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 (ϳ27.8-and ϳ25.6-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.The glutathione S-transferases (GSTs) (EC 2.5.1.18) are a family of multifunctional cytosolic (38) and membrane-associated microsomal (10, 36) proteins that can catalyze the conjugation of many electrophilic endogenous and xenobiotic compounds to glutathione. Most of our knowledge of the function of GSTs has come from studies of animals and plants, and there have been relatively few studies of microbes (34,40,50). The resultant glutathione conjugates are less toxic and more polar than the parent compounds and can be stored in vacuoles or excreted from the biological system, helping the organism regain normal cellular functions and reducing the cellular toxicity of reactive parent or organic intermediates. The existence of a variety of different GST isoenzymes suggests that there is a wide range of substrate specificity (17). GSTs are grouped into seven distinct classes, including the ...

Here, we report about a unique aquatic fungus Mucor hiemalis EH8 that can remove toxic ionic mercury from water by intracellular accumulation and reduction into elemental mercury (Hg0). EH8 was isolated from a microbial biofilm grown in sulfidic‐reducing spring water sourced at a Marching's site located downhill from hop cultivation areas with a history of mercury use. A thorough biodiversity survey and mercury‐removal function analyses were undertaken in an area of about 200 km2 in Bavaria (Germany) to find the key biofilm and microbe for mercury removal. After a systematic search using metal removal assays we identified Marching spring's biofilm out of 18 different sulfidic springs' biofilms as the only one that was capable of removing ionic Hg from water. EH8 was selected, due to its molecular biological identification as the key microorganism of this biofilm with the capability of mercury removal, and cultivated as a pure culture on solid and in liquid media to produce germinating sporangiospores. They removed 99% of mercury from water within 10–48 h after initial exposure to Hg(II). Scanning electron microscopy demonstrated occurrence of intracellular mercury in germinating sporangiospores exposed to mercury. Not only associated with intracellular components, but mercury was also found to be released and deposited as metallic‐shiny nanospheres. Electron‐dispersive x‐ray analysis of such a nanosphere confirmed presence of mercury by the HgMα peak at 2.195 keV. Thus, a first aquatic eukaryotic microbe has been found that is able to grow even at low temperature under sulfur‐reducing conditions with promising performance in mercury removal to safeguard our environment from mercury pollution.

Abstract- Ultraviolet-light screening potential of Norway spruce (Picea abies [L.] Karst.) needles was investigated by UV-spectroscopic, microscopic, fluorescence spectroscopic techniques as well as by HPLC, mass spectrometry and NMR spectroscopy. Results showed four potential barriers of UV screening by Norway spruce needles: (1) UV-light screening via reflectance of UV/violet light by epidermis, (2) UV-light screening via reduction of transmission of UV light by special anatomical arrangement of the epidermal cells containing the UV-screening allomelanins as well as by the light-reflecting hyaline hypodermal cells, (3) conversion of UV light by epidermis into photosynthetically active radiation (PAR; blue and red spectral bands) via fluorescence and (4) UV-light screening by absorption of UV light by UV-screening substances contained in the epidermis, whereby the latter was found to be the most important UV-screening mechanism. Staining of needle cross sections with Naturstoffreagenz A showed the localization of bound flavonoids and its derivatives in the cell walls of the outer epidermal cell layer as revealed by confocal laser scanning microscopy. By fluorescence spectroscopy and confocal laser scanning microscopy, the conversion of UVA light into PAR in the epidermis was related to various UV-screening substances contained in the epidermis. The methanol-soluble UV-absorbing substances were found to create novel UV-screening barrier zones: UVC, >200-253 nm; UVC/UVB, >253-300/303 nm; and UVB/UVA, >300-362/368 nm in epidermis as well as in mesophyll (±vascular bundles) tissues, suggesting the protective functions of epidermis for the underlying mesophyll as well as of mesophyll for the underlying vascular bundles. The following sequence of efficiency of UV-screening barrier zones of the methanol-soluble extracts of the needle epidermis and mesophyll (± vascular bundles) for various UV-spectral bands was detected: UVC screening at less than 265 nm > UVC screening at 265-280 nm > UVB screening at 280-320 nm > UVA screening at 280-320 nm, whereby the UV screening at 280-320 nm was suggested as the most relevant barrier against enhanced UVB radiation. A blend of various UV-screening substances occurred in the methanol-soluble fractions of needle epidermis, whereby p-hydroxybenzoic acid 4-O-β-D-glucopyranoside, picein, (+)-catechin, p-hydroxyacetophenone, benzoic acid and astragalin were identified as UVC/UVB-screening substances; picein, (+)-catechin, astringin, p-hydroxyacetophenone and astragalin(s) as UVB-screening substances and astragalin(s) as UVA/B-screening substances. Alkaline hydrolysis of methanol-insoluble epidermal cell wall fractions released p-coumaric acid, ferulic acid and as-tragalin(s) as major UVB-screening substances. Loss of vitality of Norway spruce trees (forest decline disease) led to a significant reduction of UVB (315 nm)-screening ability of methanol-soluble fractions from epidermis, mesophyll (±vascular bundles) and whole needles. The HPLC analysis showed that the loss of vitality is du...

For the first time we here present the unambiguous identification of the formyl radical (CHO) by EPR (Electron Paramagnetic Resonance) spectroscopy and mass spectrometry (MS) using DMPO (5,5-dimethyl-1-pyrroline N-oxide) as spin trap at ambient temperature without using any catalyst(s). The CHO was continuously generated by UV photolysis in closed anoxic environment from pure formaldehyde (HCHO) in aqueous solution. The isotropic hyperfine structure constants ofCHO were determined as a = 15.72G and a = 21.27G. The signals were deconvoluted and split by simulation in their single adduct components: DMPO-CHO, DMPO-H and DMPO-OH. We verified our results at first using MNP (2-methyl-2-nitroso-propane) as spin trap with known literature data and then mass spectrometry. Similarly the MNP adduct components MNP-CHO, MNP-H as well as its own adduct, the MNP-2-methyl-2-propyl (MNP-MP) were deconvoluted. Due to the low signal intensities, we had to accumulate single measurements for both spin traps. Using MS we got the exact mass of the reduced CHO adduct independently confirming the result of EPR detection of formyl radical.