title: 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.
authors.affiliation: Physical and Life Science Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA. mayali1@llnl.gov
authors.forename: Xavier
authors.initials: X
authors.lastname: Mayali
authors.forename: Peter K
authors.initials: PK
authors.lastname: Weber
authors.forename: Eoin L
authors.initials: EL
authors.lastname: Brodie
authors.forename: Shalini
authors.initials: S
authors.lastname: Mabery
authors.forename: Paul D
authors.initials: PD
authors.lastname: Hoeprich
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pett-Ridge
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D002247:Carbon Isotopes / Q000032:analysis; D063366:Estuaries; D023281:Genomics; D057166:High-Throughput Screening Assays; D009587:Nitrogen Isotopes / Q000032:analysis; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010802:Phylogeny; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D015203:Reproducibility of Results; D014871:Water Microbiology
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S