Introduction

Stable-isotope probing (SIP) of nucleic acids has become a focal method in microbial ecology since it can identify microorganisms being involved in the metabolism of specific substrates [1-7]. The ¹³C-labelled substrate has been typically used in this technique to incorporate ¹³C into deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and phospholipid-derived fatty acid (PLFA) [5,7]. The isolation of the ¹³C-labeled DNA or RNA can provide the solid information of nutrient cycling in relation to phylogeny and function of uncultivated microorganisms in the natural environment [8].

Two requirements have to be satisfied in a successful isolation of the ¹³C-labeled DNA (¹³C-DNA). The first one is that the labeled DNA should be distinguished above a background of abundant unlabeled molecules, which depends on the minimum substrate concentration and duration of the incubation. The other is the avoidance of too much substrate that may lead to cross-feeding of the substrate and enrichment bias [5,9]. Therefore, identifying the proper concentration of substrate is the key for an acceptable SIP experiment that can reflect the nature of microorganisms incorporating the substrate turnover.

However, various concentrations of ¹³C labeled substrate were used in the SIP studies. For example, Radajewski et al. [8] stated that approximately 15-20 atom% ¹³C was necessary for separating ¹³C-DNA from ¹²C-DNA and analyzing the link between identity and function of microorganisms, while around 30 atom% ¹³C substrate was recommended for DNA-SIP in other studies [10,11]. Fan et al. [10] reported that the maize residue with 31 atom% ¹³C was necessary to effectively identify the active microorganisms which decomposed the residues. In a study of commercial strains isolation, 32 atom% ¹³C of biphenyl was used to successfully probe polychlorinated biphenyls (PCB)-degrading populations in PCB-contaminated river sediment [11]. Although the protocol of the DNA-SIP technology per se have been well documented [12,4], the critical level of the ¹³C enrichment in substrate for differentiating ¹³C-labeled DNA has not been quantitatively tested. Narrowing such knowledge gap will be helpful to precisely assessing the metabolic “functional” genes on the specific substrates. In the present study, we incubated the Escherichia coli (E.coli) with a serial of ¹³C enrichments in glucose to identify the critical level of ¹³C enrichment in the substrate for the effective separation of ¹³C-DNA. It is predicted that the minimum enrichment of ¹³C labeled substrate for the distinguishable detection of ¹³C-DNA would be in a range of 10-30 atom% ¹³C substrate.

Materials and Methods

Incubation of the E.coli with ¹³C-glucose

Six levels of ¹³C enrichment in glucose were designed in this study. They were 0, 2, 5, 10, 20 and 50 atom% ¹³C in glucose (Sigma-Aldrich) as substrate. Each level comprised 3 replications. Using M9 medium with 4% of glucose [13,14], 15µl of E. coli solution (optical density at the 600 nm, OD600 = 0.8) was added to the medium and incubated at 37°C for 96 hrs on an end-over-end shaker. The OD600 values of the medium were determined at the beginning and the end of incubation with a spectrometer.

Extraction of E.coli DNA

DNA of E.coli was extracted from each treatment according to Casas and Rohwer [15]. DNA samples were visualized by electrophoresis in a 1% agarose gel. The concentration of DNA was measured using a NanoDrop 1000 (Thermo Scientific, Wilmington, DE) and the δ¹³C of the total E.coli DNA was determined by the Mat 253 isotope ratio mass spectrometer (Thermo Fisher, Germany). ¹³C enrichments were expressed relative to Pee Dee Belemnite standard as either δ¹³C or atom fraction ¹³C excess.

