Quantifying 3H-thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria (Bacteriodetes phylum).
The rate of [(3)H-methyl] thymidine ((3)H-TdR) incorporation into DNA has been applied extensively to measure cell production by bacterial communities in aquatic environments. Here we describe a method to quantify (3)H-TdR incorporation by specific, phylogenetically defined members of the bacterial community. The method involves selectively capturing DNA from targeted groups of bacteria and then quantifying its (3)H radioactivity. The method was applied to measure (3)H-TdR incorporation by the members of the phylum Bacteriodetes whose members, which include the Cytophaga-Flavobacter cluster, are ubiquitous in coastal waters. (3)H-labelled DNA from Bacteriodetes was selectively biotinylated in PCR-like reactions that contained a Bacteriodetes-specific 16S rRNA gene primer, thermostable DNA polymerase and biotinylated dUTP. The biotinylated DNA was then captured on streptavidin-coated beads and its (3)H radioactivity determined by scintillation counting. We have termed this method 'selective nucleic acid polymerase-biotinylation and capture' or 'SNAP-BAC'. Internal (33)P-labelled DNA standards were used to quantify the recovery of (3)H-labelled DNA from the SNAP-BAC reactions. The method was verified by successfully targeting Bacteriodetes in simple laboratory mixtures of (3)H-labelled DNA extracted from pure cultures of Bacteriodetes and gamma-proteobacteria. Field application of this method in Puget Sound and off the Washington coast determined that Bacteriodetes were responsible for 56 +/- 17% and 32 +/- 5% of community (3)H-TdR incorporation (1.3 +/- 0.3 and 9.9 +/- 1.7 pmol l(-1) h(-1)) at these two locations.
van Mooy
Benjamin A S
BA
School of Oceanography, University of Washington, BOX 355351, Seattle, Washington 98195-5351, USA. bvanmooy@whoi.edu
Devol
Allan H
AH
Keil
Richard G
RG
D041963:Bacteroidetes / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D007700:Kinetics; D009792:Oceans and Seas; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012588:Scintillation Counting; D013936:Thymidine / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism; D014861:Washington
D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D014316:Tritium; D004259:DNA-Directed DNA Polymerase; D013936:Thymidine