Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester

Abstract

During growth on L-cysteine ethylester, Chlorella fisca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide.This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulide when incubated with mono-and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative Xmax at 263 nm was found. The chemical structre was confirmed by mass spectrometry showing a molecular weight of 256 m/ e for the S% configuration. No labeled elemental sulfur was detected when the cells were grown on [Sjsulfate and L-cysteine ethylester indicating the origin of elemental sulfur from L-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-L-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella.Phototrophic organisms form elemental sulfur (S°) under certain growth conditions. This was observed for Rhodospirillaceaelike Rhodopseudomonas and Rhodospirillum (13), Chromatiaceae-like Chromatium (12), and Chlorobiaceae-like Chlorobium (12). In blue green algae such as Oscillatoria (23) in 10-L bottles at light intensities of 7000 lux. For growth on radio-labeled sulfate carrier-free sulfate was added to the growth medium to reach a specific activity of 1725 cpm/nmol sulfate. Growth on [35S]sulfate was performed in 400-ml glass bottles connected to a Zn-acetate trap for labeled volatile sulfur compounds.Isolation of Bound Sulfide. Bound sulfide was isolated from cultures growing in 10-L bottles on sulfate and L-cysteine ethylester when the culture reached an optical density of 0.6 at 680 nm. Cells were harvested by centrifugation and washed once with distilled H20. Bound sulfide was extracted with hot methanol. After incubation in methanol at 80°C for 10 min, the cells were centrifuged for 10 min at 5000g and the pellet was extracted again with hot methanol. The combined supernatants were evaporated to drynes and resuspended in a small volume of petrolether. This crude extract was spotted on thin layer plates (Silica GF254, 0.5 mm) and developed with petrolether/diethylether (99.5/0.5) (solvent system 1). Bound sulfide with an RF of 0.84 was scraped off the plates, eluted with hot methanol, and spotted on a second plate which was developed with the solvent system 2 (acetone/water, 1/9) where bound sulfide stayed at the origin. The bound sulfide was eluted from the gel and spotted again on a TLC plate which was developed with ethylacetate (solvent system 3; RF = 0.86). The last plate was prerun with acetone and ethylacetate to remove impurities. This material was used ...

Characterization of Elemental Sulfur in Isolated Intact Spinach Chloroplasts

“…Together, our observations are in agreement with those of Krauss et al (16), who demonstrated that S8 accumulated and was rapidly used by Chlorella grown in presence of cysteine ethylester. Thus, it is probable that spinach chloroplasts contain enzymes involved in the formation and metabolization of elemental sulfur.…”

“…Thus, it is probable that spinach chloroplasts contain enzymes involved in the formation and metabolization of elemental sulfur. For example, cysteine synthase isolated from spinach catalyzed the in vitro synthesis ofcysteine from elemental sulfur (in a bound form isolated from Chlorella) and O-acetyl-Lserine, provided that a thiol was present in the incubation mixture (16). Interestingly, Legris-Delaporte et al (17) have demonstrated that elemental sulfur was metabolized in wheat leaves consecutive to its foliar application as micronized S.…”

“…In chemoautotrophic bacteria, elemental sulfur formation takes place during oxidative processes of H2S and thiosulfate (18,22,28). Elemental sulfur was characterized only in a few eukaryotic cells, such as fungi (21), and there are only three reports on its formation within eukaryotic photosynthetic organisms: two red algae, Erythrophyllum (12) and Ceramium (1 1), and one green alga, Chlorella (16). However, the main difference between prokaryotes and eukaryotes is that sulfate reduction operates with bound intermediates in eukaryotes and free intermediates in prokaryotes (1, 23, 24, 26; 1 1 lipids together with the conditions used to obtain mass spectra were responsible for the release of bound elemental sulfur and its conversion into its most stable form (viz.…”

Metabolization of Elemental Sulfur in Wheat Leaves Consecutive to Its Foliar Application

“…In fungi, elemental S is reduced at the level of the respiratory chain (17), producing H2S and causing death. In the alga Chlorella, elemental S is metabolized as the reduced form (8); this pathway takes place also when the alga are fed with cysteine methyl-or ethyl-ester. In higher plants some experiments have provided evidence for its metbolization in leaves (R Pezet, P Zuccaroni, personal communication).…”

Distribution and Mobilization of Sulfur during Soybean Reproduction

“…They concluded that this S was formed by oxidation of sulfide. Similarly, Krauss et aL (1984) found elemental S in …”