DNA-SIP

Cesium trifluoroacetate (CsTFA, Amersham Pharmacia Biotech) density gradient centrifugation was performed to separate the ¹³C-labled DNA from total E.coli DNA [16]. Control gradients were run with the DNA from the unlabeled E.coli for each course to calibrate the centrifugation system. Approximately 7000 ng of each DNA sample was loaded into a centrifuge solution with a starting buoyant density of 1.60 g ml^-1. This centrifugation medium consisted of 3.2 ml of a 1.99 g ml^-1 the CsTFA solution and 1.8 ml of gradient buffer (GB). The CsTFA solution with DNA was transferred to 4.9 ml Ultracrimp tubes (Beckman, USA) using 5ml syringes. The tubes were centrifuged in a vertical rotor (Vti 65.2, Beckman) at 179,000 g (43,500 rpm) for 40 hrs at 20°C. The gradients were fractionated into 14 fractions by being displaced with water at a flow rate of 11.3 µl/s using a syringe pump (New Era Pump Systems, Inc. New York, USA). An AR200 digital refractometer (Reichert Inc., Depew, New York, USA) in the nD-TC mode was used to measure the buoyant density of gradient fractions. DNA in each fraction was precipitated with isopropanol (885 µl) and 1/10 volume (30 µl) of 3 M sodium acetate (pH 5.2). The DNA pellets were then washed and redissolved in ddH₂O. The redissolved DNA was amplified using a universal primer pair forthe16S rDNA gene, i.e. 357f (5’- CCT ACG GGA GGC AGC AG -3’) and 517r (5’- ATT ACC GCG GCT GCT GG -3’) [17]. The cycling profile was 95°C for 10 min and 28 cycles of 95°C for 15 s, 60°C for 10s and 72°C for 20 s, and then 72°C for 10 min. The PCR products from the 1^st to 10^th fractions were run on a 1% agarose gel.

The F (fractional abundance) was calculated as followed [18]:

Where R_PDB = 0.0112372 (the absolute isotope ratio of the PDB ¹³C standard) [19].>

Using SAS (SAS Institute, 1994), data were analyzed statistically. The difference of ¹³C enrichment in the extract DNA between treatments was assessed with protected ANOVA tests at P< 0.05 level [20].

Since ultracentrifuge creates gradient fractions, the stability of buyout densities between treatments greatly affects the quality of the ¹³C labeled DNA fractionation [12]. This stability was determined by calculating the coefficient of variability (CV) of buyout densities across treatments. The smaller the CV, the more consistent the fractionation of ¹³C-DNA was between treatments.

Results

E.coli growth supplied with ¹³C substrate and DNA extraction

After 96 hrs of incubation, there was no significant difference in optical density (OD600) of M9 medium between treatments (Figure 1). Relatively pure DNA was extracted from the E.coli and the concentrations of DNA were in a range from 441 to 760ng/µl (Figure 2). The δ¹³C of DNA increased from significantly with the increase of ¹³C enrichment in glucose. The F values had the same trend (Table 1).

The buyout density (Table 2) ranged from 1.582 g ml^-1 to 1.622 g ml^-1. The density in each fraction was consistent across treatments with small CV. This suggested that the performance of ultracentrifugation was consistent and successful between courses.

PCR products of the fractions

For the unlabeled control, the DNA band was visible from the 8^th to 10^th fraction (Figure 3). However, appearance of DNA band started in the 7^th fraction under the 2 atom% ¹³C treatment. The distinguishable DNA band moved towards smaller fractions with the increase of ¹³C enrichment. At 50 atom% ¹³C, DNA band was clearly observed in the 2^nd-10^th fraction.

Discussion

The insignificant OD600 values between treatments (Figure 1) suggest that the growth of E.coli was not affected by the addition of glucose with different ¹³C enrichments. Furthermore, the relatively high concentration of DNA and the clear bands of total DNA indicate that the extracted DNA (Figure 2) was appropriate for the DNA-SIP process.

In this study, the DNA band in the 7^th fraction appeared for the 2 atom% ¹³C-glucose treatment, while non DNA band in the same fraction for the unlabeled control, revealing that the detectable level of ¹³C substrate for ¹³C-DNA-SIP under the pure culture condition can be reduced to 2 atom% ¹³C in glucose. However, the better separation of ¹³C-DNA was achieved in the ≥10 atom% ¹³C treatment since the DNA band was visible in the 5^th, 6^th and 7^th fraction under that treatment.

