Chapter 2 : RNA Stable Isotope Probing

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $15.00

Preview this chapter:
Zoom in

RNA Stable Isotope Probing, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816896/9781555815370_Chap02-1.gif /docserver/preview/fulltext/10.1128/9781555816896/9781555815370_Chap02-2.gif


This chapter focuses on RNA-based stable isotope probing (RNA-SIP) that was developed to take advantage of the features of RNA that make it an excellent biomarker for linking environmental processes—rapid turnover rates independent of cellular replication, coupled to in-depth phylogenetic information within the molecule itself. The primary aim of an RNA extraction protocol for RNA-SIP is to generate over 1µg of quantifiable RNA. Nucleic acids appear in almost all gradient fractions as revealed by high-sensitivity methods for detecting them, such as reverse transcription PCR (RT-PCR) or PCR. The chapter also talks about the RNA-SIP- directed investigation that ultimately led to the isolation of a novel strain responsible for the observed phenol degradation and to confirmation that it could be used to revive sludge that had lost phenol-degrading activity. These findings changed one's understanding of the microbes responsible for phenol degradation in aerated sludge, revealed the pitfalls of both conventional culturing and basic molecular approaches, and highlighted the importance of methods linking function with phylogeny. Manefield used RNA-SIP to compare the communities dominating the assimilation of carbon from phenol in near-identical wastewater treatment bioreactors that were operated in the same manner but differed in wastewater treatment performance. This study revealed that species were responsible for phenol degradation and the poor performance of one reactor was associated with two different populations, while the strong performance of the other was associated with a single dominant lineage.

Citation: Manefield M, Gutierrez-Zamora M, Whiteley A. 2011. RNA Stable Isotope Probing, p 25-36. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch2

Key Concept Ranking

Denaturing Gradient Gel Electrophoresis
Microbial Ecology
Bacteria and Archaea
Environmental Microbiology
Restriction Fragment Length Polymorphism
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of FIGURE 1.

Citations of the original publication of the RNA-SIP method applied to the identification of bacteria dominating the assimilation of carbon from phenol in an industrial wastewater treatment plant located in northeast England. Over seven years the manuscript has been cited a total of 165 times (source, ISI Web of Knowledge).

Citation: Manefield M, Gutierrez-Zamora M, Whiteley A. 2011. RNA Stable Isotope Probing, p 25-36. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2.

Conceptual relationship between apparent distribution of nucleic acids in equilibrium density gradients using methods with different levels of sensitivity. (A) Apparent distribution of nucleic acids in equilibrium density centrifuge tube if a method with low sensitivity is used (e.g., ethidium bromide staining of nucleic acids within the centrifuge tube). (B) Actual distribution of labeled and unlabeled nucleic acids in equilibrium density gradients as determined by high-sensitivity detection methods (e.g., quantitative PCR or RTPCR). (C) Apparent distribution of nucleic acids in equilibrium density gradient using a detection method with moderate sensitivity (e.g., concentration of nucleic acids by precipitation followed by agarose gel electrophoresis with SYBR gold staining).

Citation: Manefield M, Gutierrez-Zamora M, Whiteley A. 2011. RNA Stable Isotope Probing, p 25-36. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch2
Permissions and Reprints Request Permissions
Download as Powerpoint


