High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use

Abstract

Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation--and therefore substrate use--by secondary ion mass spectrometer imaging (NanoSIMS). Laboratory experiments demonstrated that Chip-SIP can detect isotopic enrichment of 0.5 atom % (13)C and 0.1 atom % (15)N, thus permitting experiments with short incubation times and low substrate concentrations. We applied Chip-SIP analysis to a natural estuarine community and quantified amino acid, nucleic acid or fatty acid incorporation by 81 distinct microbial taxa, thus demonstrating that resource partitioning occurs with relatively simple organic substrates. The Chip-SIP approach expands the repertoire of stable isotope-enabled methods available to microbial ecologists and provides a means to test genomics-generated hypotheses about biogeochemical function in any natural environment.

Figure 1

(a) Fluorescence image and (b) NanoSIMS ¹³C isotope ratio image montage of an ITO microarray hybridized with RNA from a single bacterial strain (Pseudomonas stutzeri) grown on ¹³C glucose; (c) the relationship between fluorescence and enrichment for each probe spot (referred to as ‘hybridization corrected enrichment', HCE) is significant and different between RNA from cultures with 100% ¹³C (blue diamonds)- versus 5% ¹³C-enriched cultures (red squares). Units for the NanoSIMS color scale are per mil (‰), and range from natural abundance (black) to highly enriched (white).

Figure 2

Chip-SIP analysis of mixtures of two bacterial strains labeled with differential isotopic enrichment; HCE, hybridization-corrected enrichment (slope of per mil enrichment and fluorescence). Each point represents an individual probe spot's fluorescence intensity value plotted against its isotopic enrichment. Comparisons included dual labeled V. cholera and B. cereus (a, b), ¹⁵N labeled P. stutzeri and unlabeled B. cereus (c), and differentially ¹³C labeled V. cholerae and P. stutzeri (d).

Figure 3

Substrate incorporation detected by Chip-SIP for a SF Bay estuarine microbial community incubated with ¹⁵N ammonium and ¹³C glucose for 24 h; (a) ammonium and (c) glucose incorporation for 3 taxa within the family Rhodobacteriaceae; each point is derived from a single probe spot's isotopic enrichment value plotted against fluorescence; (panel c) relationship between ammonium and glucose incorporation for 16 taxa from 2 bacterial families; HCE, hybridization-corrected enrichment; arrows indicate taxa plotted in panels a and b.

Figure 4

Chip-SIP analysis of the uptake patterns of three organic substrates by different bacterial taxa in SF Bay, identifying substrate specialists and generalists; the thicknesses of the lines are proportional to the substrate incorporation rates based on HCE calculations (Flavo, Flavobacteriaceae; Roseo, Roseobacter; MarGrpA, Marine Group A; Sphingo, Sphingobacteria; Cyano, Cyanobacteria).

Figure 5

Relationship between substrate incorporation and 16S rRNA phylogeny for SF Bay taxa (AA, amino acids; FA, fatty acids; NA, nucleic acids); GenBank Accession numbers for targeted taxa are included in parentheses.