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Chapter 16 : RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils

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Abstract:

This chapter discusses RNA-radioisotope probing (RNA-RIP) for studying carbon metabolism in soils. Identifying the microorganisms driving bio-geochemical processes and how these are influenced by the physicochemical environment is the key to understand how microbial systems such as soils work. Thus, the use of stable isotope probing (SIP) has meant that the link between phylogeny and function for some metabolic processes can now be realized by coupling labeled biogeochemical tracers to the highly resolved, phylogenetic information held in the 16S rRNA and 18S rRNA of microbial cells. The most frequently used element in most SIP applications has been carbon in its stable isotope form of C. Generally, following isotopic enrichment and separation of “heavy” and “light” DNA or rRNA, the nucleic acids are further analyzed using molecular fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP) or denaturing gradient gel electrophoresis (DGGE), to identify and characterize the organisms that have assimilated the substrates. However, as DNA- and RNA-stable isotope probing (RNA-SIP) approaches have been applied to more ecologically relevant experiments, a new understanding is slowly emerging. These studies suggest that certain bacterial phyla can be differentiated into copiotrophic and oligotrophic categories that correspond to the r- and K-selected categories used to describe the ecological attributes of plants and animals.

Citation: O’Donnell A, Jenkins S, Whiteley A. 2011. RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils, p 317-332. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch16

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Restriction Fragment Length Polymorphism
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Denaturing Gradient Gel Electrophoresis
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Restriction Fragment Length Polymorphism
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Denaturing Gradient Gel Electrophoresis
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Image of FIGURE 1
FIGURE 1

Typical density gradient fraction analysis after 36 h of centrifugation using 6.4 ml volume and 1.8 g ml starting density CsTFA gradient on RNA extracted from soils amended with [C]glucose at a concentration of 150 g C g soil after 4 days of incubation. Points are means of triplicate fractions. Fraction divisions indicate the buoyant density ranges for the “heavy” fractions containing C-labeled RNA (solid lines), “light” fractions containing C-unlabeled RNA (dotted lines), and “intermediate” fractions containing C/C-labeled RNA (dashed lines).

Citation: O’Donnell A, Jenkins S, Whiteley A. 2011. RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils, p 317-332. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch16
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Image of FIGURE 2
FIGURE 2

Autoradiograph and plot of spot intensity showing distribution of C-labeled RNA over the first 20 density gradient fractions after 36 h of centrifugation. RNA was extracted from soils amended with [C]glucose at a concentration of 150 g C g soil after 4 days of incubation. RNA from each fraction was spotted onto a membrane and exposed to X-ray film for 2 to 3 weeks. Arrows show the fraction ranges for the “heavy” fractions (3 to 7) containing C-labeled RNA, “light” fractions (15 to 17) containing C-unlabeled RNA, and “intermediate” fractions (11 to 14) containing C/C-labeled RNA.

Citation: O’Donnell A, Jenkins S, Whiteley A. 2011. RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils, p 317-332. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch16
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Image of FIGURE 3
FIGURE 3

Distribution of 16S rRNA over the first 20 density gradient fractions after 36 h of centrifugation, estimated using real time qPCR. RNA was extracted from soils amended with [C]glucose at a concentration of 150 g C g soil after 4 days of incubation. RNA from each fraction was reverse transcribed and quantified. Arrows show the fraction ranges for the “heavy” fractions (3 to 7) containing C-labeled RNA, “light” fractions (15 to 17) containing C-unlabeled RNA, and “intermediate” fractions (11 to 14) containing C/C-labeled RNA.

Citation: O’Donnell A, Jenkins S, Whiteley A. 2011. RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils, p 317-332. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch16
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Image of FIGURE 4
FIGURE 4

RT-PCR-DGGE gels comparing the bacterial community profiles recovered from the first 20 fractions along the RNA-SIP density gradient after 36 h of centrifugation. RNA was extracted from soil in microcosms amended with different concentrations of [C]glucose (50 g C g soil) after 4 days of incubation. The “heavy” fractions (3 to 7) containing the C-labeled RNA, the “light” fractions (15 to 17) containing the C-unlabeled RNA, and the “intermediate” fractions (11 to 14) containing C/C-labeled RNA are shown. M indicates the marker lane, and numbers represent fraction number. Boxes indicate the bands that were excised from the gel, reamplified, and sequenced.

Citation: O’Donnell A, Jenkins S, Whiteley A. 2011. RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils, p 317-332. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch16
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Image of FIGURE 5a
FIGURE 5a

Histogram showing the relative band intensity for DGGE-derived 16S rRNA ribotypes recovered in both the “heavy” and “light” fractions. RNA was extracted from soil in microcosms amended with different concentrations of [C]glucose (0, 15, 50, and 150 g C g soil) after 4 days of incubation. RT-PCR-DGGE comparing the bacterial community profiles was then performed on the first 20 fractions along the RNA-SIP density gradient after 36 h of centrifugation. The “heavy” fractions (3 to 7) containing the C-labeled RNA and the “light” fractions (15 to 17) containing the C-unlabeled RNA are shown. M indicates the marker lane, and numbers represent fraction number. Bands showing marked changes in intensity were excised from the gel, reamplified, and sequenced.

Citation: O’Donnell A, Jenkins S, Whiteley A. 2011. RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils, p 317-332. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch16
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Image of FIGURE 5b
FIGURE 5b

Histogram showing the relative band intensity for DGGE-derived 16S rRNA ribotypes recovered in both the “heavy” and “light” fractions. RNA was extracted from soil in microcosms amended with different concentrations of [C]glucose (0, 15, 50, and 150 g C g soil) after 4 days of incubation. RT-PCR-DGGE comparing the bacterial community profiles was then performed on the first 20 fractions along the RNA-SIP density gradient after 36 h of centrifugation. The “heavy” fractions (3 to 7) containing the C-labeled RNA and the “light” fractions (15 to 17) containing the C-unlabeled RNA are shown. M indicates the marker lane, and numbers represent fraction number. Bands showing marked changes in intensity were excised from the gel, reamplified, and sequenced.

Citation: O’Donnell A, Jenkins S, Whiteley A. 2011. RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils, p 317-332. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch16
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Image of FIGURE 6
FIGURE 6

Relative band intensity and distribution of the DGGE 16S rRNA ribotypes over the first 20 fractions. DGGE 16S rRNA ribotypes found in the “heavy” fractions (3 to 7) contained the C-labeled RNA (black solid lines), while ribotypes found only in the “light” fractions (15 to 17) contained the C-unlabeled RNA (black dashed lines). RNA was extracted from soil microcosms amended with different concentrations of [C]glucose (0, 15, 50, and 150 g C g soil) after 4 days of incubation. RT-PCR-DGGE comparing the bacterial community profiles was then performed on the first 20 fractions along the RNA-SIP density gradient after 36 h of centrifugation. Bands showing marked changes in intensity were excised from the gel, reamplified, and sequenced.

Citation: O’Donnell A, Jenkins S, Whiteley A. 2011. RNA-Radioisotope Probing for Studying Carbon Metabolism in Soils, p 317-332. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch16
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