HR: 16:45h
AN: B14A-04    [Abstracts]
TI: Using Small Subunit Ribosomal RNA to Follow Dark Incorporation of 14C-bicarbonate by Bacteria and Archaea in Sandy Sediment
AU: * MacGregor, B J
EM: bmacgreg@unc.edu
AF: Department of Marine Sciences Univ. of North Carolina - Chapel Hill, CB#3300 349 Chapman Hall, Chapel Hill, NC 27599, United States
AU: Musat, N
EM: nmusat@mpi-bremen.de
AF: Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, Bremen, D-28359, Germany
AU: Kuypers, M M
EM: mkuypers@mpi-bremen.de
AF: Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, Bremen, D-28359, Germany
AB: Small subunit ribosomal RNA (SSU rRNA) and the genes encoding it have become the basis of modern microbial phylogeny, and of numerous methods for characterizing the composition of bacterial, archaeal, and even eukaryotic communities as they occur in nature. A limitation of this approach has been that phylogeny alone is not a reliable guide to physiology, particularly for groups with no close relatives in culture. We have been developing ways of using the SSU rRNA molecule itself to identify and (eventually) quantify the carbon sources incorporated by particular phylogenetic groups. This can be done by taking advantage of natural variations in carbon isotopic composition among growth substrates, or by following incorporation of ¹³C- or ¹⁴C-labeled compounds. ¹⁴C has the advantage that natural background levels are negligible. In the present study, our goal is to identify species responsible for non-photosynthetic CO₂ incorporation in sandy sediments of the German Wadden Sea. Sediment cores collected from the Janssand sand flats were percolated with ¹⁴C-bicarbonate at in situ temperature for 36-38h in the dark, total RNA isolated, and domain-specific oligonucleotide probes used to capture bacterial and archaeal SSU rRNA. Total and/or captured RNA was separated by denaturing polyacrylamide gel electrophoresis, and ¹⁴C detected by phosphor imager, autoradiography, or beta imager. Detection was fastest and most sensitive with the beta imager. Both Bacteria and Archaea had incorporated label, suggesting both groups may harbor non-photosynthetic autotrophs. The next step will be to use more specific capture probes. We are currently working to separate the captured domain-specific SSU rRNA on non-denaturing gels, with detection by the high-resolution mode of the beta imager, so that individual species incorporating label can be identified by RT-PCR and sequencing of labeled bands.
DE: 0410 Biodiversity
DE: 0428 Carbon cycling (4806)
DE: 0442 Estuarine and nearshore processes (4235)
DE: 0452 Instruments and techniques
DE: 0465 Microbiology: ecology, physiology and genomics (4840)
SC: Biogeosciences [B]
MN: 2007 Fall Meeting