Successive rapid reductive dehalogenation and mineralization of pentachlorophenol by the indigenous microflora of farmyard manure compost.

AIMS: To determine whether composting with animal manure can be used to effectively remediate soil from a pentachlorophenol (PCP)-contaminated site, and to establish the fate of the degraded xenobiotic. METHODS AND RESULTS: Contaminated soil from a sawmill site was mixed with farm animal manure and composted in a 0.5 m3 silo under fully aerobic conditions. The disappearance and fate of PCP was monitored by gas chromatography (GC-ECD) and extensive mineralization confirmed in experiments with 14C-radiolabelled PCP. The disappearance of PCP was rapid and virtually complete within 6 days, prior to the onset of thermophilic conditions. Dechlorination of the PCP was found to be both reductive and sequential. CONCLUSIONS: PCP removal from contaminated soil by aerobic composting with animal manure is efficient and proceeds via reductive dechlorination to virtually complete mineralization. This contrasts with other chlorophenol composting regimes in which mineralization is achieved but dechlorination intermediates do not accumulate to detectable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study demonstrate that anaerobic reductive dechlorination can proceed in an aerobic composting environment and contribute to efficient pentachlorophenol removal. Farmyard manure composts may represent a rapid, low-cost, low-technology option for treatment of chlorophenol-contaminated soils.

Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae.

Glutathione-deficient mutants (gshA) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis. Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium. Furthermore, all mutants examined showed a derepression of gamma-glutamyltranspeptidase (gamm-GT), the enzyme initiating GSH degradation. However, gamma-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted. The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the gamma-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes. Studies of enzyme regulation showed that the derepression of gamma-GT observed in the gshA strains was most probably due to an alteration of the thiol status. The effectors governing the biosynthesis of cysteine synthase and gamma-cystathionase seemed different from those playing a role in gamma-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As a consequence the endogenous pool of GSH was used in the synthesis of cysteine. GSH might, therefore, fulfil the role of a storage compound.

[Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae]. / Caractérisation d'une activité gamma-glutamylarylamidase-glutaminase chez Saccharomyces cerevisiae.

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.

Glutathione metabolism in yeast Saccharomyces cerevisiae. Evidence that gamma-glutamyltranspeptidase is a vacuolar enzyme.

In a first experiment we have shown that S. cerevisiae beta-glutamyltranspeptidase is associated with a particulate fraction obtained by differential centrifugation. We have subsequently shown that this enzyme activity followed accurately the distribution of vacuolar markers. Liberation of vacuoles was carried out by mechanical disruption of spheroplast under isotonic conditions and the vacuoles were purified by centrifugation of Ficoll gradients. Yeast beta-glutamyltranspeptidase could be implicated in the exchanges of amino acids between the cytoplasm and the vacuolar sap.

The glutathione-dependent glyoxalase pathway in the yeast Saccharomyces cerevisiae.

Glyoxalase I (EC 4.4.1.5), which catalyzes the reaction methylglyoxal + GSH leads to S-lactoylglutathione, is a ubiquitous enzyme for which no clear physiological function has been shown. In the yeast Saccharomyces cerevisiae, methylglyoxal may derive from the spontaneous decay of intracellular glyceraldehyde-3-P, which may accumulate during growth on glycerol as the carbon source. The half-life time for the triose phosphate was found to be 4.6 h under physiological conditions (pH 6.2, 0.05 M phosphate at 30 degrees C). Glyoxalase I is induced by growth on glycerol or by the addition of methylglyoxal to the growth medium. The enzyme is also subject to carbon catabolite repression. A mutant strain, fully defective in glyoxalase I and bearing only one nuclear mutation, was obtained. The strain, which is killed by exposure to glycerol, excretes methylglyoxal into the medium. Growth of the mutant on glucose as carbon source appears to be similar to that of the wild type strain. This investigation has clearly demonstrated a physiological role of glyoxalase I in a eucaryotic cell.