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Isolation and purification of Candidatus Glomeribacter gigasporarum from Gigaspora margarita

Symbiotic associations between endocellular bacteria and eukaryotic cells are common among plants and animals. In fungi, the presence of endocellular bacteria has only been reported in some Glomeromycota species (arbuscular mycorrhizal fungi and Geosiphon pyriforme).

In spite of the obligate endosymbiotic nature of the fungal host, the difficulty to access large fungal biomass and the relatively small endocellular bacterial density within the fungus, we have been able to isolate endocellular Candidatus G. gigasporarum bacteria from the arbuscular mycorrhizal fungus Gigaspora margarita.

The isolation protocol is based on buffers containing sucrose 250mM and on centrifugations at different speeds. The crucial point was the use of a separation buffer containing Percoll, Ficoll and PEG that allowed satisfactory separation of bacteria from most cellular debris. The number of living isolated bacteria was estimated by using a Thoma cell and conventional fluorescence microscopy. Bacteria were stained with the Live/Dead BacLight bacterial viability kit (Molecular Probes). An average of ca 13 000 bacteria per fungal spore was obtained.

A natural heterogeneity in the number of bacteria per fungal spore was observed ranging from 3 700 to 26 000. Electron microscopic observations of the isolated bacteria revealed that most bacteria are rod-shaped (0.8-1.2 by 1.5-2.0µm in size) with a laminated cell wall typical of Gram-negative bacteria and a cytoplasm rich in ribosomes. An electron transparent area, probably corresponding to the chromosome was often observed. Cell surface was particularly complex with a fibrillar coat but flagella or pili were not visible.

To investigate the free living capacities of Candidatus G. gigasporarum, several media, suitable to sustain growth of a large spectrum of different microorganisms, supplemented with various vitamins or amino-acids, were tested. Candidatus G. gigasporarum growth was never observed in any of the tested media and chosen conditions. However, Candidatus G. gigasporarum is able to survive several weeks out of its fungal host.

More information on the Genomyca -project can be found at:

Reported by

Université Paul Sabatier, UMR 5546 CNRS
Equipe de Mycologie Vegetale, Chemin de Borde-Rouge, BP 17 Auzeville
31326 Castanet-Tolosan
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