title: 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.
abstract: 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 mum 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 mum) 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.
authors.affiliation: School of Biology, TU Kaiserslautern, Erwin-Schroedinger-Str 14, Kaiserslautern, Germany.
authors.forename: Karolina
authors.initials: K
authors.lastname: Kolodziej
authors.forename: Thorsten
authors.initials: T
authors.lastname: Stoeck
mesh_terms: D000818:Animals; D016844:Blastocystis / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification*; D056890:Eukaryota / Q000145:classification* / Q000235:genetics / Q000648:ultrastructure*; D005407:Flagella / Q000648:ultrastructure; D017404:In Situ Hybridization, Fluorescence; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D009664:Norway; D010802:Phylogeny; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014867:Water
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S; D014867:Water