Assignment of 16S rDNA Sequences to the Corresponding Ectosymbiotic Bacterial Morphotypes

By using cell envelope preparations for in situ fluorescence hybridization, an interference with the fluorescence of wood particles was avoided. In addition, the fluorescence signals of the specific Cy3-labelled probes could be enhanced by applying helper oligonucleotides (Fuchs et al. 2000) and the binding of the probes was facilitated by performing a denaturing step.

• Spirochaeta stenostrepta — Spirochete clone MDS1 -Spirochete clone mpsp15

-100 Spirochete clone sp40-7

-Spirochete clone mpsp2

Spirochete clone mp1 - Spirochete clone sp40-8 ■ Spirochete clone mp3 • Spirochete clone sp5-18 Spirochete clone mp5 Spirochete clone mp4 Spirochaeta zulzerae

-Treponema maltophilum

-Treponema pallidum

-Leptospira Mini

Fig. 5.5. Phylogenetic relationship of Mixotricha paradoxa-associated spirochete clones (bold letters). The relationship was determined by neighbor joining analysis. The data set contained 15 alignment positions and Leptospira illini as the outgroup. The bootstrap values (100 runs), obtained by using the program SEQBOOT from the PHYLIP program package (Felsenstein 1985, 1993), are inserted at the respective branching points. Bootstrap values under 50% are not shown. The bar represents 10 changes per 100 nucleotide positions (from Wenzel et al. 2003; with permission)

Fluorescence in situ hybridization was performed with specific Cy3-labelled probes derived from 16S rDNA amplificates obtained from the ectosymbiotic spirochetes and rod-shaped bacterium clone B6. Three spirochetal clones could be localized on the cell surface, clone mpsp15 at the anterior and clones mp1 and mp3 at the posterior part. Clone mp1 occurred in a lower number than clone mp3 (Fig. 5.7). The spirochetal clones that could not be localized on the cell surface form probably no dense population on the cell surface and their weak fluorescence signals might have been overlooked. Cleveland and Grimstone (1964) described two morphotypes of spirochetes, shorter ones which were attached to the brackets and longer ones which only appeared sporadically and were not linked to the brackets. In the light microscope we also observed the long spirochetes on living flagellate cells. They were not found on the isolated cell envelopes, indicating that they are not tightly bound

Fig. 5.6. Phylogenetic relationship of rod-shaped clone B6. 16S rDNA sequences were obtained from the EMBL-database (Stoesser et al. 2001). The relationship was determined by neighbor joining analysis. The data set contained nine alignment positions and Burkholderia pseudomallei as the outgroup. The bootstrap values (100 runs), obtained by using the program SEQBOOT, are inserted at the respective branching points. Bootstrap values under 50% are not shown. The bar represents 10 changes per 100 nucleotide position (from Wenzel et al. 2003; with permission)

Fig. 5.6. Phylogenetic relationship of rod-shaped clone B6. 16S rDNA sequences were obtained from the EMBL-database (Stoesser et al. 2001). The relationship was determined by neighbor joining analysis. The data set contained nine alignment positions and Burkholderia pseudomallei as the outgroup. The bootstrap values (100 runs), obtained by using the program SEQBOOT, are inserted at the respective branching points. Bootstrap values under 50% are not shown. The bar represents 10 changes per 100 nucleotide position (from Wenzel et al. 2003; with permission)

to the cell surface. In contrast, the smaller morphotypes remained at the surface of the envelopes. Spirochete clone mpsp15 possesses one flagellum at each cell pole (Wenzel et al. 2003). After Margulis and Hinkle (1992) the characteristic flagella array is 1:2:1 corresponding to one flagellum at the end of the cell and two overlapping flagella in the middle of the cell. The other 16S rDNA clones localized at the posterior part of Mixotrichaparadoxa have to be assigned to certain morphotypes in a future work.

Rod-shaped bacteria (length: 0.8-1.1 |m; width: 0.3 |m) are attached to cell surface brackets in a regular pattern (Cleveland and Grimstone 1964; König and Breunig 1997) perpendicular to the cell of Mixotricha paradoxa. The distance between the cells in a row is about 0.9 | m, and between two adjacent rows is 0.5 |im. The individual rods in the rows are staggered. The fluorescent probes B6.1 and B6.2 were specific for the Bacteroides sp-related clone B6. Positive hybridization results showed that clone B6 is spread all over the surface of Mixotricha paradoxa in a similar regular pattern as found in electron micrographs.

When the cell envelopes were incubated with the probes B6.1 and B6.2 or with the Cy3-labelled universal probe Eubac 338 (Amann et al. 1990) the same regular pattern of a rod-shaped bacterium was obtained indicating that clone B6 was the only rod associated with the cell surface (Fig. 5.7)

Fig. 5.7. Schematic drawing showing the proposed distribution of the bacterial ectosymbionts on the cell surface of Mixotricha paradoxa (from Wenzel et al. 2003; with permission). A anterior part, P posterior part, F flagella. I ingestive zone

Mixotricha paradoxa, a trichomonad from the hindgut of the Australian termite Mastotermes darwiniensis Froggatt, is a rare example of a movement symbiosis between eukaryotic and prokaryotic microorganisms (Cleveland and Grimstone 1964). It is known that a lot of symbiotic relationships of protozoa and spirochetes play no role in the locomotion of the protist cell (Bloodgood and Fitzharris 1976; Breznak 1984; Iida et al. 2000). This indicates that spirochetes must fulfill also other functions. Leadbetter et al. (1999) discussed the possibility that the spirochetes consume H2 and CO2 that is produced by the flagellates. They form acetate as end product, which is consumed by the termites, while other isolates live heterotrophic (Graber et al. 2004 a,b; Dröge et al. 2006).

From the movement symbiosis between spirochetes and the flagellate Mixotricha paradoxa the hypothesis was derived that eukaryotic locomotory organelles such as flagella and cilia originated from spirochetes (Bermudes et al. 1987; Margulis 1993), while the cytoplasm is assumed to be of archaeal origin. Our studies showed that several spirochete species synergistically contrive the movement of Mixotricha paradoxa. Since Mixotricha paradoxa belongs to the early branching flagellates the movement symbiosis should be an early invention during the evolution of the eukaryotic cell. If the above-mentioned hypothesis is correct, then the ancestor of locomotory organelles should have been a relative of Treponema. Since the other large symbiotic flagellates of Mastotermes darwiniensis (e.g. Koruga bonita) are relatively closely related to Mixotricha paradoxa it seems not likely that their flagella originated from spirochetes. The symbiosis between Mixotricha paradoxa and its spirochetes seems to be a unique example of movement symbiosis.

Acknowledgements We thank H. Hertel (BAM, Berlin) for termite cultures.

Was this article helpful?

0 0

Post a comment