Development of Alternate ssu-rRNA Probing Strategies for Characterizing Aquatic Microbial Communities

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  2. Dr Charles Knapp
  3. Professor David Graham
Author(s)Knapp CW, Graham DW
Publication type Article
JournalJournal of Microbiological Methods
Year2004
Volume56
Issue3
Pages323-330
ISSN (print)0167-7012
ISSN (electronic)1872-8359
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Plastids in phytoplankton retain prokaryote-like DNA sequences that may generate false-positive signals from eubacterial small-subunit (ssu) rRNA oligonucleotide probes resulting in the overestimation of bacterial activity in aquatic microbial communities. To assess the extent of possible plastid-associated binding to eubacterial signals, we performed an extensive database search, flask experiments using algal and cyanobacterial pure cultures, and field trials on five common eubacterial probes: S-D-Bact-008-a-A-19, S-D-Bact-338-a-A-18, S-D-Bact-785-a-A-19, S-D-Bact-927-a-A-17, and S-D-Bact-1088-a-A-20. The database search and laboratory tests showed significant potential for binding among most bacterial probes and organelle ssu-rRNA. However, we propose two probing strategies to overcome this problem. First, one could use Bact-785 and Bact-338 in tandem, with the plastid component being estimated as the difference between the two signals (Bact-338 has ~70% overlap with known plastid sequences). Alternately, one might use Bact-338 as the primary eubacterial probe, but then use Cyan-785-a-A-19 (a probe that binds significantly to plastid rRNA) to correct for the plastid-associated false-positive signal. Both strategies would use a eukaryotic probe (S-D-Euca-1379-a-A-16) and Cyan-785-b-A-19 (a probe for most cyanobacteria) to further segregate rRNA signals. Trials were successfully performed using the strategies on samples from a recent field study.
URLhttp://dx.doi.org/10.1016/j.mimet.2003.10.017
DOI10.1016/j.mimet.2003.10.017
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