Mitochondrial Function in Cell Wall Glycoprotein Synthesis in Saccharomyces cerevisiae NCYC 625 (Wild Type) and [rho0] Mutants

We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S. diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [rho⁰] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [rho⁰] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [rho⁰] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs.     

Biogenesis of Mitochondria: Analysis of Deletion of Mitochondrial Antibiotic Resistance Markers in Petite Mutants of Saccharomyces cerevisiae

Yeast strains carrying markers in several mitochondrial antibiotic resistance loci have been employed in a study of the retention and deletion of mitochondrial genes in cytoplasmic petite mutants. An assessment is made of the results in terms of the probable arrangement and linkage of mitochondrial genetic markers. The results are indicative of the retention of continuous stretches of the mitochondrial genome in most petite mutants, and it is therefore possible to propose a gene order based on co-retention of different markers. The order par, mik1, oli1 is suggested from the petite studies in the case of three markers not previously assigned an unambiguous order by analysis of mitochondrial gene recombination. The frequency of separation of markers by deletion in petites was of an order similar to that obtained by recombination in polar crosses, except in the case of the ery1 and cap1 loci, which were rarely separated in petite mutants. The deletion or retention of the locus determining polarity of recombination (ω) was also demonstrated and shown to coincide with deletion or retention of the ery1, cap1 region of the mitochondrial genome. Petites retaining this region, when crossed with rho⁺ strains, display features of polarity of recombination and transmission similar to the parent rho⁺ strain. By contrast a petite determined to have lost the ω⁺ locus did not show normal polarity of marker transmission. Differences were observed in the relative frequency of retention of markers in a number of strains and also when comparing petites derived spontaneously with those obtained after ultraviolet light mutagenesis. By contrast, a similar pattern of marker retention was seen when comparing spontaneous with ethidium bromide-induced petites.     

Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m⁻¹ s⁻¹) were significantly higher than that measured for H₂O₂ (1.5 m⁻¹ s⁻¹). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 10³ m⁻¹ s⁻¹. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems.     

Maintenance and Integrity of the Mitochondrial Genome: a Plethora of Nuclear Genes in the Budding Yeast

Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho⁺ mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho⁰ petites) and/or lead to truncated forms (rho⁻) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho⁺ genome depends on a centromere-like structure dispensable for the maintenance of rho⁻ mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.     

Glutathione Deficiency Leads to Riboflavin Oversynthesis in the Yeast Pichia guilliermondii

The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3–4 and 5–6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism.     

Low-level chemiluminescence of hydroperoxide-supplemented cytochrome c

Ferricytochrome c showed low-level chemiluminescence, with a light-emission measured of about 1×10³–3×10³ counts/s, when supplemented with organic hydroperoxides. Tertiary hydroperoxides (cumene hydroperoxide and t-butyl hydroperoxide) showed a saturation behaviour at about 5mm-hydroperoxide, whereas primary hydroperoxides showed a quadratic dependence on the hydroperoxide concentration. Chemiluminescence depended linearly on cytochrome c concentration, and optimal light-emission was observed at [t-butyl hydroperoxide]/[ferricytochrome c] ratios of 160–500. Hydroperoxide-supplemented ferricytochrome c consumed O₂ at a rate of 1.0μmol/min per μmol of cytochrome c; the rate of O₂ uptake was linearly related to the concentration of cytochrome c. The Soret absorption band of ferricytochrome c decreased about 64% after incubation with t-butyl hydroperoxide, whereas the 530nm band was almost totally abolished. Light-emission was (a) inhibited competitively by cyanide. (b) inhibited by singlet-oxygen quenchers (e.g. β-carotene), scavengers (e.g. dimethylfuran) and traps (e.g. histidine and tryptophan) and (c) increased by singlet-oxygen-chemiluminescence enhancer 1,4-diazabicyclo[2.2.2]-octane. Superoxide dismutase had no effect on the present system. The participation of free radicals is suggested by the effect of the radical trap 2,5-di-t-butylquinol. Singlet-oxygen dimol emission seems to be mainly responsible for the observed light-emission; a mechanism that can account for the major part of the present experimental observations is proposed.     

Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V_max, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V_max was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min⁻¹ for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min⁻¹ (U₀ = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Purification and characterization of selenium-glutathione peroxidase from hamster liver

Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 μmol cumene hydroperoxide reduced/min/mg of protein at 37 °C, pH 7.6, and 0.25 mm GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 °C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k₁ (t-butyl hydroperoxide), 7.06 × 10⁵ mm min^?1; k₁ (cumene hydroperoxide), 1.04 × 10⁶ mm^?1 min^?1; k₁ (p-menthane hydroperoxide), 1.2 × 10⁶ mm^?1 min^?1; k₁ (diisopropylbenzene hydroperoxide), 1.7 × 10⁶ mm^?1 min^?1; k₁ (linoleic acid hydroperoxide), 2.36 × 10⁶ mm^?1 min^?1; k₁ (ethyl hydroperoxide), 2.5 × 10⁶ mm^?1 min^?1; and k₁ (hydrogen peroxide), 2.98 × 10⁶ mm^?1 min^?1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.     

Rat liver cytosolic glutathione peroxidase: reactivity with linoleic acid hydroperoxide and cumene hydroperoxide.

The reactivity of rat liver glutathione (GSH) peroxidase with two hydroperoxides was determined using integrated rate equations. The bimolecular rate constant for the reaction of GSH peroxidase with linoleic acid hydroperoxide is approximately four times the rate constant with cumene hydroperoxide. The reactivity toward reduced glutathione is not altered by different hydroperoxides. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for lipid hydroperoxide in rat liver is approximated at 9.5 × 10^?5 min.     

The relative effectiveness of human plasma glutathione peroxidase as a catalyst for the reduction of hydroperoxides by glutathione

To reveal clues to the function of human plasma glutathione peroxidase (GPx), we investigated its catalytic effectiveness with a variety of hydroperoxides. Comparisons of hydroperoxides as substrates for plasma GPx based on the ratio ofV _max /K _m were blocked by the limited solubility of the organic hydroperoxides, which prevented kinetic saturation of the enzyme at the chosen glutathione concentration. Therefore, we compared the hydroperoxides by the fold increase in the apparent first-order rate constants of their reactions with glutathione owing to catalysis by plasma GPx. The reductions of aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide,t-amyl hydroperoxide,t-butyl hydroperoxide, paramenthane hydroperoxide) were better catalyzed by plasma GPx than were reductions of the more “physiological” substrates (linoleic acid hydroperoxide, hydrogen peroxide, peroxidized plasma lipids, and oxidized cholesterol).     

Inverse correlation between cyclic GMP and tyrosine aminotransferase degradation in rat hepatoma tissue culture cells

[1,2-³H]Cholesterol was epoxidized to radioactive cholesterol α- and β-epoxides (5,6α-epoxy-5α- and 5,6β-epoxy-5β-cholestan-3β-ols) in the ratio 1:4 by hepatic microsomal lipid hydroperoxides (MsOOH, 1 mM as active oxygen) in the presence of ferrous ion. MsOOH could be replaced by methyl linoleate hydroperoxides (MOOH) under the same conditions although the latter was less effective than the former. None of cumene hydroperoxide, t-butyl hydroperoxide, and hydrogen peroxide was an effective oxidant even at 10 mM. Neither ADP nor EDTA had an effect on the epoxidation of cholesterol by MsOOH as well as by MOOH. Ferrous ion could not be replaced by ferric ion in the hydroperoxide-mediated epoxidation. Cyanide anion potentially inhibited the reaction.     

Farnesol-Induced Generation of Reactive Oxygen Species via Indirect Inhibition of the Mitochondrial Electron Transport Chain in the Yeast Saccharomyces cerevisiae

The mechanism of farnesol (FOH)-induced growth inhibition of Saccharomyces cerevisiae was studied in terms of its promotive effect on generation of reactive oxygen species (ROS). The level of ROS generation in FOH-treated cells increased five- to eightfold upon the initial 30-min incubation, while cells treated with other isoprenoid compounds, like geraniol, geranylgeraniol, and squalene, showed no ROS-generating response. The dependence of FOH-induced growth inhibition on such an oxidative stress was confirmed by the protection against such growth inhibition in the presence of an antioxidant such as α-tocopherol, probucol, or N-acetylcysteine. FOH could accelerate ROS generation only in cells of the wild-type grande strain, not in those of the respiration-deficient petite mutant ([rho⁰]), which illustrates the role of the mitochondrial electron transport chain as its origin. Among the respiratory chain inhibitors, ROS generation could be effectively eliminated with myxothiazol, which inhibits oxidation of ubiquinol to the ubisemiquinone radical by the Rieske iron-sulfur center of complex III, but not with antimycin A, an inhibitor of electron transport that is functional in further oxidation of the ubisemiquinone radical to ubiquinone in the Q cycle of complex III. Cellular oxygen consumption was inhibited immediately upon extracellular addition of FOH, whereas FOH and its possible metabolites failed to directly inhibit any oxidase activities detected with the isolated mitochondrial preparation. A protein kinase C (PKC)-dependent mechanism was suggested to exist in the inhibition of mitochondrial electron transport since FOH-induced ROS generation could be effectively eliminated with a membrane-permeable diacylglycerol analog which can activate PKC. The present study supports the idea that FOH inhibits the ability of the electron transport chain to accelerate ROS production via interference with a phosphatidylinositol type of signal.     

