RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2005; 19: 1424–1428
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1908
Technical considerations for the use of 15N-DNA
stable-isotope probing for functional microbial
activity in soils{
Georg Cadisch1,2*, Mingrelia Espana1,3, Rachel Causey4, Michael Richter2,
Eve Shaw4, J. Alun W. Morgan4, Clive Rahn4 and Gary D. Bending4
1
Institute of Plant Production and Agroecology in the Tropics and Subtropics (380a), University of Hohenheim, 70593 Stuttgart, Germany
Department of Agricultural Sciences, Imperial College London, Wye Campus, Wye TN25 5AH, UK
3
National Institute of Agricultural Research—CENIAP, A.P. 4648, Maracay, Venezuela
4
Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK
2
Received 1 November 2004; Revised 6 March 2005; Accepted 6 March 2005
Stable-isotope DNA probing is a culture-independent technique that may provide a link between
function and phylogeny of active microorganisms. The technique has been used in association with
13
C substrates while here we evaluate feasibility and limitations of 15N-DNA stable-isotope probing (SIP) using labelled and unlabelled pure microbial cultures or soil extracts. Our results showed
that 15N-DNA probing is feasible for cultures as well as soil samples. Limitations of 15N-DNA-SIP
are (a) the need for relatively large quantities of DNA to visualise bands (although molecular resolution is much higher) and (b) 15N-DNA enrichment needed to ideally be >50 at%; however, this
requirement can be lowered to approx. 40 atom% 15N with pure cultures using a modified CsCl
centrifugation method (140K g for 69 h). These advances in 15N-DNA-SIP methodology open
new opportunities to trace active microbial populations utilising specific N substrates in situ.
Copyright # 2005 John Wiley & Sons, Ltd.
Advances in molecular techniques provide increasingly
more sophisticated ways to characterise the diversity of total
active microbial polulations in soil or other environments.
Combining stable isotopes (13C, 15N) with these advanced
molecular techniques provides new approaches to achieve
a better understanding of the processes and organisms that
drive C and N cycling in soil-plant systems.1 Stable-isotope
probing (SIP) is a culture-independent technique that enables
taxonomic identity to be linked with function in the environment by providing a highly enriched microbial substrate
leading to isotopically labelled microbial DNA which can
be separated on density gradients.2 The isolated isotopically
enriched DNA can then be used as a template for polymerase
chain reaction (PCR) using general microbial primers or specific primers for functional groups (e.g. diazotroph group,
proteobacteria, cyanobacteria). Amplification products can
then be used for direct cloning and sequencing for phylogenetic identification and position analysis or community profiling analyses (T-RFLP, DGGE) for treatment comparisons.
Alternatively, assessing isotope assimilation into RNA may
be a more sensitive approach since it is not dependent on
cell replication while copy number in cells is higher than
for DNA and incorporation of stable isotopes into RNA is faster than for DNA.3
*Correspondence to: G. Cadisch, Institute of Plant Production and
Agroecology in the Tropics and Subtropics (380a), University of
Hohenheim, 70593 Stuttgart, Germany.
E-mail: cadisch@uni-hohenheim.de
{
Presented at the Joint European Stable Isotope Users Group
Meeting, Vienna, 30 August–3 September, 2004.
While most work on SIP to date has been performed using
enriched 13C substrates,3–5 no applications have so far shown
its applicability for 15N-labelled substrates. Here we assess
some of the technical issues related to the application of 15NDNA-SIP. In particular we investigated (a) the amount of
DNA required for its visualisation in CsCl isopycnic
centrifugation gradients, (b) isotopic enrichment required
to obtain a clear separation of bands, and (c) testing some
modifications to increase the sensitivity of the centrifugation
CsCl method.
EXPERIMENTAL
Determination of quantities of DNA required
for visualisation
Azospirillum lipoferum CRT1 was grown in a mineral salts
medium (Azospirillum-medium) 221, DSMZ (Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH,
Germay6). Cultures were grown on an orbital shaker at
100 rpm for 20 h at 308C. DNA was extracted in subsamples
containing approx. 1 109 cells using the DNeasy1 DNApurification kit (Qiagen). Quantification of DNA concentration was performed using a NanoDrop ND-1000 spectrophotometer. DNA extracts were mixed with 0.5 TE buffer
to obtain the following amounts of DNA in 4.5 mL Tris0.5 mM EDTA (TE): 53, 26, 13, 3 and 2 mg; thereafter, CsCl
was added at 1 g mL1 solution followed by 200 mL ethidium
bromide solution (10 mg mL1). CsCl density gradient centrifugation was performed using a Sorvall centrifuge with rotor
Copyright # 2005 John Wiley & Sons, Ltd.
