Comparison of rRNA and polar-lipid-derived fatty acid biomarkers for assessment of 13C-substrate incorporation by microorganisms in marine sediments

Abstract

We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in 13C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of 13C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the 13C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the 13C-labeled compounds. Similar results were obtained with the 13C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the 13C analysis of the captured SSU rRNA.

FIG. 1.

Examples of helper probes used for bead capture hybridization. The unlabeled upstream and downstream Bact338 helper probes (Table 1) were used at a 1:1:1 ratio with the biotin-labeled Bact338 capture probe. Note that the gill samples included both prokaryotic and eukaryotic rRNAs; Bathymodiolus azoricus gills harbor symbiotic bacteria (7).

FIG. 2.

(A) Examples of sediment-extracted total RNA. The RNA in each of the sample lanes represents approximately 7.5 mg of sediment, out of a total of approximately 30 g. The pure-culture reference RNAs were Saccharomyces cerevisiae RNA (S.c.) and Escherichia coli RNA (E.c.) (approximately 200 ng each). (B) Examples of magnetic bead capture hybridization with the Bact338 and Euk1379 probes. LSU, long subunit; euk., eukaryote; prok., prokaryote; LMW, low molecular weight.

FIG. 3.

PLFA concentrations. The values are averages ± standard deviations for all samples (n = 16). DW, dry weight; PUFA, polyunsaturated fatty acid.

FIG. 4.

Labeling of rRNA and pool average PLFA expressed as Δδ¹³C values. (A and B) Results of the [¹³C]acetate time series for bacterial (A) and eukaryotic (B) markers. (C and D) Comparison of different ¹³C-labeled substrates. Bact, bacterial; Euk, eukaryotic.

FIG. 5.

¹³C incorporation into two main bacterial PLFA, as determined in the [¹³C]acetate time series experiment. DW, dry weight.

FIG. 6.

Percentage of the added label incorporated into bacterial PLFA and bacterial rRNA for the [¹³C]acetate time series (A) and for the different substrates after 6 h (B). Note the difference the in y axis scales. Bact, bacterial.