For RNA SIP Cesium Tri Fluoro Acetate is routinely used as a media to
set up a density gradient. Cesium chloride will precipitate from the media if the samples
are spun too fast. The graph above shows the relationships, at different temperatures
between the density of the cesium chloride solution and how fast the samples are spun.
Cesium chloride will not precipitate at any point below these curves. Precipitation of
cesium chloride during isopycnic centrifugation should be avoided as it will impact
separation of labeled and non-labeled DNA.
The speed at which a rotor is spun generates varying levels of RCF in different rotor.
The graph shows the relationship between RCF and rpm for the TLN-100 rotor
23
The speed at which samples are spun, in rotations per minute, is not very
informative in SIP studies. The density gradient that forms is dependent on the amount
of g-force, often expressed in rotational centrifugal force (RCF).
While RCF is
dependent on how fast the samples are spun in rpm the relationship between rpm and
rcf varies between different rotors. Therefore, SIP publications should always report
isopycnic procedures in rcf and not rpm.
One of the challenges in SIP is to separate labeled DNA from non-labeled DNA
even though there may be only small differences in buoyant density. This is especially
the case when the nucleic acids are not fully labeled with the isotopes because nonlabeled substrate was present in the incubation. The speed at which the centrifuge is
spun will determine the difference in densities from the top of the centrifuge tube to the
bottom. If the samples are spun very fast, there will be a large difference in densities
along the gradient, resulting in relatively tight nucleic acid bands that are positioned
close together in the tube. If the samples are spun more slowly the bands will be more
diffuse but also positioned further apart. It may require more time to set up a fully
formed gradient when samples are spun more slowly.
24
The faster the samples are spun the steeper the density gradient becomes.
This graph shows the relationship between centrifugation speed and the
steepness of the density gradient for the TLN-100 rotor
25
Fixed Angle Rotor
In the fixed angle rotor the tubes are positioned at more of an angle than in the
near vertical rotor. In the case of the TLA-110 rotor, a rotor commonly used in SIP, the
tubes are placed at a 28 degree angle. This allows contaminating compounds to pellet
more readily from the samples.
Position of tubes in a fixed angle rotor. The Beckman TLA-110 is shown
26
If the samples are spun too fast CsCl will precipitate in the centrifuge tubes.
This graph shows how fast the TLA-100 rotor can be spun without having CsCl
precipitate
27
The faster the samples are spun the steeper the density gradient becomes.
This graph shows the relationship between centrifugation speed and the
steepness of the density gradient for the TLA-110 rotor
28
The compromise between speed and length of centrifuge spin
The faster the samples are spun the greater the difference in buoyant density
between the top and bottom of the centrifuge tubes. As a result compounds that differ
only slightly in buoyant density such as partially labeled nucleic acids and non-labeled
nucleic acids will position very close together along the density gradient if the centrifuge
tube is spun fast. However, if the tubes are spun very slowly it may take a relatively long
time to form the density gradient, so that the researcher must make a compromise
between length of spin and degree of separation between labeled and non-labeled
nucleic acids.
Guanine/cytosine content affects density of DNA
Incorporation of heavy isotopes is not the only determinant of buoyant density of
nucleic acids. Nucleotide composition, specifically the ratio of guanidine cytosine (GC)
over adenosine thymidine (AT) nucleotides, will also affect the buoyant density of
nucleic acids by as much as 0.05 g ml-1. Nucleic acids with high GC content are more
dense that nucleic acids with high AT content. The GC content can vary substantially
between different microbial genomes. Consequently non-labeled DNA extracted from
soil will spread over a range of densities. The impact of GC content on SIP results
requires the inclusion of a non-labeled but identically treated control. The scientist
must ascertain that the high concentrations of heavy DNA formed during a SIP
incubation is due to assimilation of the isotopically labeled substrate and not because a
microbial population grew that contained a genome with a high GC content.
DNA binding dyes may be used to exaggerate the impact of GC content
Several studies have used DNA binding dyes, such as bis-benzimide, to
preferentially bind AT rich regions to manipulate buoyant density of DNA.