15
N-DNA stable isotopes to probe functional microbial activity in soils
TV-689UA at 265 000 g for 20 h at 208C. DNA bands were
visualised with UV light.
Preparation and separation of DNA from
P. putida cultured on media containing 0–99%
15
N enrichment
Pseudomonas putida paw8 was grown in a mineral salts7 medium to which 100 (99.63) atom % 14N- and 99 atom % 15Nlabelled NH4NO3 were added to provide 1 mg N mL1 and
10 at% 15N enrichment increments between 0 (0.3663) and
99 at% 15N. Cells were washed off overnight P. putida cultures
growing on Luria-Bertani agar plates, and inoculated into
5 mL aliquots of each medium. Cultures were grown overnight at 358C. DNA was extracted from the cells following
lysis with lysozyme and treatment with proteinase and
RNase followed by precipitation by isopropanol. DNA
from the P. putida cultures was quantified using a NanoDrop
ND-1000 spectrophotometer. Approximately 10 mg of P. putida DNA labelled with between 10 and 99 at% 15N was added
to 10 mg 100 (99.63) at% 14N-labelled P. putida DNA. The DNA
was mixed with a CsCl solution (1 g mL1 TE buffer) and ethidium bromide (10 mg mL1) in 10 mM TE buffer. Centrifugation was performed using a BeckmanVTi 65.2 rotor at
265 000 g for 18 h at 208C,1 or 140 000 g for 69 h.8 DNA bands
were visualised under UV light.
Preparation and collection of
from soil samples
15
15
N-labelled DNA
N-Labelled DNA from soil was extracted from an experiment where highly 15N-enriched (>90 at%) ryegrass (Lolium
perenne L.) was incubated in a topsoil collected from Long
Close field on the site at Warwick HRI, Wellesbourne. The
soil is a sandy-loam of the Wick Series with 14% clay, a pH
of 6.5, and an organic C content of 0.8%.9 Labelled ryegrass
was obtained by propagation in a coarse sand/clay mixture
under controlled glasshouse conditions (16 h day length,
maximum day temperature 258C, minimum night temperature 158C). The plants were nourished with Hewitt’s nutrient
solution,10 in which the sole N source was 99 at% 15NH4
15
NO3. Three replicates, each consisting of 500 g fresh weight
(FW) portions of soil mixed with 25 g FW ryegrass, were set
up, and incubated at 158C in the dark for 7 days.
DNA was extracted from 1 g FW portions of soil by beadbeating using a Fast Prep DNA spin kit for soil (Bio 101). DNA
was centrifuged in CsCl as described above, at 265 000 g for
18 h at 208C. After visualisation under UV light, the base of
the DNA gradient was identified. A hypodermic needle was
inserted below the lower band and another at the top of the
tube. Pressure was applied through the upper needle to force
the gradient out of the lower needle. Fractions (ca. 100 mL)
were removed from just below the lower band to above the
top band in the gradient. Ethidium bromide was removed
from the DNA fractions using isoamyl alcohol saturated with
H2O and NaCl. Samples were dialysed on a nitrocellulose
membrane for 20 min to remove residual CsCl, and then
suspended in TE buffer. The 15N enrichment of DNA
fractions was determined by spiking 30 ng DNA with 20 mg
unlabelled NH4SO4 and analysed using an automated CN
analyzer (Robprep) coupled to a 20–20 Europa mass spectrometer (PDZ Europa Ltd, Crewe, UK).
Copyright # 2005 John Wiley & Sons, Ltd.
