Uptake and Continued Metabolic Activity of Azotobacter Within Fungal Protoplasts

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Abstract
Uptake of vegetative cells of Azotobacter vinelandii into protoplasts of the mycorrhizal fungus Rhizopogon sp. can be induced by treatment with polyethylene glycol (molecular weight, 6000). An L-form of the bacteria has been selected for within the differentiated fungal mycelium which is capable of acetylene reduction and nitrogen fixation, as confirmed by nitrogen-15 assays; this allows the fungus to grow on media lacking any combined nitrogen. The fungus grows and reduces acetylene on concentrations of antibiotics that prevent the growth and activity of free-living Azotobacter. Electron microscopy has revealed modified mitochondrial forms or included bacterial L-forms surrounded by an extra fungal membrane within the hyphae of the modified strains. Poly-β-hydroxybutyric acid, a storage product of Azotobacter cysts, has also been identified in the hyphae. This would appear to be the first report of the transgenosis for acetylene reduction activity and nitrogen fixation into a eukaryote cell.