29
This
approach can be exploited in SIP where GC rich DNA can contaminate labeled DNA. By
including a second spin in which the DNA binding dye is included GC rich DNA can be
separated from labeled DNA. This experimental approach is especially useful when 15N
is the isotope used in SIP experiments.
Preparing the ultracentrifuge tubes for isopycnic centrifugation
5. Weigh 1 mL of CsCl solution to determine that it has the correct density. A saturated
CsCl solution will have a density of 1.9 g/mL. Be certain that no crystals remain in the
solution. By holding the solution up to the light it is possible to see any remaining
transparent CsCl crystals.
6. Add a saturated solution of CsCl solution to each centrifuge tube placed on a scale to
confirm that each tube receives an identical amount of CsCl. The amount added to the
tube depends on the volume of the tube used. In the case of 4.7 ml TLA-110 rotor tubes
we add 3.6 ml of saturated CsCl solution. The final density of the tube contents, after
the DNA and gradient buffer are added should be approximately 1.72 g/mL.
7. If the researcher wants to visualize the DNA after isopycnic centrifugation, add 0.5 μL
of fresh SYBR Green I DNA stain to the DNA extracted from soil. Older SYBR I stain can
increase the time required to get good DNA separation along the cesium chloride
gradient. The DNA should still be in the microcentrifuge tube used to elute the DNA
from the column in the final step of DNA extraction (Step 4).
Alternatively, the
researcher may elect to not include a DNA binding dye, in which case the DNA cannot
be visualized and no photograph of the tube will be taken after centrifugation.
8. Add the DNA/SYBR-Green mixture to the CsCl solution in the centrifuge tubes. Add
300 μL of gradient buffer (200mM Tris pH 8.0, 200mM KCl, 2mM EDTA) to each tube.
30
Fill each centrifuge tube to the top with H2O and invert the tubes several times to
thoroughly mix the contents.
9. Weigh each tube to ensure that they all weigh within 0.01 g of each other. If
necessary, add sterile H2O to balance the tubes.
10. Load the tubes in the TLA-110 rotor and centrifuge in an ultracentrifuge at
approximately 176,000 X g for approximately 72 h.
11. Decelerate the centrifuge as slowly as possible. When the rotor is removed from the
centrifuge and the tubes are extracted from the rotor, extreme care needs to be taken to
avoid bumps, so that the gradients established in the tubes are not disturbed.
Photographing DNA Bands Within the Centrifuge Tubes
12. Carefully transfer the tubes from the rotor into a rack that has been placed on top of
a UV transilluminator in a dark room. The two bands formed during centrifugation
should be readily visible.
13. Photograph the tubes at this time using an exposure greater than 1 sec. Because of
the long exposure time, immobilize the camera on a test tube rack or camera stand.
Fractionation of centrifuge tube contents
Once the isopycnic centrifugation is complete and labeled DNA has separated
from non-labeled DNA the fractions will still need to be collected. A needle is used to
puncture the bottom of the tube while a second needle is used to pierce the tube on top.
The gradient media will drip from the bottom of the tube and 16 to 50 fractions of 100 to
250 microliter each may be collected. The density of each fraction is measured with a
digital refractometer before the DNA is precipitated from each fraction.
31
Purifying DNA from CsCl
Solution
16. Add ~500 μL of H2O and ten μL
of glycogen solution (10 µg/µL) to
each DNA fraction and shake the
tubes by hand.
17. Add 1 mL of isopropanol to each
tube and vigorously mix the contents.
18.
Centrifuge
the
tubes
in
a
microcentrifuge at full speed for 30
min.
19. Discard the supernatant from the
Harvesting of fractions from an ultracentrifuge
tube
tube, taking care not to dislodge the
pellet.
20. Add 500 μL of 70% ethanol to the tube and shake gently. Centrifuge at full speed for
5 min.
21. Discard the supernatant from the tube into a waste beaker.
22. Remove the last bit of liquid from the bottom of the tube with a pipette. Air dry the
pellet for ~30 min.
23. Dissolve the pellet in sterile H2O or TE buffer. The DNA is now ready for subsequent
analysis including production of clone libraries, generation of T-RFLP patterns, or realtime PCR analysis.
