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 [13C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-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.

FIG. 1.

RNA-SIP analysis of a defined, mixed bacterial culture. (A) GC-clamped 16S rRNA (V3 region) RT-PCR products from pure cultures of a phenol-degrading isolate (P. putida; lane 1) and a nondegrading isolate (P. chlororaphis; lane 2) on a denaturing gradient gel. (B) RT-PCR products from fractions 5, 7, 9, and 11 (lanes 1 to 4, respectively) of an equilibrium density gradient loaded with RNA extracted at 24 h from a mixed P. putida, P. chlororaphis culture grown on phenol-¹³C₆. RNA from both species was detected in fractions 9 and 11 (associated by buoyant density with unlabeled RNA). The 16S rRNA sequence of P. putida alone was detectable in fractions 5 and 7 (¹³C-labeled RNA), demonstrating specific access to ¹³C according to metabolic activity. (C) RT-PCR products from fractions 5, 7, 9, and 11 (lanes 1 to 4, respectively) of a gradient loaded with RNA extracted after 72 h from the mixed culture. RNA from both species was detectable in all four fractions, indicative of ¹³C cross-feeding between species. These results are representative of two separate experiments. Lanes labeled M were loaded with an unrelated DGGE marker.

FIG. 2.

Degradation of phenol-¹³C₆ and increase in ¹³C atom percent of RNA and DNA in the microbial community from an industrial bioreactor. Bioreactor samples were pulsed with 500 μg of phenol-¹³C₆/ml and were assessed hourly for phenol concentration (triangles). The phenol concentration remained constant over the initial 2 h but then dropped rapidly over the following 5 h. A reduced phenol degradation rate preceded the complete removal of the pulse by 8 h. Samples were assayed for phenol in triplicate. Error bars represent standard errors. These data are representative of three separate experiments. Total community DNA and RNA samples were extracted at 0, 1, 2, 4, and 8 h and were subjected to IRMS analysis. The ¹³C atom percent of RNA (•) increased more than 10-fold as the labeled phenol pulse was utilized, while that of DNA (▪) increased just over 2-fold. Nucleic acid samples were analyzed in triplicate from a single experiment. Error bars representing standard deviation were too small to be visible.

FIG. 3.

RNA-SIP profiles relating community function to specific microbial community components. DGGE profile of community RNA present in fractions 4 to 13 from equilibrium density gradients loaded with RNA extracted 1 (A) and 8 h (B) after the phenol-¹³C₆ pulse was administered. Lane numbers correspond to fraction numbers. Five of the most dominant bands are labeled A to E (B). After 1 h the profile suggests that RNA from all detectable species have the same buoyant density prior to phenol degradation. After 8 h the profile suggests that RNA from particular community members (bands A, B, and D) have increased in buoyant density subsequent to phenol degradation. Lanes labeled M were loaded with an unrelated DGGE marker.

FIG. 4.

Increasing buoyant density of the dominant band in 16S rRNA community profiles throughout the duration of the phenol-¹³C₆ pulse. (A) The intensity of the dominant DGGE band remains constant in fraction 10 over time. (B) The intensity of the dominant DGGE band increases in fraction 6 over time, confirming an increase in the buoyant density of RNA from this species as phenol-¹³C₆ is consumed.