RNA Stable Isotope Probing, a Novel Means of Linking Microbial Community Function to Phylogeny

Abstract

Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [ 13 C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13 C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus. Our results suggest that this organism is important for the function of this bioreactor.Our dependence upon laboratory-based studies of culturable bacterial species is acknowledged to be a primary limitation to progress in microbial ecology (2). Traditionally, it has been difficult to attribute a recognized microbially mediated process to the organism(s) responsible for that process in situ (12). Recently, however, culture-independent approaches to linking microbial community function with the genetic identity of key organisms have begun to emerge (7,18,23). Among the limited number of elegant methodologies capable of identifying microorganisms responsible for particular biogeochemical processes, stable-isotope probing (SIP) holds considerable promise.SIP involves the incorporation of stable-isotope-labeled substrates into cellular biomarkers that can be used to identify organisms assimilating the substrate (6). Stable isotopes were first used in this capacity to identify the microbial community component responsible for acetate oxidation in aquatic sediments (7). A 13 C-labeled acetate pulse was administered to sediments, with the resulting 13 C-enriched polar-lipid-derived fatty acid (PLFA) signature profiles subsequently being preferentially analyzed. Other studies utilizing the same approach have followed suit (9, 24).The utility of PLFA-based SIP may be limited, because resolving PLFA profiles composed of multiple species can be problematic (26). This limitation has been overcome with the demonstration that stable-isotope-labeled DNA can be recovered from total community nucleic acid extractions on the basis of its increased buoyant density and can be used as an alternative, unambiguous biomarker (21, 26, 32).Because DNA-based SIP relies on the isolation of labeled DNA by density centrifugation, the degree of isotopic enrichment is crucial. Of the many factors determining the success of an enrichment, including the duration of the pulse and the presence of unlabeled substrate inherent to the system, the rate of DNA synthesis in situ plays a pivotal role. We speculate that DNA synthesi...

Shifts in soil biodiversity—A forensic comparison between Sus scrofa domesticus and vegetation decomposition

“…Triplicate PCR amplifications used GC 388F -530R to target the 16S rRNA gene [25] and the nested primer sets NS1/NS8 and NS1/NS210-GC for the 18S rRNA gene [26]. Subsequent to checking on 1.5% (w/v) agarose gels as described above, the amplicons (20 μl) were separated on 10% (w/v) polyacrylamide gels (acrylamide/bisacrylamide gel 37.5:1) with a 30% to 70% denaturing gradient for the 16S rRNA, and 6% (w/v) polyacrylamide gels with a 25% to 45% denaturing gradient for the 18S rRNA with a PHOR-U (X2) Ingeny system (Leiden, Netherlands) at 60 °C and 110 V for 18 hours.…”

Changes to soil bacterial profiles as a result of Sus scrofa domesticus decomposition

“…The V3 region of the bacterial 16S rRNA gene from position 356 to 519 was targeted for amplification according to Manefield et al (2002) [23], with the forward primer …”

Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures

“…The metabolically active species assimilate the labelled substrate, which can then be separated from the non-labelled biomass by ultracentrifugation. By this approach, the metabolically active species can be identified using DNA (Friedrich, 2006) or RNA (Manefield et al, 2002;Whiteley et al, 2006) as target compounds. The drawbacks of this technique are the low resolution of the labelled and non-labelled DNA/RNA during ultracentrifugation and thereby the difficulty to quantify the uptake of the stable isotopes into DNA/RNA.…”