24 Quantify the DNA in each fraction using a QUBIT from Invitrogen inc.
32
The SIP experiment is successful if significantly more heavy DNA is
present in the labeled substrate treatment than the non-labeled substrate
treatment.
Most of the DNA isolated from the centrifuge tubes will have a lower buoyant
density (1.68 to 1.72 g/mL) but in the labeled treatment there should also be DNA with a
higher buoyant density. The fractions with a density greater than 1.72 g/mL and that
contain significantly more DNA in the labeled treatments than in the control nonlabeled treatments contain the genomes of microorganisms that assimilated the added
substrate.
0.3 not labeled DNA
labeled DNA
Fraction of total DNA
0.25
0.2
sample 2
sample 4
sample 6
sample 4W
0.15
0.1
0.05
0
1.6
1.7
1.8
density (g/ml)
1.9
This graph shows the DNA concentrations in different fractions taken from the tube.
In a the heavy isotope treatments (samples 2, 4 and 6, represented by filled symbols)
there is DNA in heavy fractions that is absent from the non-labeled control
treatments (sample 4W represented by open circles)
33
Analysis of DNA fractions
The labeled fractions may be combined into one labeled DNA pool or, alternatively each
fraction can be analyzed separately.
Any type of DNA analysis is feasible but
quantitative PCR and next generation sequencing are the two most common types of
analyses.
Quantitative PCR
In quantitative PCR the fluorescence generated by dyes binding to PCR product is
measured after each cycle. The resulting sigmoidal curve can be related to a set of
standard curves to determine the original abundance of gene copies in a DNA fraction.
This approach quantifies the growth or the extent to which a pre-determined microbial
population has assimilated the added substrate. The benefit from this approach is that
it is highly quantitative.
The downside is that it can only be used for microbial
populations or functional genes for which PCR primers have been developed.
Sequencing of fractions
The DNA in the fractions may also be used in next generation sequencing, including
pyrosequencing and illumina based sequencing.
Usually an amplicon, such as a
fragment of the bacterial 16S rRNA gene, is analyzed. However, increasingly scientist
are shot gun sequencing all the DNA in SIP samples in order to identify booth functional
genes and genes indicative of taxonomic structure. However, the quantity of DNA
obtained through SIP analysis is not sufficient to analyze directly in shotgun sequencing.
Therefore the DNA has to first be used in whole genome amplification. Commonly
multiple displacement amplification, a non-PCR based amplification technique, is used
to amplify small amounts of DNA to obtain sufficient quantities for genomic analyses.
PCR based amplification approaches are likely to introduce biases into the analysis
34
Use of SIP in elucidating the role of microorganisms in nutrient cycling
and soil quality
The majority of SIP studies have focused on degradation of pollutants in
environmental samples or assimilation of simple carbon compounds.
Studies of
nutrient cycling or soil quality, however, require understanding of how complex
structures with multiple compounds, such as plant biomass, are assimilated by
microorganisms in soil.
There are two possible approaches to identifying
microorganisms that assimilate complex organic structures. The first is to label the
plant with 13CO2. The challenge is that for SIP to work the majority of C atoms in the
plant need to be of the
13C
variety, thus the plant should be grown from a seed in an
atmosphere consisting predominately of
13CO2.
This is expensive, though not
impossible. In the second approach newly growing organisms are labeled with
18O
during 18O-water incubations. In this approach the growing microorganisms in soil with
only 18O-water are compared to the growing microorganisms in soil with
18O-water
and
plant litter. This approach is likely cheaper and allows comparison of many different
kinds of naturally grown litter but does not provide a direct connection between litter
and labeled organisms. Rather the organisms that are labeled in the litter treatment but
absent from labeled DNA in the treatment without litter are identified as the litter
assimilators.
