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

Abstract

Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope ((13)C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 muM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.

FIG. 1.

Degradation of [¹²C]- and [¹³C]naphthalene (3.8 μM) in flasks inoculated with groundwater. Open squares and triangles represent [¹²C]- and [¹³C]naphthalene degradation, respectively; open diamonds and circles represent filter-sterilized groundwater with and without naphthalene, respectively. Naphthalene was completely degraded within 72 h, as analyzed by gas chromatography-mass spectrometry, and biological confirmation of this was provided by salicylate (metabolic intermediate) accumulation after 40 h, as determined by using a lux-based salicylate-specific bacterial biosensor (inset). In the inset, triangles and circles represent the groundwater sample with [¹³C]naphthalene and filtered sterilized groundwater with naphthalene, respectively.

FIG. 2.

Digitized DGGE profiles showing the effect of 36 h of exposure to [¹²C]naphthalene upon 16S rRNA community profiles and comparison to cultured isolates obtained from the incubations. (A) Native groundwater; (B to D) 2-log range of amended naphthalene (μM); (E to G) pure-isolate 16S rRNA profiles for comparison to in situ community structures. For comparisons, open circles represent the band peaks for the key low-affinity degrader P. fluorescens WH2 and the filled circles represent the peak obtained from RNA SIP at the ambient concentration of naphthalene and equates to that for the Acidovorax sp. high-affinity degrader.

FIG. 3.

RT-PCR of a fractionated RNA SIP gradient after exposing groundwater communities to 3.8 μM [¹³C₁₀]naphthalene for 36 h and using Comamonas-type NDO gene primers. The RT-PCR control shows that there was no PCR product without reverse transcription. Positive reactions were obtained in both light RNA fractions (fractions 11 and 12), representing unlabeled RNAs, and in heavy fractions (fractions 6 and 7), derived from ¹³C-labeled mRNA transcripts after substrate metabolism. Fraction 6 represents the lower portion of the centrifuge tube, occupying buoyant densities of around 1.83 g ml⁻¹, while fraction 11 represents the upper portion of the gradient, occupying buoyant densities around 1.79 g ml⁻¹. C represents a positive control for a Comamonas-type NDO.

FIG. 4.

RT-PCR analysis of NDO mRNA expression in 1 μg total RNA extracted from groundwater incubations with a range of amended naphthalene. Comamonas-type NDO expression was consistently detected across all amendments and within native groundwater (GW), whereas Pseudomonas-type NDOs were expressed only at higher concentrations of amended naphthalene (30 μM and higher). In each row, +C was an appropriate positive control for each primer set and −C1 and −C2 were RT-PCR-negative controls.

FIG. 5.

FISH images of total bacterial cells in groundwater. (A) Cells hybridized with EUB338 (false-colored purple); (B) specifically probed subpopulations. For specific subpopulations Acidovorax sp. cells were hybridized with a Cy3-labeled probe (red) and Pseudomonas sp. cells were hybridized with a fluorescein isothiocyanate probe (green). Scale bar, 10 μm. (C) Atom% ¹³C incorporation into individual cells, calculated using Raman peak shift measures (see Fig. S6B in the supplemental material) under two [¹³C]naphthalene labeling concentrations. The dashed line represents the baseline values derived from [¹²C]naphthalene calibrations versus spectral shifts performed upon cultured isolates and represents the baseline for the analysis. ¹³C calibrations were also performed against Raman peak shift values obtained for cultures grown under a range of naphthalene concentrations as in the supplementary information. Numbers indicated within the plots represent the numbers of cells analyzed per treatment for each Acidovorax or pseudomonad subpopulation delimited by specific FISH probes.