RESUMO
A cobalt-resistant wall-less mutant (slime) of Neurospora crassa was obtained by repeated sub-culturing of the sensitive wall-less mutant (W-sl) on agar medium containing toxic concentrations of cobalt. Resistance was stable on culturing Cor-sl on cobalt-free medium up to 15 weekly subcultures. Cor-sl is 10-fold more resistant to cobalt when compared to W-sl. It is also cross-resistant to Cu (10-fold) and Ni (3-fold). Cobalt accumulated by Cor-sl during growth and in short-term uptake experiments was lower when compared to W-sl. Cells previously loaded with cobalt was released into medium in both mutants, while in case of Cor-sl most of cobalt taken up (> 80%), was released back into the medium when compared to W-sl. Metabolic inhibitor (Sodium azide) and magnesium ions inhibited cobalt uptake in both the mutants. Fractionation of cell-free extracts showed that most of the cobalt (70%) taken up by Cor-sl was bound to an inducible protein fraction which bound to DEAE-Cellulose, while in W-sl only 20% of cobalt was associated with this fraction. Subcellular localization of cobalt in W-sl indicated most of it to be cytoplasmic (70%) while nuclei and mitochondria had 10% and 5% respectively. In case of Cor-sl, mitochondrial cobalt accounted for only 2% while no significant differences were noted for other fractions. Our data implicate both transport block and intracellular sequestration of cobalt to play a major role in resistance.