35
Assimilation of complex organic structures such as
plant litter can be studies with 18O-water SIP. In
this approach the labeled DNA of an
environmental sample without plant litter is
compared to the labeled DNA of a sample with
plant litter. The microorganisms present in the
labeled DNA from the litter treatment but absent in
the control represent the organisms that grew due
to the presence of plant litter
36
There have been several SIP studies
that relate to soil quality and nutrient
cycling and these are described in the sections below:
Carbon Cycle
The few SIP studies that focused on the carbon cycle have employed either simple
sugars such as glucose or more complex polymers such as cellulose in order to identify
the organisms that assimilate and therefore decompose these carbon compounds in soil.
To date there have been no shotgun sequencing studies of SIP fractions in order to
identify genes involved in carbon catabolism.
Nitrogen Cycle
Nitrogen Fixation
SIP with
15N
is more difficult than SIP with
13C
or
18O
because of the small
changes in buoyant density caused by incorporation of 15N into nucleic acids. The list of
publications that employed
15N
are shown earlier in the report and several of these
studies investigated nitrogen fixers in environmental samples via SIP with
15N.
However, considering the large interest in nitrogen fixation among the soil microbial
ecology community, it is surprising not more reports have followed these initial
publications.
It is likely that nitrogen fixation studies with
15N-SIP
are extremely
challenging.
Ammonia oxidation
Ammonia oxidizers catalyze the first rate limiting step in nitrification, converting
ammonia to nitrite. These organisms may be bacterial or archaeal and are autotrophic
37
so that their nucleic acids can be labeled with 13CO2. Alternatively, it is feasible to study
the impact of environmental parameters such as ammonia availability on ammonia
oxidizers with 18O-water SIP. Here the abundance of ammonia oxidizing genes such as
bacterial amoA or archaeal amoA is compared in the labeled DNA between
environmental treatments of elevated ammonia and ambient ammonia.
Food web analysis
If environmental samples are exposed to labeled substrates for extended periods of time
the isotope will turn over, first being incorporated into biomolecules of the organism
that assimilated the substrate but subsequently becoming part of organisms that feed on
the original assimilating microorganisms. This can be an artifact if the objective is to
identify the organism that assimilated the labeled substrate. But it can also be exploited
in studies of the soil food web. Here samples are taken over time and the isotope is
followed into nucleic acids of different groups of organisms. There are very few SIP
studies of soil food webs but it is likely that SIP will provide new insights into the soil
food web.
Characterization of microorganisms in the plants rhizosphere
Plants interact with a large number of microorganisms in the rhizosphere,
releasing root exudates which may consist of simple sugars and amino acids and
forming symbiotic relationships with mycorrhizal fungi to obtain nutrient and/or water
from soil. Several studies have tried to follow organic carbon from the plant into nucleic
acids of microorganisms in the rhizosphere. Plants are exposed to high concentrations
of 13CO2 before soil attached to roots is used for nucleic acid extraction. One challenge
38
with this experimental approach is that often the plant contains large amounts of
12C-
organic compounds including sugars that will be released as exudate. Consequently it is
difficult to label the nucleic acids of microorganisms in the rhizopshere sufficiently for
SIP. There have been successful RNA-SIP studies of the rhizosphere but DNA-SIP
studies have required extensive periods of exposing the plant to labeled 13CO2, often in
excess of a month.
As a result it is not possible to determine if the organisms
represented in the labeled DNA are rhizosphere organisms or of they feed on
rhizosphere organisms. To date there have been no metagenomic studies of SIP –DNA
obtained from rhizosphere organisms.
39
List of SIP studies that employ 15N labeled molecules
Bell, T. H., Yergeau, E., Martineau, C., Juck, D., Whyte, L. G., & Greer, C. W. (2011).
Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils
by using [15N]DNA-based stable isotope probing and pyrosequencing. Applied and
environmental microbiology, 77(12), 4163–71. doi:10.1128/AEM.00172-11
Buckley, D. H., Huangyutitham, V., Hsu, S.-F., & Nelson, T. A. (2007a). Stable isotope
probing with 15N2 reveals novel noncultivated diazotrophs in soil. Applied and
environmental microbiology, 73(10), 3196–204. doi:10.1128/AEM.02610-06
Buckley, D. H., Huangyutitham, V., Hsu, S.-F., & Nelson, T. A. (2007b). Stable isotope
probing with 15N achieved by disentangling the effects of genome G+C content and
isotope enrichment on DNA density. Applied and environmental microbiology, 73(10),
3189–95. doi:10.1128/AEM.02609-06
Cadisch, G., Espana, M., Causey, R., Richter, M., Shaw, E., Morgan, J. A. W., Rahn, C.,
et al. (2005). Technical considerations for the use of 15N-DNA stable-isotope probing for
functional microbial activity in soils. Rapid communications in mass spectrometry :
RCM, 19(11), 1424–8. doi:10.1002/rcm.1908
Cupples, A. M., Shaffer, E. A., Chee-Sanford, J. C., & Sims, G. K. (2007). DNA buoyant
density shifts during 15N-DNA stable isotope probing. Microbiological research, 162(4),
328–34. doi:10.1016/j.micres.2006.01.016
Gallagher, E. M., Young, L. Y., McGuinness, L. M., & Kerkhof, L. J. (2010). Detection of
2,4,6-trinitrotoluene-utilizing anaerobic bacteria by 15N and 13C incorporation. Applied
and environmental microbiology, 76(5), 1695–8. doi:10.1128/AEM.02274-09
Roh, H., Yu, C.-P., Fuller, M. E., & Chu, K.-H. (2009). Identification of hexahydro-1,3,5trinitro-1,3,5-triazine-degrading microorganisms via 15N-stable isotope probing.
Environmental science & technology, 43(7), 2505–11. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/19452908
A list of SIP studies that employed 18O isotopes
40
Aanderud, Z. T., & Lennon, J. T. (2011). Validation of heavy-water stable isotope
probing for the characterization of rapidly responding soil bacteria. Applied and
environmental microbiology, 77(13), 4589–96. doi:10.1128/AEM.02735-10
Adair, K., & Schwartz, E. (2011). Stable isotope probing with 18O-water to investigate
growth and mortality of ammonia oxidizing bacteria and archaea in soil. Methods in
enzymology, 486, 155–69. doi:10.1016/B978-0-12-381294-0.00007-9
Schwartz, E. (2007). Characterization of growing microorganisms in soil by stable
isotope probing with H218O. Applied and environmental microbiology, 73(8), 2541–6.
doi:10.1128/AEM.02021-06
Schwartz, E. (2009). Analyzing microorganisms in environmental samples using stable
isotope probing with H2(18)O. Cold Spring Harbor protocols, 2009(12), pdb.prot5341.
doi:10.1101/pdb.prot5341
Woods, A., Watwood, M., & Schwartz, E. (2011). Identification of a toluene-degrading
bacterium from a soil sample through H218O DNA stable isotope probing. Applied and
environmental microbiology, 77(17), 5995–9. doi:10.1128/AEM.05689-11
A list of SIP publications related to the soil carbon cycle:
Degelmann, D. M., Kolb, S., Dumont, M., Murrell, J. C., & Drake, H. L. (2009).
Enterobacteriaceae facilitate the anaerobic degradation of glucose by a forest soil. FEMS
microbiology ecology, 68(3), 312–9. doi:10.1111/j.1574-6941.2009.00681.x
Eichorst, S. A., & Kuske, C. R. (2012). Identification of cellulose-responsive bacterial
and fungal communities in geographically and edaphically different soils by using stable
isotope probing. Applied and environmental microbiology, 78(7), 2316–27.
doi:10.1128/AEM.07313-11
Gan, Y., Qiu, Q., Liu, P., Rui, J., & Lu, Y. (2012). Syntrophic oxidation of propionate in
rice field soil at 15 and 30°C under methanogenic conditions. Applied and
environmental microbiology, 78(14), 4923–32. doi:10.1128/AEM.00688-12
41
Haichar, F. E. Z., Achouak, W., Christen, R., Heulin, T., Marol, C., Marais, M.-F.,
Mougel, C., et al. (2007). Identification of cellulolytic bacteria in soil by stable isotope
probing. Environmental microbiology, 9(3), 625–34. doi:10.1111/j.14622920.2006.01182.x
Hori, T., Müller, A., Igarashi, Y., Conrad, R., & Friedrich, M. W. (2010). Identification of
iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing. The
ISME journal, 4(2), 267–78. doi:10.1038/ismej.2009.100
Monard, C., Binet, F., & Vandenkoornhuyse, P. (2008). Short-term response of soil
bacteria to carbon enrichment in different soil microsites. Applied and environmental
microbiology, 74(17), 5589–92. doi:10.1128/AEM.00333-08
Murase, J., & Frenzel, P. (2007). A methane-driven microbial food web in a wetland rice
soil. Environmental microbiology, 9(12), 3025–34. doi:10.1111/j.14622920.2007.01414.x
Murase, J., Shibata, M., Lee, C. G., Watanabe, T., Asakawa, S., & Kimura, M. (2012).
