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Appl. Environ. Microbiol. doi:10.1128/AEM.02786-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Induction of Glutathione S-Transferase in Biofilms and Germinating Spores of Mucor hiemalis Strain EH5 from Cold Sulfidic Spring Waters

GSF - National Research Center for Environment and Health, Institute of Groundwater Ecology, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Leibniz Institut für Gewässerökologie und Binnenfischerei, Arbeitsgruppe Biochemische Regulation, Müggelseedamm 301, 12561 Berlin, Germany

^* To whom correspondence should be addressed. Email: hoque{at}gsf.de.

	Abstract

The occurrence and activation of glutathione S-transferase (GST) as well as the GST activity in biofilms of cold sulfidic spring waters were compared to those of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time there from. Using fluorescently labelled, polyclonal antiGST antibodies and GST activity measurements we demonstrated the strong in situ occurrence of GST in natural biofilms and pure cultures of strain EH5. Measurements of microsomal and cytosolic soluble GST activity using the different xenobiotic substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNP), 1-iodo-2,4-dinitrobenzene (IDNB) and fluorodifen showed the overall biotransforming ability of biofilms to be at least 6-fold higher than that of the strain EH5 only. Increasing levels of sodium thiosulfate (STS) in the medium stimulated microsomal and cytosolic CDNB-GST activity of strain EH5 by about 44- and 94-fold, respectively, as compared to control. Microsomal GST activity towards fluorodifen was induced by STS strongly linear, but the initial strong linear increment of cytosolic fluorodifen-GST activity showed saturation-like effects at concentrations higher than approximately 1 mM STS. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labelled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High performance size-exclusion chromatography and SDS-PAGE electrophoresis indicated the presence of at least two main GSTs (sub-units 27.8 and 25.6 kDa) in the cytosol of EH5, whereas the major sub-unit 27.8 kDa was the only GST in microsomes. It is suggested that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to the understanding of induction of GST by sulfurous compounds as well as to the immuno-fluorescence visualization of GST in aquatic fungus and fungal-bacterial biofilms.