Comparative studies on lignin and polycyclic aromatic hydrocarbons degradation by basidiomycetes fungi

A total of 130 wild basidiomycetes fungi were collected and identified. The polycyclic aromatic hydrocarbons (PAHs) degradation by the potential Phellinus sp., Polyporus sulphureus (in liquid state fermentation (LSF), solid state fermentation (SSF), in soil) and lignin biodegradation were compared with those of a bacterial isolate and their corresponding cocultures. The PAHs degradation was higher in LSF and the efficiency of the organisms declined in SSF and in soil treatment. Phellinus sp. showed better degradation in SSF and in soil. Bacillus pumilus showed higher degradation in LSF. B. pumilus was seen to have lower lignin degradation than the fungal cultures and the cocultures could not enhance the degradation. Phellinus sp. which had higher PAHs and lignin degradation showed higher biosurfactant production than other organism. Manganese peroxidase (MnP) was the predominant enzyme in Phellinus sp. while lignin peroxidase (Lip) was predominant in P. sulphureus.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

Degradation of polycyclic aromatic hydrocarbons with three to seven aromatic rings by higher fungi in sterile and unsterile soils

Seven commercial 3- to 7-ring (R) polycyclic aromatic hydrocarbons (PAH) as well as PAH derived from lignite tar were spiked into 3 soils (0.8 to 9.7% of organic carbon). The disappearance of the original PAH was determined for the freshly spiked soils, for soils incubated for up to 287 d with their indigenous microflora, and for autoclaved, unsterile and pasteurized soils inoculated with basidiomycetous and ascomycetous fungi. Three to 12 d after spiking, 22 to 38% of the PAH could no longer be recovered from the soils. At 287 d, 88.5 to 92.7%, 83.4 to 87.4%, and 22.0 to 42.1% of the 3-, 4-, and 5- to 7-R PAH, respectively, had disappeared from the unsterile, uninoculated soils. In 2 organic-rich sterile soils, the groups of wood- and straw-degrading, terricolous, and ectomycorrhizal fungi reduced the concentration of 5 PAH by 12.6, 37.9, and 9.4% in 287 d. Five- to 7-R PAH were degraded as efficiently as most of the 3- to 4-R PAH. In organic-rich unsterile soils inoculated with wood- and straw-degrading fungi, the degradation of 3- to 4-R PAH was not accelerated by the presence of fungi.The 5- to 7-R PAH, which were not attacked by bacteria, were degraded by fungi to 29 to 42% in optimum combinations of fungal species and soil type. In organic-poor unsterile soil, these same fungi delayed the net degradation of PAH possibly for 2 reasons. Mycelia of Pleurotus killed most of the indigenous soil bacteria expected to take part in the degradation of PAH, whereas those of Hypholoma and Stropharia promoted the development of opportunistic bacteria in the soil, which must not necessarily be PAH degraders. Contemporarily, the contribution of the fungi themselves to PAH degradation may be negligible in the absence of soil organic matter due to the lower production of ligninolytic enzymes. It is concluded that fungi degrade PAH irrespective of their molecular size in organic-rich and wood chip-amended soils which promote fungal oxidative enzyme production.     

Occurrence and community composition of fast-growing Mycobacterium in soils contaminated with polycyclic aromatic hydrocarbons

