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

Abstract

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 13 C 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-isotopelabeled [13 C]glucose, [ 13 C]propionate, or [ 13 C]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 13 C labeling can be much lower, to the extent that ␦ 13 C ratios can be studied even at their natural abundances.

Tracing carbon flow from microphytobenthos to major bacterial groups in an intertidal marine sediment by using an in situ 13C pulse‐chase method

“…The DELTA495a probe was used in combination with a competitor probe, cDELTA495a (Macalady et al 2006), to avoid capture of Gammaproteobacteria, which have only one different base in the target region of DELTA495a. The family Desulfobacteraceae was covered with the probe Dbact653 (Miyatake et al 2009) and the probe BG553 was used for Gammaproteobacteria (Miyatake et al 2013). Both the Dbact653 and BG553 probes were used in combination with unlabeled helper probes complementary to the consensus sequence upstream and downstream of the probe target in order to increase yield (Fuchs et al 2000;MacGregor et al 2002).…”

“…Hybridization of 16S rRNA with oligonucleotide probes and the isotope analysis of captured 16S rRNA were done as described in Miyatake et al (2009). In short, total RNA was extracted from the sediment with the chloroformphenol method and 20-40 mg total RNA was hybridized with biotin-labeled probes.…”

“…MacGregor et al (2002) developed a method where 16S rRNA from defined phylogenetic groups was isolated by using specific oligonucleotide probes attached to paramagnetic beads for subsequent stable isotope analysis. Miyatake et al (2009Miyatake et al ( , 2013 further improved the stable isotope probing with magnetic bead capture hybridization (Mag-SIP) and demonstrated the utilization of organic substrates in marine sediments by certain microbial groups at the family level.…”

From Structure to Function: the Ecology of Host-Associated Microbial Communities

“…One of these is the use of stable-isotope probing (SIP), which allows for the identification of community members capable of utilizing a substrate of interest by detecting stable isotopes that have been incorporated into cellular components such as nucleic acids. This technique involves introduction of a stable isotope-labeled substrate, usually containing 13 C, to a community, followed by FISH, nucleic acid extraction, and sequence analysis, T-RFLP analysis, or some other method of determining community composition (76,131,144,226,265). In one example, Pseudomonas fluorescens, Pseudomonas putida, and an uncultured Acidovorax sp.…”

Use of immunomagnetic separation for the detection of Desulfovibrio vulgaris from environmental samples

“…Immunology based detection systems, including but not limited to enzyme-linked immunosorbent assay (ELISA), colony immunblotting, direct immunofluorescent filter techniques, and several immunocapture techniques have gained in popularity over the years as a viable screening method towards detection of target microorganisms, especially since they do not destroy the sample being tested. IMS has been applied recently in various combinations of modern methods, for example with bioluminescence (Bushon et al 2009;Su and Li, 2004), microfluidics (Qiu et al, 2009;Sivagnanam et al, 2010), PCR (Ayaz et al, 2009;Lindqvist et al, 1998;Mercanoglu and Griffiths, 2005), laboratory-on-a-chip systems (Beyor et al, 2009), or stable isotope probing (Miyatake et al, 2009).…”