Optimizing ultracentrifugation conditions for DNA-based stable isotope probing (DNA-SIP)
Introduction
Since the DNA-based stable isotope probing (DNA-SIP) technique was first used in microbial ecological studies (Radajewski et al., 2000), it has been employed universally as a tool to link the identification of microorganisms with specific environmental processes in recent years (Coyotzi et al., 2016; Da Costa et al., 2018; Neufeld et al., 2007b). Nowadays, a series of 13C (seldom 15N/18O)-labelled organic and inorganic compounds are chosen to find those microorganisms that prefer to utilize these materials, such as carbon sources (glucose, methanol, butyrate, xylose, cellulose), organic contaminants (isoprene, PCP, PAHs), plant residues, root exudates, CO2, CH4 or urea (Esson et al., 2016; Haichar et al., 2012; Li et al., 2015a; Li et al., 2015b; Long et al., 2018; Nicholson et al., 2009; Pepe-Ranney et al., 2016; Radajewski et al., 2000; Song et al., 2016; Zhao et al., 2015).
Except for a small number of pure culture experiments, most studies have been conducted in complicated ecosystems, such as soil, sediments, waters and bioreactors (Jameson et al., 2019; Li et al., 2019; Niu et al., 2019; Sultana et al., 2019; Xu et al., 2019). Generally, after the sample has been incubated with these substrates for several hours to weeks (Jiang et al., 2015; Zhang et al., 2015), DNA of the soil microbes is extracted and mixed with CsCl and gradient buffer. Ideally, the DNA of microorganisms assimilating the 13C-labelled substrates would show up in relatively heavier density fractions after isopycnic ultracentrifugation compared to corresponding natural 13C abundance substrates. Subsequently, the labelled DNA is picked out and analyzed by clone library or high throughput sequencing to identify the specific microbial population metabolizing the related carbon source.
The key step in these procedures is to identify the labelled fractions after ultracentrifugation. In the first report of DNA-SIP for ecological studies, two-step ultracentrifugation, combined with ethidium bromide (EtBr) staining, was adopted to distinguish the DNA in different fractions visibly (Radajewski et al., 2000). Considering the accuracy and sensitivity, Lueders et al. (2004) selected fluorometry and real-time PCR to in order to detect the ‘heavy’ nucleic acids in a short time. Although the experiment was based on pure cultures, this approach was also widely used in soil microbial studies (Gkarmiri et al., 2017; Hannula et al., 2017; Sun et al., 2018). Due to the complexity of soil microbial community structure and function, it is not so easy to find the labelled fractions. It is significant that it was observed that almost 8 gradient fractions shifted after ultracentrifugation for pure cultures (Lueders et al., 2004) but only 1––4 for environmental samples (Hu and Lu, 2015). Therefore, DNA-SIP for environmental samples should be implemented more cautiously than for pure culture. Otherwise, there was a puzzling phenomenon that labelled 16S rRNA gene or DNA abundance could be identified after labelling root exudates for 14 days or 8 days in a pulse experiment, or even labelling with CO2 for 28 days (Beulig et al., 2015; Da Costa et al., 2018; Gschwendtner et al., 2016), while it was hard to separate the curves of qPCR between the 13C and 12C treatments after continuously labelling root exudates for 21 days with different soils and plants in our laboratory (Wang et al., 2019). Although the condition of soils and plants were clearly important factors, the labelling amount of soil microbes by continuous labelling was theoretically higher than pulse labelling of plant and 13CO2 of bulk soil. In this case, the failure in detecting the presence of labelled DNA in Wang et al. (2019) was unlikely due to the insufficient labelling amount of DNAs but maybe caused by nonoptimal ultracentrifugation procedures.