Fast-growing mycobacteria are considered essential members of the polycyclic aromatic hydrocarbons (PAH) degrading bacterial community in PAH-contaminated soils. To study the natural role and diversity of the Mycobacterium community in contaminated soils, a culture-independent fingerprinting method based on PCR combined with denaturing gradient gel electrophoresis (DGGE) was developed. New PCR primers were selected which specifically targeted the 16S rRNA genes of fast-growing mycobacteria, and single-band DGGE profiles of amplicons were obtained for most Mycobacterium strains tested. Strains belonging to the same species revealed identical DGGE fingerprints, and in most cases, but not all, these fingerprints were typical for one species, allowing partial differentiation between species in a Mycobacterium community. Mycobacterium strains inoculated in soil were detected with a detection limit of 10(6) CFU g(-1) of soil using the new primer set as such, or approximately 10(2) CFU g(-1) in a nested PCR approach combining eubacterial and the Mycobacterium specific primers. Using the PCR-DGGE method, different species could be individually recognized in a mixed Mycobacterium community. This approach was used to rapidly assess the Mycobacterium community structure of several PAH-contaminated soils of diverse origin with different overall contamination profiles, pollution concentrations and chemical-physical soil characteristics. In the non-contaminated soil, most of the recovered 16SrRNA gene sequence did not match with previous described PAH-degrading Mycobacterium strains. In most PAH-contaminated soils, mycobacteria were detected which were closely related to fast-growing species such as Mycobacterium frederiksbergense and Mycobacterium austroafricanum, species that are known to include strains with PAH-degrading capacities. Interestingly, 16S rRNA genes related to M. tusciae sequences, a Mycobacterium species so far not reported in relation to biodegradation of PAHs, were detected in all contaminated soils.     

Degradation of polycyclic aromatic hydrocarbons in soil by a two-step sequential treatment

The objectives of this work were to isolate the microorganisms responsible for a previously observed degradation of polycyclic aromatic hydrocarbons (PAH) in soil and to test a method for cleaning a PAH-contaminated soil. An efficient PAH degrader was isolated from an agricultural soil and designated as Mycobacterium LP1. In liquid culture, it degraded phenanthrene (58%), pyrene (24%), anthracene (21%) and benzo(a)pyrene (10%) present in mixture (initial concentration 50 μg ml⁻¹ each) and phenanthrene (92%) and pyrene (94%) as sole carbon sources after 14 days of incubation at 30°C. In soil, Mycobacterium LP1 mineralised ¹⁴C-phenanthrene (45%) and ¹⁴C-pyrene (65%) after 10 days. The good ability of this Mycobacterium was combined with the benzo(a)pyrene oxidation effect obtained by 1% w/w rapeseed oil in a sequential treatment of a PAH-spiked soil (total PAH concentration 200 mg kg⁻¹). The first step was incubation with the bacterium for 12 days and the second step was the addition of the rapeseed oil after this time and a further incubation of 22 days. Phenanthrene (99%), pyrene (95%) and anthracene (99%) were mainly degraded in the first 12 days and a total of 85% of benzo(a)pyrene was transformed during the whole process. The feasibility of the method is discussed.     

Expression profiles of seven glutathione S-transferase (GST) genes in cadmium-exposed river pufferfish (Takifugu obscurus)

Glutathione S-transferase (GST; EC 2.5.1.18) plays a critical role in detoxification pathways. In this study, we report cloning and expression of seven genes of the GST family of the pufferfish Takifugu obscurus together with mRNA tissue distribution pattern and time-course of expression in response to exposure to cadmium. At basal levels of tissue expression, GST-Mu is highly expressed in liver compared with other tissues. When fish were exposed to cadmium (5 mg/L for 96 h), expression of GST-MAPEG, GST-Mu, GST-Omega, and GST-Zeta was greatly increased, whereas GST-Alpha and GST-Kappa genes showed no significant response. These findings suggest that gene expression of a number of GST isoforms in T. obscurus is modulated in response to exposure to cadmium. We propose GST-Mu, GST-Theta, and GST-Zeta as candidate biomarkers for heavy metal exposure in this fish.     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.     

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.     

