Assessing soil extracellular DNA decomposition dynamics through plasmid amendment coupled with real-time PCR
- 101 Downloads
Determining soil extracellular DNA decomposition dynamics is essential to assessing lateral gene transfer possibility, nutrient-cycling efficiency, and the reliability of DNA-based methods for examining microbes in soils. The existing methods based on competent cell transformation and stable isotope probes are generally inefficient and not strictly quantitative. Therefore, this study aimed to establish a rigorously quantitative and efficient approach to monitor the decomposition dynamics of the soil extracellular DNA.
Materials and methods
A soil was collected from a Tibetan alpine meadow. Extracellular DNA was simulated by modified exogenous plasmids. The plasmid solution was sprayed onto the fresh soil and thoroughly homogenized. Then, the soil was incubated for 4 weeks, during which they were sampled and immediately stored at − 20 °C on days 0, 0.5, 1, 2, 4, 8, 16, and 28 of the incubation. Finally, the total soil DNA was extracted, and the exogenous plasmid copies remained in the soils were determined using real-time PCR. Additionally, another similar experiment was conducted with a sterilized soil to assess the abiotic influences on the changes in the exogenous plasmid copies.
Results and discussion
In the fresh soil, the exogenous plasmid DNA copies decreased quickly in the first 12 h of the incubation, remained stable in the following 36 h, and gradually dropped to 1.10–5.20% of the initial plasmid copies at sampling time 0 after being incubated for 4 weeks. The variations in the soil plasmid DNA copies fitted well with the modified exponential decay model. As for the sterilized soil, the exogenous plasmid copies remained stable during the 16 day’s incubation. However, they dramatically dropped after being incubated for 28 days, which was probably elicited by the recolonization of microbes in the soil. Collectively, the decrease in the exogenous plasmid DNA copies could be mainly attributed to biological activities.
Extracellular DNA can persist in soil for more than 4 weeks. Exogenous plasmid amendment coupled with real-time PCR provides a convenient and rigorously quantitative approach for monitoring extracellular DNA degradation in soils.
KeywordsAntibiotic resistance genes DNA degradation Quantitative approach Environmental DNA eDNA
This work was supported by the National Key Research and Development Program of China (2016YFC0501800), the Strategic Priority Research Program (A) of the Chinese Academy of Sciences (XDA20050103), and the National Natural Science Foundation of China (31570518).
- Alef K, Nannipieri P (1995) Methods in applied soil microbiology and biochemistry. Academic Press, LondonGoogle Scholar
- Berendonk TU, Manaia CM, Merlin C, Fatta-Kassinos D, Cytryn E, Walsh F, Burgmann H, Sorum H, Norstrom M, Pons MN, Kreuzinger N, Huovinen P, Stefani S, Schwartz T, Kisand V, Baquero F, Martinez JL (2015) Tackling antibiotic resistance: the environmental framework. Nat Rev Microbiol 13:310–317CrossRefGoogle Scholar
- Frostegård Å, Courtois S, Ramisse V, Clerc S, Bernillon D, Le Gall F, Jeannin P, Nesme X, Simonet P (1999) Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbiol 65:5409–5420Google Scholar
- Kandhavelu M, Vennison SJ (2008) Persistence of plasmid DNA in different soils. Afr J Biotechnol 7:2543–2546Google Scholar
- Mićić M, Whyte JD, Karsten V (2016) High performance bead beating based lysing, homogenization and grinding for DNA, RNA and proteins extraction with FastPrep® systems, sample preparation techniques for soil, plant, and animal samples. Springer Protocols Handbooks. Humana Press, New York, pp 99–116Google Scholar
- Romanowski G, Lorenz MG, Sayler G, Wackernagel W (1992) Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay. Appl Environ Microbiol 58:3012–3019Google Scholar
- Romanowski G, Lorenz MG, Wackernagel W (1993a) Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils. Appl Environ Microbiol 59:3438–3446Google Scholar
- Romanowski G, Lorenz MG, Wackernagel W (1993b) Plasmid DNA in a groundwater aquifer microcosm-adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis. Microb Ecol 2:171–181Google Scholar
- Tuominen L, Kairesalo T, Hartikainen H (1994) Comparison of methods for inhibiting bacterial activity in sediment. Appl Environ Microbiol 60:3454–3457Google Scholar
- Vishnivetskaya TA, Layton AC, Lau MCY, Chauhan A, Cheng KR, Meyers AJ, Murphy JR, Rogers AW, Saarunya GS, Williams DE, Pfiffner SM, Biggerstaff JP, Stackhouse BT, Phelps TJ, Whyte L, Sayler GS, Onstott TC (2014) Commercial DNA extraction kits impact observed microbial community composition in permafrost samples. FEMS Microbiol Ecol 87:217–230CrossRefGoogle Scholar
- WRB (2006) World reference base for soil resources 2006. FAO/ISRIC/ISSS, ItalyGoogle Scholar