Incorporation of plant residue-derived carbon into the microeukaryotic community in a
rice field soil revealed by DNA stable-isotope probing. FEMS microbiology ecology,
79(2), 371–9. doi:10.1111/j.1574-6941.2011.01224.x
Schellenberger, S., Kolb, S., & Drake, H. L. (2010). Metabolic responses of novel
cellulolytic and saccharolytic agricultural soil Bacteria to oxygen. Environmental
microbiology, 12(4), 845–61. doi:10.1111/j.1462-2920.2009.02128.x
A list of SIP studies that investigate ammonia oxidizers in environmental
samples:
Adair, K., & Schwartz, E. (2011). Stable isotope probing with 18O-water to investigate
growth and mortality of ammonia oxidizing bacteria and archaea in soil. Methods in
enzymology, 486, 155–69. doi:10.1016/B978-0-12-381294-0.00007-9
42
Avrahami, S., Jia, Z., Neufeld, J. D., Murrell, J. C., Conrad, R., & Küsel, K. (2011). Active
autotrophic ammonia-oxidizing bacteria in biofilm enrichments from simulated creek
ecosystems at two ammonium concentrations respond to temperature manipulation.
Applied and environmental microbiology, 77(20), 7329–38. doi:10.1128/AEM.05864-11
Pratscher, J., Dumont, M. G., & Conrad, R. (2011). Ammonia oxidation coupled to CO2
fixation by archaea and bacteria in an agricultural soil. Proceedings of the National
Academy of Sciences of the United States of America, 108(10), 4170–5.
doi:10.1073/pnas.1010981108
Xia, W., Zhang, C., Zeng, X., Feng, Y., Weng, J., Lin, X., Zhu, J., et al. (2011).
Autotrophic growth of nitrifying community in an agricultural soil. The ISME journal,
5(7), 1226–36. doi:10.1038/ismej.2011.5
Zhang, L.-M., Hu, H.-W., Shen, J.-P., & He, J.-Z. (2012). Ammonia-oxidizing archaea
have more important role than ammonia-oxidizing bacteria in ammonia oxidation of
strongly acidic soils. The ISME journal, 6(5), 1032–45. doi:10.1038/ismej.2011.168
A list of SIP studies related to food webs:
Bernard, L., Chapuis-Lardy, L., Razafimbelo, T., Razafindrakoto, M., Pablo, A.-L.,
Legname, E., Poulain, J., et al. (2012). Endogeic earthworms shape bacterial functional
communities and affect organic matter mineralization in a tropical soil. The ISME
journal, 6(1), 213–22. doi:10.1038/ismej.2011.87
Wüst, P. K., Horn, M. A., & Drake, H. L. (2011). Clostridiaceae and Enterobacteriaceae
as active fermenters in earthworm gut content. The ISME journal, 5(1), 92–106.
doi:10.1038/ismej.2010.99
A list of SIP studies focused on the rhizosphere
43
Bressan, M., Roncato, M.-A., Bellvert, F., Comte, G., Haichar, F. Z., Achouak, W., &
Berge, O. (2009). Exogenous glucosinolate produced by Arabidopsis thaliana has an
impact on microbes in the rhizosphere and plant roots. The ISME journal, 3(11), 1243–
57. doi:10.1038/ismej.2009.68
Drigo, B., Pijl, A. S., Duyts, H., Kielak, A. M., Gamper, H. A., Houtekamer, M. J.,
Boschker, H. T. S., et al. (2010). Shifting carbon flow from roots into associated
microbial communities in response to elevated atmospheric CO2. Proceedings of the
National Academy of Sciences of the United States of America, 107(24), 10938–42.
