Maria José Magalhães Trópia

Bioleaching of zinc and nickel from silicates using Aspergillus niger cultures

In this work, we investigated the role of bacteria from the genera Bacillus and Pseudomonas and fungi from the genera Aspergillus and Penicillium in the leaching process of two different silicates (calamine and garnierite). Since the results obtained with A. niger were better than those with different bacteria, a more detailed investigation of the leaching process with this microorganism was conducted. Moreover, although it is clear that the citric acid generated by fungi could be an important leaching agent acting in the solubilization of the used silicates, other products of metabolism could be involved. Related to this, the results obtained with chemical leaching using low concentrations of citric acid (lower than 10 mM) showed, for both calamine and garnierite, that the respective dissolution of zinc and nickel was much lower when compared to those processes in which cultures or supernatant liquor of A. niger cultures were used and in which the maximum concentration of citric acid was 8 mM. The results obtained also suggest that the type of mineral (and/or the metal present in it) presents a different susceptibility to the bioleaching process and also demonstrate that depending of the situation, the presence of the fungi cells seem to improve the leaching process. From a practical point of view, the high yield rate of extracting metals from silicates obtained by using for example, supernatant liquors of A. niger cultures, is noteworthy. This bioleaching process present two advantages as compared to conventional chemical leaching processes: (a) the very low concentrations of organic compounds present in such a situation represent a lower ecological risk; and (b) even with a lower final yield, the economical cost of a such process. Both characteristics could facilitate its industrial application.

New aspects of the glucose activation of the H(+)-ATPase in the yeast Saccharomyces cerevisiae.

The glucose-induced activation of plasma membrane ATPase from Saccharomyces cerevisiae was first described by Serrano in 1983. Many aspects of this signal transduction pathway are still obscure. In this paper, evidence is presented for the involvement of Snf3p as the glucose sensor related to this activation process. It is shown that, in addition to glucose detection by Snf3p, sugar transport is also necessary for activation of the ATPase. The participation of the G protein, Gpa2p, in transducing the internal signal (phosphorylated sugars) is also demonstrated. Moreover, the involvement of protein kinase C in the regulation of ATPase activity is confirmed. Finally, a model pathway is presented for sensing and transmission of the glucose activation signal of the yeast H(+)-ATPase.

The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells.

Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H(+)-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H(+)-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca(2+)-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H(+)-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H(+)-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP(3). Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H(+)-ATPase activation.

Carbonyl cyanide m‐chlorophenylhydrazone induced calcium signaling and activation of plasma membrane H+‐ATPase in the yeast Saccharomyces cerevisiae

The plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H(+)-ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H(+)-ATPase, suggesting a possible connection between calcium signaling and activation of ATPase.

Protective effect of ions against cell death induced by acid stress in Saccharomyces

Saccharomyces boulardii is a probiotic used to prevent or treat antibiotic-induced gastrointestinal disorders and acute enteritis. For probiotics to be effective they must first be able to survive the harsh gastrointestinal environment. In this work, we show that S. boulardii displayed the greatest tolerance to simulated gastric environments compared with several Saccharomyces cerevisiae strains tested. Under these conditions, a pH 2.0 was the main factor responsible for decreased cell viability. Importantly, the addition of low concentrations of sodium chloride (NaCl) protected cells in acidic conditions more effectively than other salts. In the absence of S. boulardii mutants, the protective effects of Na(+) in yeast viability in acidic conditions was tested using S. cerevisiae Na(+)-ATPases (ena1-4), Na(+)/H(+) antiporter (nha1Delta) and Na(+)/H(+) antiporter prevacuolar (nhx1Delta) null mutants, respectively. Moreover, we provide evidence suggesting that this protection is determined by the plasma membrane potential, once altered by low pH and low NaCl concentrations. Additionally, the absence or low expression/activity of Ena proteins seems to be closely related to the basal membrane potential of the cells.

Detailed search for protein kinase(s) involved in plasma membrane H + −ATPase activity regulation of yeast cells

This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

Yeast genes YOL002C and SUL1 are involved in neomycin resistance

In previous studies we suggested the importance of the control of plasma membrane H+-ATPase by a phosphatidylinositol-like pathway for cellular proton extrusion in Saccharomyces cerevisiae (Brandão et al. 1994; Coccetti et al. 1998). The observations that provided the model above include the inhibition of the glucose-induced activation of the plasma membrane H+-ATPase as well as the inhibition of the glucose-induced external acidification by neomycin, a known inhibitor of the phosphatidylinositol turnover in eukaryotic cells. In this work, using two libraries, we isolated two yeast clones that were able to prevent the inhibition of glucose-induced activation of the H+-ATPase by neomycin. We show that the YOL002C gene, which encodes a protein of unknown function, and the SUL1 gene, which is a sulphate transporter belonging to the major facilitator superfamily, suppress growth inhibition by neomycin. However, they are not required for glucose-induced activation of the plasma membrane H+-ATPase. The resistance of the clones to neomycin is probably related to the level and/or activity of proteins functioning as drug extrusion pumps.

Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells

In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that maintains the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase accessible for phosphorylation by at least one protein kinase.