Characterization of the glutathione‐dependent reduction of the peroxiredoxin 5 homolog PfAOP from Plasmodium falciparum

Peroxiredoxins use a variety of thiols to rapidly reduce hydroperoxides and peroxynitrite. While the oxidation kinetics of peroxiredoxins have been studied in great detail, enzyme‐specific differences regarding peroxiredoxin reduction and the overall rate‐limiting step under physiological conditions often remain to be deciphered. The 1‐Cys peroxiredoxin 5 homolog PfAOP from the malaria parasite Plasmodium falciparum is an established model enzyme for glutathione/glutaredoxin‐dependent peroxiredoxins. Here, we reconstituted the catalytic cycle of PfAOP in vitro and analyzed the reaction between oxidized PfAOP and reduced glutathione (GSH) using molecular docking and stopped‐flow measurements. Molecular docking revealed that oxidized PfAOP has to adopt a locally unfolded conformation to react with GSH. Furthermore, we determined a second‐order rate constant of 6 × 10⁵ M⁻¹ s⁻¹ at 25°C and thermodynamic activation parameters ΔH ^‡, ΔS ^‡, and ΔG ^‡ of 39.8 kJ/mol, −0.8 J/mol, and 40.0 kJ/mol, respectively. The gain‐of‐function mutant PfAOP^L109M had almost identical reaction parameters. Taking into account physiological hydroperoxide and GSH concentrations, we suggest (a) that the reaction between oxidized PfAOP and GSH might be even faster than the formation of the sulfenic acid in vivo, and (b) that conformational changes are likely rate limiting for PfAOP catalysis. In summary, we characterized and quantified the reaction between GSH and the model enzyme PfAOP, thus providing detailed insights regarding the reactivity of its sulfenic acid and the versatile chemistry of peroxiredoxins.     

Influence of Osmotic and Nutritional Stress on Physiology of Penicillium fellutanum in Removal of Phosphocholine and Modification of Phospho-1-O-[N-Peptidyl-(2-Aminoethanol)] Phosphodiesters of Peptidophosphogalactomannan Species

Addition of 3 M NaCl to 72-h cultures of Penicillium fellutanum in 2 mM phosphate resulted in an increase in percentage of extracellular peptidophosphogalactomannan III (pP_xGMⁱⁱⁱ) and a decrease in that of pP_xGMⁱⁱ. The magnitude of ³¹P nuclear magnetic resonance signals at 1.47 and 1.33 ppm of phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiesters pP_xGMⁱⁱ and pP_xGMⁱⁱⁱ decreased compared with controls. The data suggest that serine, glycine, and threonine residues from the 3-kDa peptide and from galactofuranosyl-6-O-phospho-1′-O-[N-peptidyl-(2-aminoethanol)] residues were the precursors of the needed choline-derived osmolytes.     

Glutathione peroxidase activity of glutathione-S-transferases purified from rat liver

Gel filtration chromatography demonstrated the presence of two peaks of glutathione peroxidase activity assayed with cumene hydroperoxide in the soluble fraction of rat liver, brain, kidney, and testis. The peak with an approximate molecular weight of 45,000 (GSH-Px II) was purified from rat liver labeled $\text{in vivo}$ with Na₂⁷⁵SeO₃. Chromatography on DEAE-cellulose, Sephadex G-150, DEAE-cellulose, and CM-cellulose resulted in the co-purification of glutathione-S-transferase activity measured with 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity assayed with cumene hydroperoxide, and in the removal of all detectable ⁷⁵Se. Studies on GSH-Px II indicated that the apparent K_m for both cumene and t-butyl hydroperoxides was considerably higher than that for purified seleno-glutathione peroxidase. The V_max estimated with cumene hydroperoxide was only $\text{1}\text{300}$ of that determined for the selenoenzyme at pH 7.5 and with 1 mM GSH.     