doi:10.1073/pnas.0912421107
Haichar, F. el Z., Marol, C., Berge, O., Rangel-Castro, J. I., Prosser, J. I., Balesdent, J.,
Heulin, T., et al. (2008). Plant host habitat and root exudates shape soil bacterial
community structure. The ISME journal, 2(12), 1221–30. doi:10.1038/ismej.2008.80
Haichar, F. el Z., Roncato, M.-A., & Achouak, W. (2012). Stable isotope probing of
bacterial community structure and gene expression in the rhizosphere of Arabidopsis
thaliana. FEMS microbiology ecology, 81(2), 291–302. doi:10.1111/j.15746941.2012.01345.x
Lu, Y., & Conrad, R. (2005). In situ stable isotope probing of methanogenic archaea in
the rice rhizosphere. Science (New York, N.Y.), 309(5737), 1088–90.
doi:10.1126/science.1113435
Lu, Y., Rosencrantz, D., Liesack, W., & Conrad, R. (2006). Structure and activity of
bacterial community inhabiting rice roots and the rhizosphere. Environmental
microbiology, 8(8), 1351–60. doi:10.1111/j.1462-2920.2006.01028.x
Prosser, J. I., Rangel-Castro, J. I., & Killham, K. (2006). Studying plant-microbe
interactions using stable isotope technologies. Current opinion in biotechnology, 17(1),
98–102. doi:10.1016/j.copbio.2006.01.001
Rangel-Castro, J. I., Killham, K., Ostle, N., Nicol, G. W., Anderson, I. C., Scrimgeour, C.
M., Ineson, P., et al. (2005). Stable isotope probing analysis of the influence of liming on
root exudate utilization by soil microorganisms. Environmental microbiology, 7(6),
828–38. doi:10.1111/j.1462-2920.2005.00756.x
44
Alphabetical List of Published SIP Studies
Aanderud, Z. T., & Lennon, J. T. (2011). Validation of heavy-water stable isotope
probing for the characterization of rapidly responding soil bacteria. Applied and
environmental microbiology, 77(13), 4589–96. doi:10.1128/AEM.02735-10
Adair, K., & Schwartz, E. (2011). Stable isotope probing with 18O-water to investigate
growth and mortality of ammonia oxidizing bacteria and archaea in soil. Methods in
enzymology, 486, 155–69. doi:10.1016/B978-0-12-381294-0.00007-9
Addison, S. L., McDonald, I. R., & Lloyd-Jones, G. (2010). Stable isotope probing:
technical considerations when resolving (15)N-labeled RNA in gradients. Journal of
microbiological methods, 80(1), 70–5. doi:10.1016/j.mimet.2009.11.002
Akob, D. M., Kerkhof, L., Küsel, K., Watson, D. B., Palumbo, A. V, & Kostka, J. E. (2011).
Linking specific heterotrophic bacterial populations to bioreduction of uranium and
nitrate in contaminated subsurface sediments by using stable isotope probing.
Applied and environmental microbiology, 77(22), 8197–200.
doi:10.1128/AEM.05247-11
Andeer, P., Strand, S. E., & Stahl, D. A. (2012). High-sensitivity stable-isotope probing
by a quantitative terminal restriction fragment length polymorphism protocol.
Applied and environmental microbiology, 78(1), 163–9. doi:10.1128/AEM.0597311
Anderson, R., Wylezich, C., Glaubitz, S., Labrenz, M., & Jürgens, K. (2013). Impact of
protist grazing on a key bacterial group for biogeochemical cycling in Baltic Sea
pelagic oxic/anoxic interfaces. Environmental microbiology. doi:10.1111/14622920.12078
Andreoni, V., & Gianfreda, L. (2007). Bioremediation and monitoring of aromaticpolluted habitats. Applied microbiology and biotechnology, 76(2), 287–308.
doi:10.1007/s00253-007-1018-5
Antony, C. P., Kumaresan, D., Ferrando, L., Boden, R., Moussard, H., Scavino, A. F.,
Shouche, Y. S., et al. (2010). Active methylotrophs in the sediments of Lonar Lake, a
saline and alkaline ecosystem formed by meteor impact. The ISME journal, 4(11),
1470–80. doi:10.1038/ismej.2010.70
Aslett, D., Haas, J., & Hyman, M. (2011). Identification of tertiary butyl alcohol (TBA)utilizing organisms in BioGAC reactors using 13C-DNA stable isotope probing.
