Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry

McInnes, AS; Shepard, AK; Raes, EJ; Waite, AM; Quigg, A

Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry

McInnes, AS

Shepard, AK Raes, EJ Waite, AM Quigg, A

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Publication Type:: Journal Article
Citation:: Applied and Environmental Microbiology, 2014, 80 (21), pp. 6750 - 6759
Issue Date:: 2014-01-01

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Field	Value	Language
dc.contributor.author	McInnes, AS https://orcid.org/0000-0002-8340-3133	en_US
dc.contributor.author	Shepard, AK	en_US
dc.contributor.author	Raes, EJ	en_US
dc.contributor.author	Waite, AM	en_US
dc.contributor.author	Quigg, A	en_US
dc.date.issued	2014-01-01	en_US
dc.identifier.citation	Applied and Environmental Microbiology, 2014, 80 (21), pp. 6750 - 6759	en_US
dc.identifier.issn	0099-2240	en_US
dc.identifier.uri	http://hdl.handle.net/10453/115710
dc.description.abstract	© 2014, American Society for Microbiology. 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 14C/15N 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.	en_US
dc.relation.ispartof	Applied and Environmental Microbiology	en_US
dc.relation.isbasedon	10.1128/AEM.01962-14	en_US
dc.subject.classification	Microbiology	en_US
dc.subject.mesh	Nitrogen	en_US
dc.subject.mesh	Ribulose-Bisphosphate Carboxylase	en_US
dc.subject.mesh	Oxidoreductases	en_US
dc.subject.mesh	Flow Cytometry	en_US
dc.subject.mesh	In Situ Hybridization	en_US
dc.subject.mesh	Environmental Microbiology	en_US
dc.subject.mesh	Nitrogen Fixation	en_US
dc.subject.mesh	Carbon Cycle	en_US
dc.title	Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry	en_US
dc.type	Journal Article
utslib.citation.volume	21	en_US
utslib.citation.volume	80	en_US
utslib.for	0605 Microbiology	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
utslib.copyright.status	open_access
pubs.issue	21	en_US
pubs.publication-status	Published	en_US
pubs.volume	80	en_US

Abstract:

© 2014, American Society for Microbiology. 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 14C/15N 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.

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http://hdl.handle.net/10453/115710