1425
RESULTS AND DISCUSSION
Visualisation of DNA in CsCl
The successful application of the SIP methodology ideally
requires that DNA is visible in the CsCl medium. Staining
with ethidium bromide and exposure to UV light show sufficient quantities of DNA as clearly visible bands. In our
experiments we found that >13 mg DNA resulted in clearly
identifiable bands in the CsCl tubes (Fig. 1). Smaller quantities down to 2 mg DNA were only faintly visible. These results
reflect similar observations by other authors,11 who observed
that about 15 mg DNA was necessary for clear visualisation of
DNA using ethidium bromide in CsCl gradients. Work with
rRNA suggested that lower quantities (e.g. 500 ng) may be
sufficient for rRNA in cesium trifluoroacetate gradients.2
The relatively large quantities of DNA required using ethidium bromide in CsCl gradients limit somewhat the use of
the DNA-SIP method if only small quantities of DNA are
available. It is thus desirable to aim for larger yields, which
can be achieved by multiple DNA extractions and pooling
of samples where necessary. It is, however, possible that, if
a control 14N-DNA band is present, extraction of even very
small ‘invisible’ amounts of DNA below the 14N-DNA band
will result in sufficient DNA for PCR amplification purposes
and subsequent further community/species analysis.
Separation of
14/15
N-labelled DNA
When a mixture of 100 at% 15N-labelled and unlabelled DNA
from P. putida was subject to isopycnic centrifugation at
265K g for 18 h, two clearly separated DNA bands were visible and the distance between the two bands was approximately 4 mm (Fig. 2). These results confirm observations
Figure 1. Visualisation of varying quantities of 14N-DNA
from Azosprillum lipoferum in a CsCl gradient (256K g for
20 h).
Rapid Commun. Mass Spectrom. 2005; 19: 1424–1428
1426
G. Cadisch et al.
(average C/N ratio 2.1:1), hence affecting their respective
buoyant densities.
15
N-isotopic DNA enrichment required
In order to determine the minimum 15N-DNA enrichment
required to obtain a clear separation between labelled and
unlabelled DNA we centrifuged unlabelled and a mixture
of unlabelled and increasingly 15N-labelled DNA (10–99
at%) on a CsCl gradient at 265K g for 18 h. A clear separation
of bands was achieved with 15N-DNA enrichments 50 at%
15
N (Fig. 3(A)). Below that, bands of labelled and unlabelled
DNA were undistinguishable by eye. These results suggest
that the 15N-DNA probing method needs high 15N enrichment for successful application. The 50 at% 15N identified
in this experiment is much higher than the minimum enrichment of 20 at% suggested for application of DNA-SIP when
using 13C labelling1 or as low as 10 at% 13C for RNA-SIP.2
Evaluation of an alternative approach to enhance
separation of 14/15N-DNA bands
Figure 2. 14N (unlabelled) and 15N (99%) enriched DNA of
Pseudomonas putida or a mixture of unlabelled and highly
enriched DNA in a CsCl gradient (256K g for 18 h).
with highly 13C-labelled DNA where also a clear separation
of DNA bands in CsCl gradient was obtained using unlabelled/highly enriched DNA.12 However, whereas a separation between highly labelled/unlabelled 13C-DNA of about
1 cm was achieved,12 our results suggest that, under standard
isopycnic centrifugation conditions (265 K g for 18 h), only a
maximum 4 mm separation of 14/15N-DNA is achievable.
This is because the proportional difference of 14/15N is lower
than of 12/13C and DNA contains more C than N atoms
The fact that with the conventional centrifugation conditions
of 265K g at 18 h a maximum separation of 4 mm between
fully labelled and unlabelled bands is achieved severely
limits its application in many ecological studies where often
only low microbial enrichments are achieved unless the system is artificially highly loaded with the labelled substrate.
We tested an alternative centrifugation approach which
used much longer spinning time (69 h) at somewhat lower
speed (38K rpm or 140 K g). The results, shown in Fig. 3(B),
demonstrate that with this method the maximum separation
of unlabelled and highly 15N-labelled DNA can be extended
to 5 mm. This improved separation of differently labelled
DNA allowed a clear differentiation of enrichments as low
as 40 at% 15N. This is an improvement over the minimum
50 at% 15N identified under the higher speed but shorter centrifugation. Similar experiences have been achieved with 13Clabelled DNA,8 although the results were more encouraging
by nearly doubling the separation of differently labelled
DNA strands.
Figure 3. Separation of unlabelled (14N) and increasingly enriched (10–99 at% 15N) DNA
of Pseudomonas putida in a CsCl gradient obtained at (A) 265K g for 18 h and (B) 140K g
for 69 h.