Effect of glutathione biosynthesis-related modulators on the thiol redox state enzymes and on sclerotial differentiation of filamentous phytopathogenic fungi

In this study, sclerotial differentiation in filamentous phytopathogenic fungi, representing the four main types of sclerotia, was studied in relation to thiol redox state (TRS)-related enzymes and their substrates/products. TRS was altered by the general TRS modulator Ν-acetylcysteine (AcCSH) and by the glutathione (GSH) biosynthesis modulators l-oxo-thiazolidine-4-carboxylate (OTC), and l-buthionine-S,R-sulfoximine (BSO). This study showed that the four studied types of sclerotial differentiation are directly related with the antioxidant –SH groups of GSH and/or CSH, since the decrease of sclerotial differentiation concurred with an increase of these thiols by the GSH biosynthesis modulators AcCSH, OTC, and BSO. Supportive to that conclusion is the fact that, in general, the activities of the TRS-related enzymes GR/GPDH and Ttase decrease in the end of the undifferentiated stage due to the substitution of their antioxidant function by the antioxidant potential of the –SH group providers AcCSH and OTC. Moreover, it was found that BSO expectedly suppressed GSH biosynthesis in the tested fungi, and unexpectedly decreased their sclerotial differentiation by a dose-dependent manner typical for antioxidants. The possible antioxidant role of BSO was supported by the decrease it caused in the antioxidant enzymes GR/GPDH and Ttase. The results of this study are in accordance with our hypothesis that sclerotial differentiation in phytopathogenic fungi is induced by oxidative stress.     

Enhancement of bioconversion of high-molecular mass polycyclic aromatic hydrocarbons in contaminated non-sterile soil by litter-decomposing fungi

With the focus on alternative microbes for soil-bioremediation, 18 species of litter-decomposing basidiomycetous fungi were screened for their ability to grow on different lignocellulosic substrates including straw, flax and pine bark as well as to produce ligninolytic enzymes, namely laccase and manganese peroxidase. Following characteristics have been chosen as criteria for the strain selection: (i) the ability to grow at least on one of the mentioned materials, (ii) production of either of the ligninolytic enzymes and (iii) the ability to invade non-sterile soil. As the result, eight species were selected for a bioremediation experiment with an artificially contaminated soil (total polycyclic aromatic hydrocarbon (PAH) concentration 250 mg/kg soil). Up to 70%, 86% and 84% of benzo(a)anthracene, benzo(a)pyrene, and dibenzo(a,h)anthracene, respectively, were removed in presence of fungi while the indigenous microorganisms converted merely up to 29%, 26% and 43% of these compounds in 30 days. Low molecular-mass PAHs studied were easily degraded by soil microbes and only anthracene degradation was enhanced by the fungi as well. The agaric basidiomycetes Stropharia rugosoannulata and Stropharia coronilla were the most efficient PAH degraders among the litter-decomposing species used.     

An Arabidopsis gene with homology to glutathione S-transferases is regulated by ethylene

A cDNA clone obtained from Arabidopsis leaf RNA encodes a 24 kDa protein with homology to glutathione S-transferases (GST). It is most homologous with a tobacco GST (57% identity). In Arabidopsis, expression of GST mRNA is regulated by ethylene. Exposure of plants to ethylene increased the abundance of GST mRNA, while treatment with norbornadiene had the reverse effect. Ethylene had no effect on the mRNA level in ethylene-insensitive etr1 plants. The abundance of this mRNA increased with the age of plants. DNA hybridizations indicate that GSTs are encoded by a large multigene family in Arabidopsis.     

Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium,Nitrosomonas europaea

The soil nitrifying bacterium Nitrosomonas europaea has shown the ability to transform cometabolically naphthalene as well as other 2- and 3-ringed polycyclic aromatic hydrocarbons (PAHs) to more oxidized products. All of the observed enzymatic reactions were inhibited by acetylene, a selective inhibitor of ammonia monooxygenase (AMO). A strong inhibitory effect of naphthalene on ammonia oxidation by N. europaea was observed. Naphthalene was readily oxidized by N. europaea and 2-naphthol was detected as a major product (85%) of naphthalene oxidation. The maximum naphthol production rate was 1.65 nmole/mg protein-min in the presence of 240 M naphthalene and 10 mM NH₄ ⁺. Our results demonstrate that the oxidation between ammonia and naphthalene showed a partial competitive inhibition. The relative ratio of naphthalene and ammonia oxidation, depending on naphthalene concentrations, demonstrated that the naphthalene was oxidized 2200-fold slower than ammonia at lower concentration of naphthalene (15 M) whereas naphthalene was oxidized only 100-fold slower than ammonia oxidation. NH₄ ⁺- and N₂H₄-dependent O₂ uptake measurement demonstrated irreversible inhibitory effects of the naphthalene and subsequent oxidation products on AMO and HAO activity.     

