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Applications  |  Ecology: Functional Microbes (DNA-SIP)


Identifying functional microbial species using Stable Isotope Probing.

Introduction

Microbial species each have specific functions in ecosystems, e.g. in nutrient cycling. Identification of micro-organisms is often difficult because only a minority of the species within the system will grow on artificial media. DNA-techniques help to overcome this problem and to extend our knowledge on species diversity because non-culturable species are also detected. However, both techniques still have a major disadvantage: they cannot distinguish between active and dormant organisms, i.e. they do not tell which species are functionally active in specific processes. Stable isotopes like 13C and 15N help to tackle this problem.

Stable Isotope Solution : Stable Isotope Probing

To identify organisms involved in specific processes, their favourite substrate is supplied labelled with a stable isotope. For instance, 13C-labelled components like leaves, roots, lignin, cellulose etc. amended to (natural) growth media (like soil) can be provided as substrate. Micro-organisms actively growing on this substrate will multiply and produce intra- and extracellular end products.

13C-labelled substrate → microbial turnover → 13C-end products

Detection of species-specific end products like 13C-PFLA's (Boschker et al, 1998) and 13C-DNA (Radajewski et al, 2000) or less specific compounds like 13C-CO2 (Raghoebarsing et al, 2005) may help to identify the active species involved in certain processes.

Typical Result

After growing a mix of different microbial species on a simple 13C-labelled substrate like 13C-methanol, the 13C DNA produced by methanol utilising species can be clearly separated from the 12C DNA originating from species unable to utilize methanol. After DNA extraction from the growth medium, the newly formed ('heavy') 13C DNA can be separated from the ('light') 12C DNA by density-gradient centrifugation (Figure 1). The 13C DNA can then be identified by comparing with DNA libraries and subsequently linked to the active microbial species. This method is called Stable Isotope Probing (DNA-SIP).

Figure 1Figure 1. Equilibrium centrifugation of isotopically labelled DNA in CsCl/ethidium bromide density gradients. Pure fractions and a mixture of the DNA extracted from a Methylobacterium extorquens AM155 culture utilizing either 12C- or 13C-methanol as the sole carbon source
(redrawn after Radajewski et al., 2000).

Thanks to the application of 13C-labelled methane as a substrate, Raghoebarsing et al. (2005) could demonstrate that methane produced in peat bogs is recovered by methanotrophic symbiotic bacteria, providing CO2 for photosynthesis by Sphagnum mosses.

Figure 2

The same principle as shown in the examples above applies to much more complex 13C-labelled substrates like cellulose, lignin or even whole plant parts. Application of stable isotope probing (DNA-SIP, RNA-SIP) in ecology or nutrition has a large potential to link specific micro-organisms to metabolic and ecosystem processes.

References

Boschker HTS, SC Nold, P Wellsbury, D Bos, W de Graaf, R Pel, RJ Parkes, TE Cappenberg. 1998.
Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers.
Nature 392, 801-805.

Radajewski S, P Ineson, NR Parekh, JC Murrell. 2000.
Stable-isotope probing as a tool in microbial ecology.
Nature 403: 646-649.

Raghoebarsing AA, AJP Smolders, MC Schmid, WIC Rijpstra, M Wolters-Arts, J Derksen, MSM Jetten, S Schouten, JSS Damste, LPM Lamers, JGM Roelofs, H op den Camp, M Strous. 2005.
Methanotrophic symbionts provide carbon for photosynthesis in peat bogs.
Nature 436: 1153-1156.

 

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