Whole-cell versus total RNA extraction for analysis of microbial community structure with 16S rRNA-targeted oligonucleotide probes in salt marsh sediments.
rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% +/- 3.7% [mean +/- standard deviation]) relative to direct DAPI (4',6'-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r(2) = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.
Frischer
M E
ME
Skidaway Institute of Oceanography, Savannah, Georgia 31411, USA. frischer@skio.peachnet.edu
Danforth
J M
JM
Newton Healy
M A
MA
Saunders
F M
FM
D017753:Ecosystem; D019015:Geologic Sediments / Q000382:microbiology*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D010758:Phosphorus; D010802:Phylogeny; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011849:Radioactive Tracers; D016999:Sulfur-Reducing Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D014871:Water Microbiology
D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011849:Radioactive Tracers; D010758:Phosphorus