The detectable level of ¹³C substrate for ¹³C-DNA-SIP in this study is lower than that in a study by Radajewski et al. [8], reporting that 20 atom% ¹³C in substrate would be feasible to incorporate ¹³C into DNA, and resolve ¹³C-DNA from ¹²C-DNA. The difference is probably because the carbon source of ¹³CH₃OH used in that study could only label the methylotrophic bacterium i.e. Methylobacterium extorquens and this bacterium was not dominant in the soil microbial communities, while the ¹³C-labeled glucose was the sole C source in the pure incubation solution in this study. Thus, the threshold of ¹³C enrichment in glucose for the separation of ¹³C-DNA in this study was not as high as other studies. In addition, compared with ¹⁵N substrate for ¹⁵N-DNA-SIP, the minimum requirement of ¹³C enrichment in substrate was much lower even under the similar pure culture condition [1]. The minimum level of a ¹⁵N-labelled substrate, i.e. NH₄NO₃ has been quantified as low as 40 atom% ¹⁵N for the clear separation of ¹⁵N-DNA of Pseudomonas putida [1]. The difference is attributed to the fact that the C concentration of DNA is theoretically in a range of 41.2 to 46.1%, while the N concentration of DNA varies only between 13.9 and 15.8% [1]. This is also reflected in the shifts in buoyant density in the CsCl gradients. For ¹³C-labeled DNA this shift is approximate 0.036 g/ml, while it is only 0.013-0.016 g/ml for ¹⁵N-labeled DNA [21,22].

Nevertheless, the ¹³C enrichment in the extracted DNA for the minimum requirement of DNA-SIP is probably more practicable than that in substrates when this technology are used in the environmental experiments, especially in the soil experiments, because a number of uncertain factors such as carbon-conversion efficiency and growth rate of the target organisms [5] may lower the convert efficiency of ¹³C from substrate to DNA. A number of studies have also stated that the requirement for supplemental nutrient addition for C assimilation of microbes and the amount of carbon incorporated into nucleic acid mostly depend on the targeted microorganisms and the characteristics of the samples being analyzed [12,4]. On the point of these views, the ¹³C enrichment in the extracted DNA is likely to be more accurate for predicting the successful SIP. In this study, the detectable level of ¹³C enrichment for DNA-SIP in the extracted DNA was 1.3 atom% ¹³C (Table 1).

It is worth to note that ¹³C-DNA bands were successively distinguishable from light to heavy fractions with the increase of ¹³C enrichment in substrate (Figure 3). This indicates that ¹³C gradually and proportionally incorporates into the C frame of the DNA molecular. However, in some DNA-SIP studies [5,23], the ¹³C DNA bands were observed in the heavy fractions (the 4^th and 5^th fractions) rather than the fractions in between such as the 6^th and 7^th fractions. The difference may be attributed to the ¹³C enrichment of substrate, incubation time and microbial specificity in utilizing ¹³C substrates.

Conclusion

In this study, we confirmed that the level of ¹³C enrichment in glucose for detectable ¹³C-labeled DNA could be reduced to 2 atom% ¹³C (1.30 atom% ¹³C in DNA extract). The critical level of ¹³C for the isolation of ¹³C-DNA provides a new reference of DNA-SIP in order to trace active microbial communities utilizing specific C substrates in environments.

Acknowledgement

The project was funded by the Key Project of Chinese Academy of Sciences (KZZD-EW-TZ-16-01), the Hundred Talents Program, and the National Natural Science Foundation of China (41271261).

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  Figure 1: Optical densities at the 600 nm (OD600) for Escherichia coli that were grown in the M9 medium [1,18] for 96 hrs. The dash line indicates the initial OD600 value at the beginning of incubation.

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  Figure 2: Agarose gel electrophoresis of total DNA extracted from the Escherichia coli incubated in the M9 medium for 96 hrs. E.coli was supplied with different levels of ¹³C enrichment in glucose, i.e. 0, 2, 5, 10, 20 and 50 atom% ¹³C.

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  Figure 3: Aliquots of gradient fractions of DNA-SIP were run on 1% agarose gels and a 1-kb ladder is included as a marker. DNA samples were extracted from the M9 mediums of Escherichia coli supplied with 0, 2, 5, 10, 20 and 50 atom% 13C in glucose.

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  Table 1: The δ¹³C and F values of the DNA extracted from Escherichia coli mediums. E.coli was supplied with different levels of 13C abundance in glucose (0, 2, 5, 10, 20 and 50 atom% ¹³C)

  F indicates fractional abundance, i.e. ¹³C/(¹²C + ¹³C). Different letters represent significance of difference at P < 0.05 level.

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  Table 2: Buyout densities from the 1^st to 14^th fraction of DNA-SIP in the treatments of ¹³C-labeled glucose (0, 2, 5, 10, 20 and 50 atom% ¹³C).