DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.
1. Preparation of Reagents
DNA-SIP requires the use of reagents that should be prepared in advance of the actual procedure. The directions for preparing each reagent are listed in this section and are modified from a previous SIP protocol1.
2. Sample Incubation and DNA Extraction
For DNA-SIP incubations, samples are typically incubated with heavy-isotope carbon (13C) substrate. Incubation periods and conditions (e.g. nutrient supplementation, moisture, light) will vary depending on the type of sample that is incubated and the nature of the substrate. DNA-SIP experiments have been successfully performed using a variety of single carbon compounds 2,3, multi-carbon compounds 4,5,6, and using labelled nitrogen 7,8 or oxygen 9. However, a drawback to using 15N- or 18O-labelled compounds is the decreased physical separation of labelled nucleic acid, primarily due to the presence of fewer nitrogen and oxygen atoms in DNA and RNA relative to carbon atoms.
A critical control for DNA-SIP experiments is an identical incubation established with native (e.g. 12C) substrate. This incubation provides a subsequent comparison to ensure that any apparent labelling of nucleic acid was not an artifact of the ultracentrifugation or G+C content density differences in DNA contributing to separation 10. It is also important to keep frozen sample material for comparison to ‘light’ and ‘heavy’ DNA, and worth including a no-substrate control to assess background population changes throughout the SIP incubation.
3. Preparing Gradient Solutions for Ultracentrifugation
This procedure involves adding DNA to ultracentrifuge tubes. There are more than one type of tube and rotor so the exact protocol will vary and will depend on the manufacturer’s instructions. That said, we recommend use of a vertical-well rotor to ensure maximum possible separation of light and heavy DNA. We use a Beckman-Coulter Vti 65.2 rotor with 16 wells for holding 5.1 ml QuickSeal Polyallomer tubes and the protocol will provide the steps and considerations for these conditions.
4. Creating an EtBr control Gradient (optional)
Because EtBr is an intercalating dye that complexes with DNA making it visible under UV light, control gradients containing EtBr are helpful because they provide immediate visual confirmation of gradient formation prior to fractionation of sample tubes (e.g. Figure 1). The inclusion of a control tube containing EtBr and a mixture of both 12C-DNA and 13C-DNA (or 14N-DNA and 15N-DNA) allows for immediate visualization of band formation within the tubes upon completion of ultracentrifugation. This is important because a ruptured tube during ultracentrifugation or improperly programmed run conditions can result in failed gradient formation. Bound to DNA, EtBr lowers the density of the DNA and as a result, a different protocol is followed to prepare gradients. Note that other nucleic acid stains can be used instead of EtBr 11 but the protocol will require optimization with other fluorophores.
5. Ultracentrifugation
6. Gradient Fractionation
There are two methods that are currently used to recover DNA from the ultracentrifuge tubes: fractionation and needle extraction. This protocol will only describe the process of extracting DNA using the fractionation technique. This is because for most SIP experiments, labelled DNA cannot be visualized with EtBr and must instead be detected by comparing equivalent light and heavy fractions from multiple sample tubes. A syringe pump is highly recommended to retrieve equal density gradient fractions from ultracentrifuge tubes. We use a BSP model infusion pump (Braintree Scientific Inc.). A low-flow peristaltic pump or an HPLC pump may also be used.
7. DNA Precipitation
8. Fraction Characterization
The method used to characterize gradient fractions to assess the success of a SIP incubation will vary depending on the lab and availability of equipment. Using a fingerprinting method for targeting the 16S rRNA gene is a common approach and methods such as terminal restriction fragment length polymorphism (T-RFLP) or denaturing gradient gel electrophoresis (DGGE) are appropriate (Figure 1). Following the protocol described above, expect the light DNA to be associated with fractions 9-11 (~1.705-1.720 g ml-1) and the heavy DNA fingerprints to be associated within fractions 5-8 (~1.720-1.735 g ml-1). Unique fingerprints associated with fractions 5-8 of stable-isotope incubated samples, but not with native-substrate incubated controls provides strong evidence linking specific organisms with the metabolism of particular labelled substrate. If insufficient labelled DNA remains for some applications (hybridization, metagenomics), multiple displacement amplification may be used to produce greater quantities 13-15 but this can introduce chimeras into the amplified DNA 14,16.
9. Results
Typical DNA-SIP results will demonstrate a separation of labelled and unlabelled DNA in the gradient formed by ultracentrifugation. Ideally, complete resolution of high molecular weight genetic material (e.g. 13C, 15N) from unlabelled materials will be achieved. Resolution can be witnessed visually by observing band formation in EtBr control tubes. The concentrations of retrieved genomic DNA contained in the individual gradient fractions may also be used to confirm proper gradient formation.
