The relationship between sulfur metabolism and tolerance of hexavalent chromium in Scenedesmus acutus (Spheropleales): Role of ATP sulfurylase.

Sulfur availability and the end products of its metabolism, cysteine, glutathione and phytochelatins, play an important role in heavy metal tolerance, chromium included. Sulfate and chromate not only compete for the transporters but also for assimilation enzymes and chromium tolerance in various organisms has been associated to differences in this pathway. We investigated the mechanisms of Cr(VI)-tolerance increase induced by S-starvation focusing on the role of ATP sulfurylase (ATS) in two strains of Scenedesmus acutus with different chromium sensitivity. S-starvation enhances the defence potential by increasing sulfate uptake/assimilation and decreasing chromium uptake, thus suggesting a change in the transport system. We isolated two isoforms of the enzyme, SaATS1 and SaATS2, with different sensitivity to sulfur availability, and analysed them in S-sufficient and S-replete condition both in standard and in chromium supplemented medium. SaATS2 expression is different in the two strains and presumably marks a different sulfur perception/exploitation in the Cr-tolerant. Its induction and silencing are compatible with a role in the transient tolerance increase induced by S-starvation. This enzyme can however hardly be responsible for the large cysteine production of the Cr-tolerant strain after starvation, suggesting that cytosolic rather than chloroplastic cysteine production is differently regulated in the two strains.

Identification of S2-T A63: a cDNA fragment corresponding to a gene differentially expressed in a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus.

The gene expression of the wild type (S2-N) and a Cr-tolerant strain (S2-T) of the unicellular green alga Scenedesmus acutus has been compared in order to get more insight on their different chromium sensitivity. The RNA of the two strains was extracted after 4 days of culture in standard medium without chromium and analyzed by means of RNA differential display. The two strains showed differential gene transcription even in the absence of the heavy metal and six putatively differential amplicons were evidenced in the Cr-tolerant strain. Among the isolated amplicons, S2-T A63 was much more pronouncedly transcribed in the tolerant than in the wild type strain and was further characterized. S2-T A63 corresponding gene is present with the same copy number in the wild type and tolerant genomes and corresponds to an mRNA of about 2000 nt. The corresponding transcript is overexpressed in the Cr-tolerant strain after a 4-day culture and is not up-regulated by chromium exposure. The S2-T A63 sequence, obtained up to now, does not show significant homologies with any known gene. However, the analysis of the putative translation product reveals the presence of an interrupted fasciclin domain. This extracellular domain has been found in proteins from mammals, insects, echinoderms, plants, yeast and bacteria and is usually involved in cell adhesion. This finding suggests that the product for the S2-T A63 translation has an extracellular collocation, maybe as surface or secreted protein involved in external chromium detoxification.

Sulfur starvation and chromium tolerance in Scenedesmus acutus: a possible link between metal tolerance and the regulation of sulfur uptake/assimilation processes.

In a laboratory-selected Cr-tolerant strain of the unicellular green alga Scenedesmus acutus, the capacity to synthesize higher amounts of cysteine (Cys) and reduced glutathione (GSH) than the wild-type was demonstrated to underlie tolerance to Cd and Cr(VI). In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys, hence it was hypothesized that the sensitivity of the two strains to Cr(VI) could be modified after culturing in sulfate-deprived medium. Both strains were grown in the presence of different concentrations or in the absence of sulfate (sulfur-starved) and then assayed for Cr(VI) tolerance in standard medium. Unstarved, sulfur-starved and sulfur-replete cells (cells maintained in standard medium after S-starvation) were analysed for Cys, GSH and sulfur content. Sulfur-starved cells showed a greater tolerance to Cr(VI) than unstarved ones. The increased tolerance was ascribable to a transient physiological change and can be considered as specifically due to sulfur deprivation, since it was lost after a 3-day culture in standard medium and was not exhibited by nitrogen-starved cells. The comparison between Cys, GSH and sulfur content in sulfur-starved and sulfur-replete cells of the two strains suggests that the higher tolerance to Cr(VI) after S-starvation could depend on the up-regulation of sulfate uptake mechanisms, and that the primary reason for the higher tolerance to chromium in the selected strain could be due to greater sensitivity to the decrease in negative intracellular end-products (free Cys and GSH) leading to an earlier up-regulation of sulfate assimilation processes.

