Miyatake, T. and MacGregor, B.J. and Boschker, H.T.S. (2009) 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. Applied and Environmental Microbiology, 75, 4927-4935. ISSN 0099-2240.
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Official URL: http://dx.doi.org/10.1128/AEM.00652-09
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 level. The main improvements were a substantial decrease in the protocol blank and an approximately 10 fold increase in 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 were 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. Target specificity of the probes was examined with pure cultures of target and non-target species and by determining the phylogenetic composition of the captured sediment rRNA. 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, 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 even natural abundance 13C ratios can be studied.
|Institutes:||Nederlands Instituut voor Ecologie (NIOO)|
|Deposited On:||23 Mar 2010 01:00|
|Last Modified:||24 Apr 2012 16:45|
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