Copyright # 2005 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2005; 19: 1424–1428
15
N-DNA stable isotopes to probe functional microbial activity in soils
1427
practice given the width of DNA bands (related to their
quantity and variation in %G þ C content) in the media.
Hence, there is a need for further improvements in
techniques that lead to a better separation between DNA
strands in density gradients.
Verification of isotopic enrichment of heavy
bands from soil residue experiments
Figure 4. Estimated effect of varying nucleic composition of
DNA on separation efficiency of differently 15N-enriched
DNA. Error bars depict theoretical maximum differences in
GC content of DNA based on buoyant density, i.e. high or low
GC content.
These separations were achieved with single species DNA
composition. However, the nucleic (nitrogenous) composition of DNA can vary depending on the proportion of
adenine-thymine (AT) to cytosine-guanine (CG) combinations; the N concentration of DNA could then theoretically
vary between 13.9 and 15.8% depending on species. This also
implies that the molecular weight and buoyant density of
DNA change depending on the CG/AT composition13 as
well as with its respective labelling with 15N. For example,
with a CG content of microbial DNA varying between 35 and
70% the respective buoyant density varies between 1.69 and
1.73 g cm3.14 Thus, variation in the nucleic content of the
DNA also has a significant effect on the position of the band in
the density medium (see error bars in Fig. 4 depicting
theoretical maximum/minimum GC content) particularly
for 15N-SIP and to a lesser degree also for 13C-SIP.12 Hence, a
clear separation at 40 at% 15N-DNA enrichment, although
theoretically still possible, may be difficult to achieve in
To test the applicability of the 15N stable-isotope probing
method in a soil environment, a highly enriched L. perenne
residue was incubated in soil. After 7 days, DNA was
extracted and exposed to centrifugation (265K g for 18 h) in
a CsCl gradient. Unlike in the pure culture experiments a
clear but more widespread band appeared in the CsCl media
(Fig. 5). This was expected as it was hypothesised that not all
organisms would feed solely on the substrate and that the
turnover of 15N due to cross feeding of DNA from originally
unlabelled organisms would dilute the DNA from organisms
feeding primarily on the labelled substrate. Analysis of the
actual enrichments of the isolated DNA fractions showed
that the DNA at the bottom of the visible band was heavily
enriched with 15N of up to 90 at%. The evidence that this
highly 15N-enriched DNA was primarily of microbe and
not plant origin comes from both microbial biomass and its
15
N-enrichment measurements and specific primers/DGGE
results (data not presented) which suggested that by day 7 little if any plant DNA remained. Enrichment subsequently
declined rapidly with increasing distance from the bottom
sample. In this experiment we were not able to harvest sufficient amounts of unlabelled (100 at% 14N) DNA for isotope
ratio mass-spectrometric analysis (IRMS) (which for the current equipment needed to be around 30 mg to obtain reliable
results). However, lower levels of enriched DNA might be
detectable through the use of GC-C/IRMS or emergent technologies such as HPLC/IRMS. The reason that there was no
significant 14N-DNA band is that the added material supplied much more microbially available substrate than the
native soil organic matter and hence resulted in good 15Nenriched microbial DNA yields. These results clearly suggest
that the sampling strategy of DNA from the CsCl media is
Figure 5. Isotopic 15N-enrichment pattern of DNA bands extracted from a CsCl gradient
(position 0 starts just below visible band; data only available where sufficient DNA
recovered) 7 days after application of highly enriched Lolium perenne residue to soil.
Copyright # 2005 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2005; 19: 1424–1428
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G. Cadisch et al.
crucial and that a careful stepwise separation of DNA at different distances from the reference is advisable.
CONCLUSIONS
The current experience has shown that 15N-DNA probing is
feasible using both pure cultures and soil. This opens new
opportunities to trace active microbial populations feeding
on specific N substrates. However, several limitations of the
15
N-DNA-SIP method were identified, i.e. (a) the need for
relatively large quantities of DNA to visualise bands and
(b) 15N-DNA enrichment needs ideally to be >50 at% 15N.
This can, however, be lowered to 40 at% 15N if the modified
CsCl centrifugation method (140K g for 69 h) is applied.
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