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

Appl Environ Microbiol. 2007 Apr;73(8):2718-26. doi: 10.1128/AEM.02158-06. Epub 2007 Feb 9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blastocystis / genetics
  • DNA, Protozoan / genetics
  • DNA, Protozoan / isolation & purification*
  • DNA, Ribosomal / genetics
  • DNA, Ribosomal / isolation & purification*
  • Eukaryota / classification*
  • Eukaryota / genetics
  • Eukaryota / ultrastructure*
  • Flagella / ultrastructure
  • In Situ Hybridization, Fluorescence
  • Microscopy, Electron, Scanning
  • Molecular Sequence Data
  • Norway
  • Phylogeny
  • RNA, Ribosomal, 18S* / genetics
  • Sequence Analysis, DNA
  • Sequence Homology, Nucleic Acid
  • Water*


  • DNA, Protozoan
  • DNA, Ribosomal
  • RNA, Ribosomal, 18S
  • Water

Associated data

  • GENBANK/EF219381