Biodegradation of soil-adsorbed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus

The white rot fungus, Pleurotus ostreatus, metabolized four soil adsorbed polycyclic aromatic hydrocarbons: 50% of pyrene (0.1 mg g^–1 dry soil), 68% of anthracene and 63% of phenanthrene were mineralized after 21 d. Biodegradation was increased to 75%, 80% and 75%, respectively of the initial concentration when 0.15% Tween 40 was added. Biodegradation of pyrene in the presence of surfactant and H₂O₂ (1.0 mM) was 90%. Benz[a]pyrene was also oxidized by Pleurotus ostreatus but it is not mineralized.     

Application of microscopic fungi isolated from polluted industrial areas for polycyclic aromatic hydrocarbons and pentachlorophenol reduction

The growth abilities of fifteen fungal strains isolated fromcontaminated areas, in the presence of xenobiotics compounds mixture (overworked cuttingfluid, crude and waste oil) were examined. Strains with the richest growth were chosen for anthracene, phenanthrene and pentachlorophenol biodegradation in Sabouraudmedium (with initial xenobiotic concentration 250 mg/l in cultures with polycyclicaromatic hydrocarbons and 10 mg/l for the chlorinated substrate). Strains IM 1063and IM 6325 were able to attack phenanthrene forming its derivative 9-phenanthrenolwith the yields 5.22 mg/l and 2.82 mg/l, respectively. Strain IM 1063 and IM 6325 transformed pentachlorophenol to an intermediatecompound – pentachloromethoxybenzene. Final content of pentachloromethoxybenzene reached 3.46 mg/l and3.2 mg/l, respectively. Strain IM 6203 (contrary to other strains) released an intermediateproduct of pentachlorophenol metabolism – 2,3,5,6-tetrachlorohydroquinone(8.73 mg/l substrate remaining and 1.2 mg/l 2,3,5,6-tetrachlorohydroquinone forming).The IM 6203 strain was identified as Mucor ramosissimus. The chlorinatedpesticide degradation by M. ramossimus was improved significantly on a medium with overworked oil. Only 8.3% of pentachlorophenol and 4.3% of 2,3,5,6-tetrachlorohydroquinone in relation to the introduced substrate (10 mg/l) were found, after7 days of incubation. The growth of M. ramosissimus on medium with overworked oil in pentachlorophenol presence was associated with oil emulgation,which enhanced fungal growth and the pesticide degradation.     

The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light/dark cycles involves genes that represent direct responses to light and photosynthesis

In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)⁺ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40 000 clones in each library revealed 44 clones which hybridize preferentially with a [³²P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [³²P] cDNA probe synthesized from poly(A)⁺ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.     

Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.     

Implication of two glutathione S-transferases in the optimal metabolism of m-toluate by Sphingomonas yanoikuyae B1

A putative glutathione S-transferase (GST) gene (bphK) was identified in the meta-cleavage operon for the degradation of m-toluate by Sphingomonas yanoikuyae B1. Disruption of bphK resulted in the loss of GST activity against 1-chloro-2,4-dinitrobenzene and a much increased lag time of the mutant strain MB3 (bphK::Km) following subculture into m-toluate medium. In contrast, an increased lag time was not observed when MB3 was grown on biphenyl or m-xylene and MB3 showed normal growth on m-toluate when complemented with a subclone containing the bphK gene only. Furthermore, an additional GST activity was detected in MB3. The induction timing of this second GST activity coincided with the beginning of the exponential growth phase of MB3 on m-toluate, reached maximal activity within three hours, and then dropped sharply to the basal level. Thus, it is apparent that BphK and/or the second GST are necessary for optimal growth of B1 on m-toluate. This revised version was published online in June 2006 with corrections to the Cover Date.