title: Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry.
abstract: Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and (14)C/(15)N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean.
authors.affiliation: Department of Oceanography, Texas A&M University, College Station, Texas, USA allison.mcinnes@uwa.edu.au.
authors.forename: Allison S
authors.initials: AS
authors.lastname: McInnes
authors.affiliation: Department of Oceanography, Texas A&M University, College Station, Texas, USA.
authors.forename: Alicia K
authors.initials: AK
authors.lastname: Shepard
authors.affiliation: The Oceans Institute, University of Western Australia, Crawley, WA, Australia.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Raes
authors.affiliation: The Oceans Institute, University of Western Australia, Crawley, WA, Australia The School of Civil, Environmental and Mining Engineering, University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Anya M
authors.initials: AM
authors.lastname: Waite
authors.affiliation: Department of Oceanography, Texas A&M University, College Station, Texas, USA Department of Marine Biology, Texas A&M University at Galveston, Galveston, Texas, USA.
authors.forename: Antonietta
authors.initials: A
authors.lastname: Quigg
mesh_terms: D057486:Carbon Cycle; D004783:Environmental Microbiology; D005434:Flow Cytometry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D009584:Nitrogen / Q000378:metabolism; D009586:Nitrogen Fixation; D010088:Oxidoreductases / Q000032:analysis* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis* / Q000235:genetics
chemical_list: D010088:Oxidoreductases; C029130:nitrogenase reductase; D012273:Ribulose-Bisphosphate Carboxylase; D009584:Nitrogen