As known, the separation degree of labelled nucleic acid fragments depends on the rotor geometry, isotopic label (13C/15N/18O), and type of nucleic acid (RNA/DNA). Here we selected the Vti 65.2 rotor to study the impact of ultracentrifugation conditions on isopyncnic gradients. Although the protocol for DNA-SIP was initially published (Neufeld et al., 2007a), there has been much divergence in ultracentrifugation procedures existing in different labs. For example, the density of the blended solution (containing DNA, CsCl, and gradient buffer) before ultracentrifugation was adjusted to 1.69 to–1.77 g ml−1, the time of ultracentrifugation ranged from 24 to 66 h, and the speeds of ultracentrifugation were set from 43,300 to 56,200 rpm (about from 140,000 to 228,000 g). In order to examine these factors, soil was incubated with 13C- glucose to ensure that the DNA of the soil was strongly labelled with 13C, then different initial densities of solution, and different speeds and times of ultracentrifugation were designed to compare the separation effect of universal genes (bacterial 16S rRNA gene and fungal 18S rRNA gene, existing in diverse microbes) and functional genes (bacterial and archaeal amoA gene, only existing in some specific microbes) in this experiment. The aim was to provide suggestions for optimum ultracentrifugation conditions for ecological studies employing DNA-SIP.
Section snippets
Soil microcosm
Soil samples were collected from Taihu (116°17′49′′E, 30°26′03′′) in Anhui Province, China, from a paddy soil planted with rice continuously for forty years. The pH and total organic carbon content were 6.8 and 1.9% respectively. After being sampled from the field (0–20 cm), the soil was air-dried and sieved through 2 mm mesh after removing small stones and root debris. Then 1 Kg soil was adjusted to 50% water holding capacity and pre-incubated for one week and then homogenized.
In order to
The effect of centrifuge time on the fractionation gradient
This showed that the DNA sample had a buoyant density of 1.646–1.748 g ml−1 after centrifugation for 36 to 60 h, regardless of the control or 13C labelled soil (Fig. 1, Fig. 2, Fig. 3, Fig. 4). For the 16S rRNA gene, the maximum abundances were mainly concentrated in the seventh fractions (sequenced from heavy to light buoyant density) except for the 13C sample centrifuged for 42 h which was in the sixth fraction and the control sample centrifuged for 48 h which was in the eighth fraction. All
The effect of centrifuge time on fractionation gradient
That the interval of buoyant density after centrifugation in the different centrifuge time experiment was mainly between 1.65 and 1.75 g ml−1 was similar to a previous study (Ruyters et al., 2013). The centrifuge conditions in the study of Ruyters et al. (2013) were 186,000 g (also with a Vti65.2 rotor), 24 h (initial buoyant density 1.69 g ml−1), and the final buoyant density was 1.66–1.74 g ml−1. It showed that different centrifuge times didn't materially change the buoyant density of
Author contributions
All authors designed the experiments, which were performed by J.W. analyzed the results and wrote the first draft, which was corrected by all authors.
Funding information
This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 41701282 and 41761134085), the National Ten-thousand Talents Program of China (201829), and the Two-hundred Talents Plan of Fujian Province, China (2018A12).
Declaration of Competing Interest
None.
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2022, Water ResearchCitation Excerpt :DNA stable isotope probing (DNA-SIP) provides an effective approach to study functional microorganisms in complex environments (Yi et al., 2020; Zhang et al., 2020). By applying this method, stable isotopes (e.g., 13C or 15N isotopes) are first labeled to substrates of interest, and microorganisms utilizing a stable isotope-labeled substrate incorporate the stable isotopes into their DNA, which can then be separated from the DNA of other bacteria by using density-gradient centrifugation (Wang et al., 2020; Ziels et al., 2018). Analyzing the separated DNA can provide detailed information about the bacteria utilizing the substrate.
Applications of DNA/RNA-stable isotope probing (SIP) in environmental microbiology
2021, Methods in MicrobiologyCitation Excerpt :For example, for a sample of DNA with a buoyant density of 1.71 g mL− 1, if the speed of the Beckman VTi 65.2 rotor is set to 45,000 (~ 184,000 g) and 55,000 (~ 275,000 g) rpm, approximately 34 and 15 h, respectively, will be required to equilibrate the DNA. However, for the Beckman VTi 90 rotor, approximately 53 and 24 h, respectively, will be required and for the TLA-110 rotor, approximately 187 and 84 h, respectively, will be required (Wang, Zhang, & Yao, 2020). Hence, some studies have set the TLA-110 rotor centrifugation speed and time to 55,000 rpm and 66 h, respectively, for DNA equilibration (Pepe-Ranney et al., 2016; Youngblut & Buckley, 2014).