Formation of Metallogenium-like structures by a manganese-oxiding fungus

Structures resembling Metallogenium spp. were observed in agar and in liquid cultures of a Mn-oxidizing basidiomycetous fungus only when Mn²⁺ was oxidized. Fungal viability was necessary for formation of the structures; Mn²⁺ concentration and the presence or absence of agar in the medium were important factors determining their morphology. Slide cultures revealed no identifiable cells in any stage of development. Fluorescent dyes that stained nucleic acids and polysaccharides in the fungal hyphae did not stain the Metallogenium-like structures. Likewise, Rhodamine 123, a fluorescent probe for membrane potential, stained fungal mitochondria, but did not stain the structures. Thin sections through the structures showed no biological membranes or other cellular features. Only the characteristic ultrastructure of biological Mn oxides were observed in serial thin sections. In agar, unfixed structures disappeared permanently during reduction of Mn oxides with hydroxylamine. Glutaraldehyde fixation stabilized these structures. Fixed structures lost most of their original phase density during reduction with hydroxylamine, but continuous microscopic observations showed that their phase density could be restored by staining with Coomassie blue. Structures that formed in liquid medium did not require stabilization with glutaraldehyde during reduction of Mn oxides. They, too, lost their original phase density during reduction with hydroxylamine; phase density could be restored by staining with cationic colloidal iron or Coomassie blue. The results suggest that the Metallogenium-like structures were formed as a result of Mn oxidation associated with exopolymers produced by the fungus.Non-standard abbreviations HEPES (N-hydroxyethylpiperazine-N-2-ethane sulfonic acid) - DAPI (4,6-diamidino-2-phenylindole) - PIPES (piperazine-N,N-bis2-ethane sulfonic acid])