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.

FIG. 1.

Bayesian inference tree for stramenopile 18S rRNA gene sequences, showing the phylogenetic position of clone KKTS_D3 retrieved from an enrichment sample of an oxygen-depleted microbial mat from a small Norwegian estuary. This clone branches within the environment-specific MAST-12 clade. The tree is based on 1,600,000 generations with two runs (each run with three hot chains and one cold chain) using a general time-reversible model. The gamma shape parameter, the rate of invariable sites, the rate matrix, and the base frequencies of the substitution model were estimated by MrBayes (35). The sample frequency was set at 1,000, resulting in 1,601 trees. The numbers at the nodes are ML bootstrap/evolutionary distance bootstrap/Bayesian posterior probability values. For ML and evolutionary distance trees, we used the TrN substitution model with a gamma shape parameter of 0.6482, a rate of invariable sites of 0.3339, and base frequencies and a rate matrix suggested by Modeltest (32). The tree is based on 906 unambiguously aligned positions.

FIG. 2.

FISH of a microbial consortium retrieved from a microbial mat sample using the KKTS_D3-specific probe MAST-12Nor2 labeled with the fluorochrome Cy3 (left panels) and DAPI stain to visualize the nuclei (right panels). (a) Positively labeled target cell. No red chlorophyll autofluorescence was detectable with UV excitation (right panel), indicating that the target cell was unpigmented (compare with panel b). (b) Chlorophyll-containing (photoautotroph) algae in a negative control without a probe, showing autofluorescence of chlorophyll with the Cy3-specific filter set. However, chlorophyll also showed red autofluorescence with the DAPI-specific filter set, which allowed us to determine if the signal obtained with the Cy3 filter could be ascribed to Cy3 labeling or a chlorophyll signal. (c) Unlabeled nontarget cells from the same filter as the cell in panel a. The exposure time used for the Cy3 image was nearly three times the exposure time used in panel a to visualize the cells. The fluorescence intensity of the nontarget cells hybridized with MAST-12Nor2 is the same order of magnitude as the fluorescence intensity of cells in negative controls (without a probe) and in hybridizations with a nonsense probe.

FIG. 3.

Images obtained at different stages of the combined FISH-SEM protocol to obtain information about the morphology of the target organism behind environmental sequence KKTS_D3. (a) FISH staining in the red channel (Cy3) using probe MAST-12Nor2. (b) DAPI staining in the UV channel to visualize the nucleus of the target cell and to confirm that the Cy3-stained structure was a eukaryotic cell. (c) Same filter area as in panels a and b examined by SEM. (d) Higher magnification of a target cell, revealing the first clues about its morphology.

FIG. 4.

SEM images of a target cell. (a and b) Overview of the target cell's general morphotype. The round regularly shaped opening is visible at the posterior of the cell, indicated by an arrow. The short flagellum (sF) is directed posteriorly and seems to be attached to the cell's surface in panel a. The long flagellum (lF) is directed anteriorly and tapers towards its end. (c) Opening at the posterior of the cell (arrow). (d) Arrangement of flagellar hairs (mastigonemes) on the long flagellum, indicated by a white arrow. The base of the flagellum does not have flagellar hairs (black arrow). (e) Detailed view of the arrangement of flagellar hairs (arrow) on the long flagellum. (f) Detailed view of the tapering tip of the long flagellum. The last 1 to 2 μm of the flagellum does not have flagellar hairs (arrow).