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Quantifying 3 H‐thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria ( Bacteriodetes phylum)
Author(s) -
Van Mooy Benjamin A. S.,
Devol Allan H.,
Keil Richard G.
Publication year - 2004
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2004.00636.x
Subject(s) - biology , biotinylation , dna , primer (cosmetics) , 16s ribosomal rna , nucleic acid , thymidine , bacteria , phylum , proteobacteria , polymerase chain reaction , microbiology and biotechnology , gene , biochemistry , genetics , chemistry , organic chemistry
Summary 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 γ‐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.