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Applied and Environmental Microbiology, August 2009, p. 4927-4935, Vol. 75, No. 15
0099-2240/09/$08.00+0     doi:10.1128/AEM.00652-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Linking Microbial Community Function to Phylogeny of Sulfate-Reducing Deltaproteobacteria in Marine Sediments by Combining Stable Isotope Probing with Magnetic-Bead Capture Hybridization of 16S rRNA{triangledown}

Tetsuro Miyatake,1 Barbara J. MacGregor,2 and Henricus T. S. Boschker1*

Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 140, 4400 AC Yerseke, The Netherlands,1 Department of Marine Sciences, University of North Carolina, 349 Chapman Hall, CB No. 3300, Chapel Hill, North Carolina 275992

Received 19 March 2009/ Accepted 28 May 2009

We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine 13C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotope-labeled [13C]glucose, [13C]propionate, or [13C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that 13C labeling can be much lower, to the extent that {delta}13C ratios can be studied even at their natural abundances.

* Corresponding author. Mailing address: Netherlands Institute of Ecology (NIOO-KNAW), Centre for Estuarine and Coastal Ecology, P.O. Box 140, 4400 AC Yerseke, The Netherlands. Phone: 31 113577300. Fax: 31 113573616. E-mail: e.boschker{at}nioo.knaw.nl

{triangledown} Published ahead of print on 5 June 2009.

Applied and Environmental Microbiology, August 2009, p. 4927-4935, Vol. 75, No. 15
0099-2240/09/$08.00+0     doi:10.1128/AEM.00652-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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