1. Addison, S. L.,, I. R. McDonald, and, G. Lloyd-Jones. 2009. Stable isotope probing: technical considerations when resolving 15N2-labeled RNA in gradients. J. Microbiol. Methods 80:7075.
2. Bastias, B. A.,, I. C. Anderson,, J. I. Rangel-Castro,, P. I. Parkin,, J. I. Prosser,, J. W. G. Cairney. 2009. Influence of repeated prescribed burning on incorporation of 13C from cellulose by forest soil fungi as determined by RNA stable isotope probing. Soil Biol. Biochem. 41:467472.
3. Brinkmann, N.,, R. Martens, and, C. C. Tebbe. 2008. Origin and diversity of metabolically active gut bacteria from laboratory-bred larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta). Appl. Environ. Microbiol. 74:71897196.
4. Degelmann, D. M.,, S. Kolb,, M. Dumont,, J. C. Murrell, and, H. L. Drake. 2009. Enterobacteriaceae facilitate the anaerobic degradation of glucose by a forest soil. FEMS Microbiol. Ecol. 68:312319.
5. Frias-Lopez, J.,, A. Thompson,, J. Waldbauer, and, S. W. Chisholm. 2009. Use of stable isotope-labeled cells to identify active grazers of picocyanobacteria in ocean surface waters. Environ. Microbiol. 11:512525.
6. Glaubitz, S.,, T. Lueders,, W. R. Abraham,, G. Jost,, K. Jürgens, and, M. Labrenz. 2009. 13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea. Environ. Microbiol. 11:326337.
7. Griffiths, R.,, M. Manefield,, N. Ostle,, N. Mc-Namara,, A. G., O'Donnel,, M. J. Bailey,, A. S. Whiteley. 2004. 13CO2 pulse labelling of plants in tandem with stable isotope probing: methodological considerations for examining microbial function in the rhizosphere. J. Microbiol. Methods 58:119129.
8. Griffiths, R. I.,, A. S. Whiteley,, A. G. O'Donnell, and, M. J. Bailey. 2000. Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl. Environ. Microbiol. 66:54885491.
9. Hamberger, A.,, M. A. Horn,, M. G. Dumont,, J. C. Murrell, and, H. L. Drake. 2008. Anaerobic consumers of monosaccharides in a moderately acidic fen. Appl. Environ. Microbiol. 74:31123120.
10. Hatamoto, M.,, H. Imachi,, Y. Yashiro,, A. Ohashi, and, H. Harada. 2008. Detection of active butyrate-degrading microorganisms in methanogenic sludges by RNA-based Stable isotope probing. Appl. Environ. Microbiol. 74:36103614.
11. Huang, W. E.,, A. Ferguson,, A. C. Singer,, K. Lawson,, I. P. Thompson,, R. M. Kalin,, M. J. Larkin,, M. J. Bailey, and, A. S. Whiteley. 2009a. Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell Raman-fluorescence in situ hybridization. Appl. Environ. Microbiol. 75:234241.
12. Huang, W. E.,, A. Ward, and, A. Whiteley. 2009b. Raman tweezers sorting of single microbial cells. Environ. Microbiol. Rep. 1:4449.
13. Kasai, Y.,, Y. Kodama,, Y. Takahata,, T. Hoaki, and, K. Watanabe. 2007. Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11. Environ. Sci. Technol. 41:62226227.
14. Kasai, Y.,, Y. Takahata,, M. Manefield, and, K. Watanabe. 2006. RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater. Appl. Environ. Microbiol. 72:35863592.
15. Kittelmann, S., and, M. W. Friedrich. 2008. Novel uncultured Chloroflexi dechlorinate perchloroethene to trans-dichloroethene in tidal flat sediments. Environ. Microbiol. 10:15571570.
16. Kovatcheva-Datchary, P.,, M. Egert,, A. Maathuis,, M. Rajili-Stojanovi,, A. A. de Graaf,, H. Smidt,, W. M. De Vos, and, K. Venema. 2009. Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing. Environ. Microbiol. 11:914926.
17. Langenheder, S., and, J. I. Prosser. 2008. Resource availability influences the diversity of a functional group of heterotrophic soil bacteria. Environ. Microbiol. 10:22452256.
18. Lear, G.,, B. Song,, A. G. Gault,, D. A. Polya, and, J. R. Lloyd. 2007. Molecular analysis of arsenate-reducing bacteria within Cambodian sediments following amendment with acetate. Appl. Environ. Microbiol. 73:10411048.
19. Lueders, T.,, M. Manefield, and, M. W. Friedrich. 2004. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ. Microbiol. 6:7378.
20. MacGregor, B. J., and, R. Amann. 2006. Single-stranded conformational polymorphism for separation of mixed rRNAS (rRNA-SSCP), a new method for profiling microbial communities. Syst. Appl. Microbiol. 29:661670.
21. Manefield, M.,, R. I. Griffiths,, M. B. Leigh,, R. Fisher, and, A. S. Whiteley. 2005. Functional and compositional comparison of two activated sludge communities remediating coking effluent. Environ. Microbiol. 7:715722.
22. Manefield, M.,, A. S. Whiteley,, R. I. Griffiths, and, M. J. Bailey. 2002b. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl. Environ. Microbiol. 68:53675373.
23. Manefield, M.,, A. S. Whiteley,, N. Ostle,, P. Ineson, and, M. J. Bailey. 2002a. Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function. Rapid Commun. Mass Spectrom. 16:21792183.
24. Monard, C.,, F. Binet, and, P. Vandenkoornhuyse. 2008. Short-term response of soil bacteria to carbon enrichment in different soil microsites. Appl. Environ. Microbiol. 74:55895592.
25. Noll, M.,, P. Frenzel, and, R. Conrad. 2008. Selective stimulation of type I methanotrophs in a rice paddy soil by urea fertilization revealed by RNA-based stable isotope probing. FEMS Microbiol. Ecol. 65:125132.
26. Ostle, N.,, A. S. Whiteley,, M. J. Bailey,, D. Sleep,, P. Ineson, and, M. J. Manefield. 2003. Microbial Turnover in a grassland soil using a CO2 'spike.' Soil Biol. Biochem. 35:877885.
27. Radajewski, S.,, P. Ineson,, N. R. Parekh, and, J. C. Murrell. 2000. Stable-isotope probing as a tool in microbial ecology. Nature 403:646649.
28. Rangel-Castro, J. I.,, K. Killham,, N. Ostle,, G. W. Nicol,, I. C. Anderson,, C. M. Scrimgeour,, P. Ineson,, A. Meharg, and, J. I. Prosser. 2005. Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms. Environ. Microbiol. 7:828838.
29. Sapp, M.,, G. Gerdts,, M. Wellinger, and, A. Wichels. 2008. Consuming algal products: trophic interactions of bacteria and a diatom species determined by RNA stable isotope probing. Helgoland Marine Res. 62:283287.
30. Sueoka, K.,, S. Hiroyasu,, O. Motoharu, and, M. Takashi. 2009. Microorganisms involved in anaerobic phenol degradation in the treatment of synthetic coke-oven wastewater detected by RNA stable-isotope probing. FEMS Microbiol. Lett. 291:169174.
31. Whiteley, A. S.,, B. Thomson,, T. Lueders, and, M. Manefield. 2007. RNA stable-isotope probing. Nature Protocols 2:838844.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error