Stable-isotope probing as a tool in microbial ecology

Abstract

Microorganisms are responsible for driving the biogeochemical cycling of elements on Earth. Despite their importance and vast diversity, the taxonomic identity of the microorganisms involved in any specific process has usually been confined to that small fraction of the microbiota that has been isolated and cultivated. The recent coupling of molecular biological methods with stable-isotope abundance in biomarkers has provided a cultivation-independent means of linking the identity of bacteria with their function in the environment. Here we show that 13C-DNA, produced during the growth of metabolically distinct microbial groups on a 13C-enriched carbon source, can be resolved from 12C-DNA by density-gradient centrifugation. DNA isolated from the target group of microorganisms can be characterized taxonomically and functionally by gene probing and sequence analysis. Application of this technique to investigate methanol-utilizing microorganisms in soil demonstrated the involvement of members of two phylogenetically distinct groups of eubacteria; the alpha-proteobacterial and Acidobacterium lineages. Stable-isotope probing thus offers a powerful new technique for identifying microorganisms that are actively involved in specific metabolic processes under conditions which approach those occurring in situ.

Targeted metagenomics of active microbial populations with stable-isotope probing

“…Since its development, DNA-SIP [10,11] has been a key technique linking the assimilation 55 of particular growth substrates with specific populations of microorganisms, regardless of our ability to cultivate these microbes in the laboratory. The recovery of genomes from microorganisms that were active under selected study conditions is a major benefit of this method.…”

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact

“…DNA stable-isotope probing (DNA-SIP) can reveal phylogenetic identity of previously unknown and uncultivated organisms that are metabolically active in a particular ecosystem (Radajewski et al, 2000;Dumont and Murrell, 2005). DNA-SIP successfully identified active methanotrophs and methylotrophs in Transbaikal soda lake sediments (Lin et al, 2004), Washington fresh water lake sediments (Nercessian et al, 2005), Colne estuary sediments (Moussard et al, 2009) and alkaline soils (pH 9) from a Chinese coal mine (Han et al, 2009).…”

The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation

“…One possible reason for this lack of success may be an inadequate mimicking of in situ growth requirements during isolation, which has motivated us to apply a culture-independent technique, nucleic acid-based stable isotope probing (SIP) for the identification of key microbes involved in degradation. SIP involves the incorporation of 13 C-label into cellular biomarkers such as nucleic acids, followed by density gradient separation of labeled nucleic acids and molecular identification of the involved microbes (Radajewski et al, 2000). Today, the SIP technique has greatly increased our understanding of the role of specific microbial community members in diverse environmental settings (Madsen, 2006) and identified key microbes utilizing various aromatic and chlorinated hydrocarbons (Manefield et al, 2002;Jeon et al, 2003;Padmanabhan et al, 2003;Mahmood et al, 2005;Yu and Chu, 2005;Kasai et al, 2006).…”