Biodegradation, 22(5), 961–72. doi:10.1007/s10532-011-9455-3
45
Auclair, J., Lépine, F., Parent, S., & Villemur, R. (2010). Dissimilatory reduction of
nitrate in seawater by a Methylophaga strain containing two highly divergent narG
sequences. The ISME journal, 4(10), 1302–13. doi:10.1038/ismej.2010.47
Avrahami, S., Jia, Z., Neufeld, J. D., Murrell, J. C., Conrad, R., & Küsel, K. (2011). Active
autotrophic ammonia-oxidizing bacteria in biofilm enrichments from simulated
creek ecosystems at two ammonium concentrations respond to temperature
manipulation. Applied and environmental microbiology, 77(20), 7329–38.
doi:10.1128/AEM.05864-11
Barclay, A. R., Morrison, D. J., & Weaver, L. T. (2008). What is the role of the metabolic
activity of the gut microbiota in inflammatory bowel disease? Probing for answers
with stable isotopes. Journal of pediatric gastroenterology and nutrition, 46(5),
486–95. doi:10.1097/MPG.0b013e3181615b3a
Barret, M., Gagnon, N., Kalmokoff, M. L., Topp, E., Verastegui, Y., Brooks, S. P. J.,
Matias, F., et al. (2013). Identification of Methanoculleus spp. as Active
Methanogens during Anoxic Incubations of Swine Manure Storage Tank Samples.
Applied and environmental microbiology, 79(2), 424–33.
doi:10.1128/AEM.02268-12
Bastida, F., Jechalke, S., Bombach, P., Franchini, A. G., Seifert, J., Von Bergen, M., Vogt,
C., et al. (2011). Assimilation of benzene carbon through multiple trophic levels
traced by different stable isotope probing methodologies. FEMS microbiology
ecology, 77(2), 357–69. doi:10.1111/j.1574-6941.2011.01118.x
Bastida, F., Rosell, M., Franchini, A. G., Seifert, J., Finsterbusch, S., Jehmlich, N.,
Jechalke, S., et al. (2010). Elucidating MTBE degradation in a mixed consortium
using a multidisciplinary approach. FEMS microbiology ecology, 73(2), 370–84.
doi:10.1111/j.1574-6941.2010.00889.x
Baytshtok, V., Kim, S., Yu, R., Park, H., & Chandran, K. (2008). Molecular and
biokinetic characterization of methylotrophic denitrification using nitrate and
nitrite as terminal electron acceptors. Water science and technology : a journal of
the International Association on Water Pollution Research, 58(2), 359–65.
doi:10.2166/wst.2008.391
Baytshtok, V., Lu, H., Park, H., Kim, S., Yu, R., & Chandran, K. (2009). Impact of
varying electron donors on the molecular microbial ecology and biokinetics of
methylotrophic denitrifying bacteria. Biotechnology and bioengineering, 102(6),
1527–36. doi:10.1002/bit.22213
Beckmann, S., Lueders, T., Krüger, M., Von Netzer, F., Engelen, B., & Cypionka, H.
(2011). Acetogens and acetoclastic methanosarcinales govern methane formation in
abandoned coal mines. Applied and environmental microbiology, 77(11), 3749–56.
doi:10.1128/AEM.02818-10
46
Bell, T. H., Yergeau, E., Martineau, C., Juck, D., Whyte, L. G., & Greer, C. W. (2011).
Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic
soils by using [15N]DNA-based stable isotope probing and pyrosequencing. Applied
and environmental microbiology, 77(12), 4163–71. doi:10.1128/AEM.00172-11
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