Mitochondria-mediated nuclear mutator phenotype in Saccharomyces cerevisiae

Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine-resistant colonies as a measure of nuclear mutator phenotype. Our data suggest that mitochondrial dysfunction leads to a nuclear mutator phenotype (i) when oxidative phosphorylation is blocked in wild-type yeast at mitochondrial complex III by antimycin A and (ii) in mutant strains lacking the entire mitochondrial genome (rho⁰) or those with deleted mitochondrial DNA (rho^–). The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho^– and rho⁰ cells were ~2- to 3-fold higher compared to untreated control and wild-type cells, respectively. Blockage of oxidative phosphorylation by antimycin A treatment led to increased intracellular levels of reactive oxygen species (ROS). In contrast, inactivation of mitochondrial activity (rho^– and rho⁰) led to decreased intracellular levels of ROS. We also demonstrate that in rho⁰ cells the REV1, REV3 and REV7 gene products, all implicated in error-prone translesion DNA synthesis (TLS), mediate mutagenesis in the nuclear genome. However, TLS was not involved in nuclear DNA mutagenesis caused by inhibition of mitochondrial function by antimycin A. Together, our data suggest that mitochondrial dysfunction is mutagenic and multiple pathways are involved in this nuclear mutator phenotype.     

Uptake of Choline and Its Conversion to Glycine Betaine by Bacteria in Estuarine Waters

The uptake and degradation of nanomolar levels of [methyl-¹⁴C]choline in estuarine water samples and in seawater filtrate cultures composed mainly of natural free-living bacteria was studied. Uptake of [¹⁴C]choline exhibited Michaelis-Menten kinetics, with K_t + S_n values of 1.7 to 2.9 nM in filtrate cultures and 1.7 to 4.1 nM in estuarine-water samples. V_max values ranged from 0.5 to 3.3 nM · h⁻¹. The uptake system for choline in natural microbial assemblages therefore displays very high affinity and appears able to scavenge this compound at the concentrations expected in seawater. Uptake of choline was inhibited by some natural structural analogs and p-chloromercuribenzoate, indicating that the transporter may be multifunctional and may involve a thiol binding site. When 11 nM [¹⁴C]choline was added to water samples, a significant fraction (>50%) of the methyl carbon was respired to CO₂ in incubations lasting 10 to 53 h. Cells taking up [¹⁴C]choline produced [¹⁴C]glycine betaine ([¹⁴C]GBT), and up to 80% of the radioactivity retained by cells was in the form of GBT, a well-known osmolyte. Alteration of the salinity in filtrate cultures affected the relative proportion of [¹⁴C]choline degraded or converted to [¹⁴C]GBT, without substantially affecting the total metabolism of choline. Increasing the salinity from 14 to 25 or 35 ppt caused more [¹⁴C]GBT to be produced from choline but less ¹⁴CO₂ to be produced than in the controls. Lowering the salinity to 7 ppt decreased [¹⁴C]GBT production and increased ¹⁴CO₂ production slightly. Intracellular accumulations of [¹⁴C]GBT in the salt-stressed cultures were osmotically significant (34 mM). Choline may be used as an energy substrate by estuarine bacteria and may also serve as a precursor of the osmoprotectant GBT, particularly as bacteria are mixed into higher-salinity waters.     

Polycyclic Aromatic Hydrocarbon Metabolism by White Rot Fungi and Oxidation by Coriolopsis gallica UAMH 8260 Laccase

We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of high levels of laccase and manganese peroxidase, was used as the growth medium. Ten fungi grown for 5 days in this medium in the presence of anthracene, pyrene, or phenanthrene, each at a concentration of 5 μg/ml could metabolize these PAHs. We studied the oxidation of 10 PAHs by using laccase purified from Coriolopsis gallica. The reaction mixtures contained 20 μM PAH, 15% acetonitrile in 60 mM phosphate buffer (pH 6), 1 mM 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and 5 U of laccase. Laccase exhibited 91% of its maximum activity in the absence of acetonitrile. The following seven PAHs were oxidized by laccase: benzo[a]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene, biphenylene, acenaphthene, and phenanthrene. There was no clear relationship between the ionization potential of the substrate and the first-order rate constant (k) for substrate loss in vitro in the presence of ABTS. The effects of mediating substrates were examined further by using anthracene as the substrate. Hydroxybenzotriazole (HBT) (1 mM) supported approximately one-half the anthracene oxidation rate (k = 2.4 h⁻¹) that ABTS (1 mM) supported (k = 5.2 h⁻¹), but 1 mM HBT plus 1 mM ABTS increased the oxidation rate ninefold compared with the oxidation rate in the presence of ABTS, to 45 h⁻¹. Laccase purified from Pleurotus ostreatus had an activity similar to that of C. gallica laccase with HBT alone, with ABTS alone, and with 1 mM HBT plus 1 mM ABTS. Mass spectra of products obtained from oxidation of anthracene and acenaphthene revealed that the dione derivatives of these compounds were present.