Induction of glutathione S-transferase in biofilms and germinating spores of Mucor hiemalis strain EH5 from cold sulfidic spring waters.

Abstract

The occurrence and activation of glutathione S-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs ( approximately 27.8- and approximately 25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to our understanding of induction of GST by sulfurous compounds, as well as to the immunofluorescence visualization of GST in aquatic fungus and fungus-bacterium biofilms.