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Applied and Environmental Microbiology, April 2007, p. 2718-2726, Vol. 73, No. 8
0099-2240/07/$08.00+0     doi:10.1128/AEM.02158-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cellular Identification of a Novel Uncultured Marine Stramenopile (MAST-12 Clade) Small-Subunit rRNA Gene Sequence from a Norwegian Estuary by Use of Fluorescence In Situ Hybridization-Scanning Electron Microscopy{triangledown}

Karolina Kolodziej and Thorsten Stoeck*

School of Biology, Technische Universität Kaiserslautern, Erwin-Schroedinger-Str. 14, D-67663 Kaiserslautern, Germany

Received 14 September 2006/ Accepted 4 February 2007

Revealing the cellular identity of organisms behind environmental eukaryote rRNA gene sequences is a major objective in microbial diversity research. We sampled an estuarine oxygen-depleted microbial mat in southwestern Norway and retrieved an 18S rRNA gene signature that branches in the MAST-12 clade, an environmental marine stramenopile clade. Detailed phylogenetic analyses revealed that MAST-12 branches among the heterotrophic stramenopiles as a sister of the free-living Bicosoecida and the parasitic genus Blastocystis. Specific sequence signatures confirmed a relationship to these two groups while excluding direct assignment. We designed a specific oligonucleotide probe for the target sequence and detected the corresponding organism in incubation samples using fluorescence in situ hybridization (FISH). Using the combined FISH-scanning electron microscopy approach (T. Stoeck, W. H. Fowle, and S. S. Epstein, Appl. Environ. Microbiol. 69:6856-6863, 2003), we determined the morphotype of the target organism among the very diverse possible morphologies of the heterotrophic stramenopiles. The unpigmented cell is spherical and about 5 µm in diameter and possesses a short flagellum and a long flagellum, both emanating anteriorly. The long flagellum bears mastigonemes in a characteristic arrangement, and its length (30 µm) distinguishes the target organism from other recognized heterotrophic stramenopiles. The short flagellum is naked and often directed posteriorly. The organism possesses neither a lorica nor a stalk. The morphological characteristics that we discovered should help isolate a representative of a novel stramenopile group, possibly at a high taxonomic level, in order to study its ultrastructure, physiological capabilities, and ecological role in the environment.

* Corresponding author. Mailing address: School of Biology, TU Kaiserslautern, Erwin-Schroedinger-Str. 14, D-67663 Kaiserslautern, Germany. Phone: 49-631-205-2502. Fax: 49-631-205-2496. E-mail: stoeck{at}rhrk.uni-kl.de

{triangledown} Published ahead of print on 9 February 2007.

Applied and Environmental Microbiology, April 2007, p. 2718-2726, Vol. 73, No. 8
0099-2240/07/$08.00+0     doi:10.1128/AEM.02158-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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