For this protocol, we include representative results of gradient ultracentrifugation performed using nucleic acid from two pure cultures (Figure 2). The fractionated gradient included here was prepared using genomic DNA extracted from S. meliloti (ATCC 1021), and 13C-labelled M. capsulatus str. Bath. Following ultracentrifugation, fractionation and DNA recovery, labelled and unlabelled genomic DNA separate into respective gradient fractions with differing densities (Figure 2A). Heavy-isotope labelled DNA can be observed in fractions 4-5, whereas unlabelled DNA is found at high concentrations in fractions 9-10. The DNA from each fraction was characterized with denaturing gradient gel electrophoresis 17 and the PCR-amplified products generated discrete banding patterns corresponding to the two organisms included in the gradient (Figure 2B). The density of the fractions ranged from ~1.580 – 1.759 g ml-1, and they are shown in order of decreasing density from left to right.
Although the separation of pure 13C- and 12C-DNA can be pronounced (Figure 2), environmental sample incubations may be more difficult to interpret. For example, we incubated tundra soils from Resolute Bay (Nunavut, Canada) with either 12C- or 13C-labelled glucose for a 14-day period at 15°C. The agarose gels of purified gradient fraction DNA demonstrated that genomic DNA was ‘smeared’ across fractions 7-10 for both 12C- and 13C-incubations (Figure 3A and 3C, respectively). In this case, 13C-enrichment of biomass from particular microbial taxa can only be determined with an approach such as DGGE of 16S rRNA genes. The 12C-glucose incubated soil DNA generates similar patterns across all gradient fractions (Figure 3B), but the 13C-glucose incubated sample generated DGGE fingerprints that are uniquely associated with fractions 5-8 (Figure 3D). Of particular interest are the conserved bands indicated by the arrows. This dominant ‘phylotype’ is consistent across all gradient fractions but shifts to heavier fractions for DNA obtained from 13C-glucose incubated soil. Subsequent DNA sequencing of this band and/or clone library analysis would confirm the identity of this particular 16S rRNA gene and guide subsequent metagenomic or cultivation-based approaches.
Figure 1. Outline of a DNA-SIP experiment involving sample incubation, DNA extraction, CsCl density gradient ultracentrifugation and DNA characterization with molecular techniques.
Figure 2. Expected results for a SIP gradient fractionation including DNA from two pure cultures. (A) Aliquots of DNA from gradient fractions 1-12 were run on a 1% agarose gel from a gradient containing 13C-labelled M. capsulatus strain Bath (fractions 4-6) and 12C-labelled S. meliloti (fractions 8-10). A 1-kb ladder is included for comparison (B) PCR-amplified DNA from the same fractions were run on a 10% DGGE gel. Fingerprint patterns reveal distinct differences between fractions 5 and 9, for example.
Figure 3. Expected results for SIP gradient fractionations from soil sample incubations. Aliquots of gradient fractions from both 12C-glucose amended soil (A) and 13C-glucose amended soil (C) were run on 1% agarose gels and a 1-kb ladder is included for comparison. Corresponding DGGE fingerprints for each of these samples are shown in (B) and (D). Fingerprinting of fractions reveals enrichment of particular bacterial taxa in the 13C-glucose amended sample in fractions 5-8 (D).
Proper design of stable-isotope probing experiments is of critical importance for obtaining labelled DNA above the background unlabelled community. Considerations related to sample incubation times, substrate concentrations, incubation conditions (e.g. nutrients, soil moisture content), cross-feeding and replication have been discussed elsewhere 10,18 and we recommend the reader consult these publications when designing a SIP incubation. Related to the current protocol, it is worth commenting on additional considerations related to the interpretation of data from SIP gradients. Due to the nature of the ultracentrifugation process, it is additionally important to include controls such as pure cultures and native-substrate incubated samples to ensure that bands appearing or disappearing in particular fractions are not artifacts of the protocol itself. For example, DNA within an ultracentrifuge gradient may not be visible in an agarose gel (Figure 2A), but may still contaminate the full length of the gradient (Figure 2B). Although M. capsulatus patterns are most distinct in the dense fractions (5-7) of the gel shown in Figure 2B, the same DGGE pattern was still observed in the lightest fraction (12). With carefully considered controls, interpretation of SIP gradient fraction data is possible.