Differential responses to Cr(VI)-induced oxidative stress between Cr-tolerant and wild-type strains of Scenedesmus acutus (Chlorophyceae).

A wild-type and a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus were used to investigate if the difference in tolerance to Cr(VI) could depend on a different response to oxidative stress in terms of free cysteine (Cys) and reduced glutathione (GSH), and in preventing membrane lipid peroxidation. The growth of the two strains in standard medium in the presence of Cr(VI) 9.6, 19.2 or 38.4microM was compared, and the content of thiobarbituric acid reactive substances (TBARS) after a 4-day treatment was determined as marker of lipid peroxidation. The Cys and GSH contents were determined in both strains treated with Cr(VI) for 24h in HEPES buffer both enriched and not with sulphate. The treated algae were also subjected to recovery test in standard medium. The growth of wild type was inhibited at all Cr(VI) concentrations, while that of the Cr-tolerant strain only at the highest one. After exposure to 19.2microM Cr(VI), TBARS levels ranging from 0.091 to 0.263micromol/g d.w. were detected in the wild type, while only a slight increase (0.090+/-0.014micromol/g d.w.) was observed in the Cr-tolerant strain. Following treatment with 38.4microM Cr(VI), TBARS levels rose significantly and were similar in the two strains. The Cys content did not vary significantly in the cells exposed to Cr(VI) in either sulphate-lacking or sulphate-enriched buffer, and the differences between the two strains were insignificant. In the wild-type strain, the GSH content showed a significant downward trend with the increase in Cr(VI) concentration in the sulphate-lacking buffer, while it remained as high as the one of control cells in the sulphate-enriched buffer. In the Cr-tolerant strain, the GSH content did not vary significantly when the treatment took place in the sulphate-lacking buffer, while it showed a significant rise with the increase in Cr(VI) concentration in the sulphate-enriched buffer. The growth of both strains during recovery was significantly faster after treatment in the sulphate-enriched than in the sulphate-lacking buffer, the Cr-tolerant strain showing a much higher recovery capacity than the wild type. It appears that the Cr-tolerant strain, when exposed to Cr(VI) in the presence of a sulphur source, can increase GSH pool to levels not achievable by the wild type, and is thus able to recover better. This first report on the role of thiol compounds in Cr tolerance in algae suggests that tolerance to Cr(VI) in S. acutus could depend on a prompt up-regulation of the pathways leading to GSH synthesis.

Bioavailability, bioaccumulation and tolerance of chromium: consequences in the food chain of freshwater ecosystems.

Many abiotic and biotic factors can influence the partitioning equilibrium of heavy metals, thus influencing metal impact on aquatic environments. Unicellular algal species release soluble organic substances able to complex metals. In our laboratory a Cr-tolerant strain was selected and isolated from a wild type strain of Scenedesmus acutus. The exudates released by the two strains counteracted the growth inhibition caused by Cr(VI) and the exudates of the Cr-tolerant strain were more effective. On the contrary, the exudates did not reduce chromium toxicity to the cladoceran Daphnia magna. The reduction of chromium effect on algae seems the consequence of an algae-specific interaction among Cr(VI), exudates and algal cells. Chromium uptake resulted to be energy-dependent since bioaccumulation rate in subdued light condition was lower than at high light intensity. The effect of Cr(VI) on algae changed depending on metabolism of the cells and in particular it seemed to be related to the bioaccumulation rate. Tolerance in the selected strain could not be ascribed to a lower uptake of chromium. The difference in sensitivity to chromium between the two strains was exploited to evaluate if tolerance acquired by algae could have consequences for Daphnia. After treatment with Cr(VI), the two strains of S. acutus were used as food source for D. magna. The results indicate that chromium is accumulated by algae in a form not available for daphnids and that Cr tolerance acquired by the algae can be of some advantage to the consumer organism.