Abstract
Aspergillus sp. P37 is an arsenate-hypertolerant fungus isolated from a river in Spain with a long history of contamination with metals. This strain is able to grow in the presence of 0.2 M arsenate, i.e. 20-fold higher than the reference strain, Aspergillus nidulans TS1. Although Aspergillus sp. P37 reduces As(V) to As(III), which is slowly pumped out of the cell, the measured efflux of oxyanions is insufficient to explain the high tolerance levels of this strain. To gain an insight into this paradox, the accumulation of acid-soluble thiol species in Aspergillus sp. P37 when exposed to arsenic was compared with that of the arsenic-sensitive A. nidulans TS1 strain. Increasing levels of arsenic in the medium did not diminish the intracellular pool of reduced glutathione in Aspergillus sp. P37, in sharp contrast with the decline of glutathione in A. nidulans under the same conditions. Furthermore, concentrations of arsenic that were inhibitory for the sensitive A. nidulans strain (e.g. 50 mM and above) provoked a massive formation of vacuoles filled with thiol species. Because the major fraction of the cellular arsenic was present as the glutathione conjugate As(GS)3, it is plausible that the arsenic-hypertolerant phenotype of Aspergillus sp. P37 is in part due to an enhanced capacity to maintain a large intracellular glutathione pool under conditions of arsenic exposure and to sequester As(GS)3 in vacuoles. High pressure liquid chromatography analysis of cell extracts revealed that the contact of Aspergillus sp. P37 (but not A. nidulans) with high arsenic concentrations (> or =150 mM) induced the production of small quantities of a distinct thiol species indistinguishable from plant phytochelatin-2. Yet, we argue that phytochelatins do not explain arsenic resistance in Aspergillus, and we advocate the role of As(GS)3 complexes in arsenic detoxification.
Highlights
Arsenic is a major environmental pollutant that typically contaminates the soil and water of areas subject to intensive mining and metallurgical activities
A simple perusal of cells grown in the presence of 50 mM As(V) under a phase-contrast microscope revealed the formation of large vacuoles to the point of bulging out the hyphal filaments and causing a deformation in cell shape [2]. This observation could not be interpreted rigorously per se, as a negative control was not feasible; the arsenic-sensitive A. nidulans reference strain could not grow at such high concentrations of the oxyanion
Further increases in the level of the metalloid in the medium resulted in a reduction of the glutathione pool of A. nidulans TS1, surely due to the toxic effect exerted by arsenic on sensitive cells (Fig. 3A)
Summary
Arsenic is a major environmental pollutant that typically contaminates the soil and water of areas subject to intensive mining and metallurgical activities. For the determination of arsenite-glutathione complexes, samples of the fungal strains under scrutiny, grown in the presence or absence of arsenate, were extracted in trifluoroacetic acid at pH 3, separated by HPLC, and detected by the post-column derivatization method with 5,5Ј-dithiobis(2-nitrobenzoic acid) as described previously [21].
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