Due to the nature of some well-designed SIP experiments (e.g. near in situ substrate concentrations, short incubation times), isotope incorporation can be very low10. In addition, most microorganisms in terrestrial or aquatic environments have long generation times compared to growth in the laboratory, and require extended incubation times to reach detectable levels of isotopic enrichment. Other populations may be capable of metabolizing a variety of substrates, and may be not grow fully on labelled substrate. There are also communities (e.g. groundwater) that may be associated with low biomass levels and generate low yields of extracted nucleic acid. In all of these cases, the quantitative retrieval of labelled nucleic acids may be challenging.
To circumvent these limitations, a variety of natural and synthetic carrier molecules exist that assist in the precipitation and recovery of DNA from CsCl gradients. Carrier molecules can be biological in origin such as glycogen or DNA from an archaeal organism 19, or synthetic in nature, such as linear polyacrylamide. The benefit of using carrier molecules such as these when performing DNA-SIP is that they can enable visualization of bands in the CsCl gradients that would not normally be visible and ensure quantitative recovery of low DNA concentrations. Successful recovery of low nanogram amounts of DNA from CsCl gradients actually requires the use of a carrier molecule 1,12. Recent research has indicated that carrier molecules obtained from biological sources can often be contaminated with DNA from the source organism 12 and the results are very difficult to distinguish from patterns associated with 13C-labelled DNA (data not shown). Therefore it is recommended that synthetic carrier molecules such as linear polyacrylamide be used for DNA-SIP. In addition, the use of multiple-displacement amplification (MDA) can generate high fidelity yields of labelled DNA for downstream molecular analyses 13,14, although chimeras may be generated by the amplification and detected in downstream molecular analyses 14.
One of the most powerful applications of DNA-SIP that has yet to be fully exploited is the potential recovery of DNA from active community members for metagenomic library analysis. We expect that major advances in enzyme discovery will result from the integration of stable-isotope probing into existing metagenomic surveys from diverse terrestrial and aquatic environments. The protocol visualized here will produce labelled DNA of sufficient quality for these discovery-based applications.
The authors have nothing to disclose.
This work was supported by Strategic Project and Discovery Grants to J.D.N. from the Natural Sciences and Engineering Research Council of Canada (NSERC).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Bromophenol Blue | Reagent | Fisher Scientific | BP115-25 | |
Cesium chloride | Reagent | Fisher Scientific | BP210-500 | |
Ethanol, reagent grade | Reagent | Sigma-Aldrich | 652261 | |
Ethidium bromide | Reagent | Sigma-Aldrich | E1510 | |
Hydrochloric acid | Reagent | Fisher Scientific | 351285212 | |
Linear polyacrylamide | Reagent | Applichem | A6587 | |
Polyethylene Glycol 6000 | Reagent | VWR | CAPX1286L-4 | |
Potassium Chloride | Reagent | Fisher Scientific | AC42409-0010 | |
Sodium Chloride | Reagent | Fisher Scientific | S2711 | |
Sodium Hydroxide pellets | Reagent | Fisher Scientific | S3181 | |
Tris base | Reagent | Fisher Scientific | BP1521 | |
Dark Reader | Equipment | Clare Chemical | DR46B | |
Microcentrifuge | Equipment | Eppendorf | 5424 000.410 | |
Nanodrop 2000 | Equipment | Fisher Scientific | 361013650 | |
Infusion pump | Equipment | Braintree Scientific | N/A | Model Number: BSP See www.braintreesci.com for ordering details. |
Tube sealer | Equipment | Beckman-Coulter | 358312 | |
Ultracentrifuge | Equipment | Beckman-Coulter | ||
Ultracentrifuge rotor | Equipment | Beckman-Coulter | 362754 | |
Ultraviolet light source | Equipment | UVP Inc. | 95-0017-09 | Any UV source will suffice |
Ultraviolet light face shield | Equipment | Fisher Scientific | 114051C | |
Butyl rubber stoppers, gray | Material | Sigma-Aldrich | 27232 | |
Centrifuge tubes | Material | Beckman-Coulter | 342412 | |
Hypodermic needle, 23 gauge, 2” length | Material | BD | 305145 | |
Microfuge tubes, 1.5 mL | Material | DiaMed | AD151-N500 | |
Open center seals, 20 mm diameter | Material | Sigma-Aldrich | 27230-U | |
Pasteur pipettes, glass | Material | Fisher Scientific | 13-678-6C | |
Pipet tips | Material | DiaMed | BPS340-1000 | Catalogue number is for 200 μl tips. 10 or 20 μl tips may be purchased from the same source |
Pump tubing 1.5 mm bore x 1.5 mm wall | Material | Appleton Woods | ||
Screw-cap tubes, 15 mL | Material | DiaMed | AD15MLP-S | |
Serum vials, 125 mL volume | Material | Sigma-Aldrich | Z114014 | |
Syringe, 60 mL | Material | BD | 309653 |