Enamul Hoque

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 (∼27.8- and ∼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.

A new mercury‐accumulating Mucor hiemalis strain EH8 from cold sulfidic spring water biofilms

Here, we report about a unique aquatic fungus Mucor hiemalisEH8 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 Marchings 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 springs 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.

High-performance size-exclusion chromatographic characterization of water-soluble polymeric substances produced by Phanerochaete chrysosporium from free and wheat cell wall bound 3,4-dichloroaniline

A high-performance size-exclusion chromatographic (HPSEC) method was developed and optimized for the separation of aq. soluble polymeric xenobiotics. The use of this method for the characterization of aq. soluble polymeric substances produced by Phanerochaete chrysosporium cultures from [ring-U-14C]-3,4-dichloroaniline ([14C]DCA) and wheat cell wall bound [ring-U-14C]-3,4-dichloroaniline was demonstrated by on-line UV detection at 280 nm and radioactivity tracing. The polymeric nature of aq. soluble lignin-like 14C-products was shown by polydispersity and correspondence of the UV signal at 280 nm with the radioactivity trace. The higher polydispersity and molecular mass of lignin-like substances in low-C cultures as compared to those in low-N cultures were found to be associated with lower mineralization rates. Results indicated that the major portion of copolymerized DCA was apparently covalently bound via labile bonds to the α-C atom of the lignin monomer side chains on native wheat cell wall.

Detection of the formyl radical by EPR spin-trapping and mass spectrometry

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 of •CHO were determined as aN = 15.72G and aH = 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.

Ecology, adaptation, and function of methane‐sulfidic spring water biofilm microorganisms, including a strain of anaerobic fungus Mucor hiemalis

Ecological aspects, adaptation, and some functions of a special biofilm and its unique key anaerobic fungus Mucor hiemalis strain EH11 isolated from a pristine spring (Künzing, Bavaria, Germany) are described. The springs pure nature is characterized by, for example, bubbling methane, marine‐salinity, mild hydrothermal (~19.1°C), sulfidic, and reductive‐anoxic (Eh: −241 to −253 mV, O2: ≤ 0.1 mg/L) conditions. It is geoecologically located at the border zone between Bavarian Forest (crystalline rocky mountains) and the moor‐like Danube River valley, where geological displacements bring the springs water from the deeper layers of former marine sources up to the surface. In the springs outflow, a special biofilm with selective microorganisms consisting of archaea, bacteria, protozoa (ciliate), and fungus was found. Typical sulfidic‐spring bryophyta and macrozoobenthos were missing, but many halo‐ and anaerotolerant diatoms and ciliate Vorticella microstoma beside EH11 were identified. Phase contrast and scanning electron microscopy revealed the existence of a stabilizing matrix in the biofilm formed by the sessile fungal hyphae and the exopolysaccharide substance (EPS) structures, which harbors other microorganisms. In response to ecological adaptation pressure caused by methane bubbles, EH11 developed an atypical spring‐like hyphal morphology, similar to the spiral stalk of ciliate V. microstoma, to rise up with methane bubbles. For the first time, it was also demonstrated that under strict anaerobic conditions EH11 changes its asexual reproduction process by forming pseudosporangia via hyphal cell divisions as well as switching its metabolism to chemoautotrophic bacteria‐like anaerobic life using acetate as an e‐donor and ferrihydrite as an e‐acceptor, all without fermentation. EH11 can be suggested to be useful for the microbial community in the Künzing biofilm not only due to its physical stabilization of the biofilms matrix but also due to its ecological functions in element recycling as well as a remover of toxic metals.

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins

ObjectivesTo investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications.ResultsCatalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M.xa0hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100xa0µg MC-LR l−1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100xa0µgxa0l−1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-l-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected.ConclusionsM. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.