Fungal Alcohol Dehydrogenases: Physiological Function, Molecular Properties, Regulation of Their Production, and Biotechnological Potential.

Fungal alcohol dehydrogenases (ADHs) participate in growth under aerobic or anaerobic conditions, morphogenetic processes, and pathogenesis of diverse fungal genera. These processes are associated with metabolic operation routes related to alcohol, aldehyde, and acid production. The number of ADH enzymes, their metabolic roles, and their functions vary within fungal species. The most studied ADHs are associated with ethanol metabolism, either as fermentative enzymes involved in the production of this alcohol or as oxidative enzymes necessary for the use of ethanol as a carbon source; other enzymes participate in survival under microaerobic conditions. The fast generation of data using genome sequencing provides an excellent opportunity to determine a correlation between the number of ADHs and fungal lifestyle. Therefore, this review aims to summarize the latest knowledge about the importance of ADH enzymes in the physiology and metabolism of fungal cells, as well as their structure, regulation, evolutionary relationships, and biotechnological potential.

Secretion of the siderophore rhizoferrin is regulated by the cAMP-PKA pathway and is involved in the virulence of Mucor lusitanicus.

Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that deletion of rfs, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in Mucor lusitanicus, led to a lower virulence in diabetic mice and nematodes. Upregulation of rfs correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H2O2 in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing M. lusitanicus in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (pkaR1), correlated with a decrease in the toxicity of SS, downregulation of rfs, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in M. lusitanicus. Moreover, rfs upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of rfs in M. lusitanicus infection.

Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence.

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1+) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1+ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

Alcohol dehydrogenase 1 participates in the Crabtree effect and connects fermentative and oxidative metabolism in the Zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of adh mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh+ strain R7B, the ADH-defective (Adh-) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD+-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the adhl gene. Complementation with the wild-type allele adh1+ by transformation of M5 remedied all the defects caused by the adh1 mutation. These findings indicate that in M. circinelloides, the product of the adh1 gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of M. circinelloides possess low mRNA levels of the ethanol assimilation genes (adl2 and acs2), which could explain their inability to grow in the alcohol.

Impact of Cr(VI) on the oxidation of polyunsaturated fatty acids in Helianthus annuus roots studied by metabolomic tools.

The impact of Cr(VI) in sunflower roots has been studied, focusing on the oxidation of polyunsaturated fatty acids. Plants were grown hydroponically in the presence of 0, 1.0, 5.0 and 25 mgCr L-1. Methanolic root extracts were analyzed by capillary liquid chromatography coupled through negative electrospray ionization to a quadrupole-time of flight mass spectrometry (capHPLC-ESI-QTOF-MS). Using partial least squares algorithm, eighteen features strongly affected by Cr(VI) were detected and annotated as linoleic acid (LA), alpha-linolenic acid (ALA) and sixteen oxidation products containing hydroperoxy-, epoxy-, keto-, epoxyketo- or hydroxy-functionalities, all of them classified as oxylipins. Inspection of the MS/MS spectra acquired for features eluting at different retention times but assigned as a sole compound, confirmed isomers formation: three hydroperoxy-octadecadienoic acids (HpODE), two oxo-octadecadienoic acids (OxoODE) and four epoxyketo-octadecenoic acids (EKODE). Around 70% of metabolites in sunflower LA metabolic pathway were affected by Cr(VI) stress and additionally, four EKODE isomers not included in this pathway were found in the exposed roots. Among ALA-derived oxylipins, 13-epi-12-oxo-phytodienoic acid (OPDA) is of relevance, because of its participation in the activation of secondary metabolism. The abundances of all oxylipins were directly dependent on the Cr(VI) concentration in medium; furthermore, autooxidation of LA to HpODE isomers was observed after incubation with Cr(VI). These results point to the direct involvement of Cr(VI) in non-enzymatic oxidation of fatty acids; since oxylipins are signaling molecules important in plant defensive response, their synthesis under Cr(VI) exposure sustains the ability of sunflower to grow in Cr(VI)-contaminated environments.

CrpP Is a Novel Ciprofloxacin-Modifying Enzyme Encoded by the Pseudomonas aeruginosa pUM505 Plasmid.

The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed crpP, for ciprofloxacin resistance protein, plasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-crpP, conferred resistance to CIP on Escherichia coli strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in P. aeruginosa, which involves phosphorylation of the antibiotic.

Changes of Metabolomic Profile in Helianthus annuus under Exposure to Chromium(VI) Studied by capHPLC-ESI-QTOF-MS and MS/MS.

The application of capHPLC-ESI-QTOF-MS and MS/MS to study the impact of Cr(VI) on metabolites profile in Helianthus annuus is reported. Germinated seeds were grown hydroponically in the presence of Cr(VI) (25 mgCr/L) and root extracts of the exposed and control plants were analyzed by untargeted metabolomic approach. The main goal was to detect which metabolite groups were mostly affected by Cr(VI) stress; two data analysis tools (ProfileAnalysis, Bruker, and online XCMS) were used under criteria of intensity threshold 5 · 104 cps, fold change ≥ 5, and p ≤ 0.01, yielding precursor ions. Molecular formulas were assigned based on data processing with two computational tools (SIRIUS and MS-Finder); annotation of candidate structures was performed by database search using CSI:FingerID and MS-Finder. Even though ultimate identification has not been achieved, it was demonstrated that secondary metabolism became activated under Cr(VI) stress. Among 42 candidate compounds returned from database search for seven molecular formulas, ten structures corresponded to isocoumarin derivatives and eleven were sesquiterpenes or sesquiterpene lactones; three benzofurans and four glycoside or pyrane derivatives of phenolic compounds were also suggested. To gain further insight on the effect of Cr(VI) in sunflower, isocoumarins and sesquiterpenes were selected as the target compounds for future study.

Cr(VI) reduction by gluconolactone and hydrogen peroxide, the reaction products of fungal glucose oxidase: Cooperative interaction with organic acids in the biotransformation of Cr(VI).

The Cr(VI) reducing capability of growing cells of the environmental A. tubingensis Ed8 strain is remarkably efficient compared to reference strains A. niger FGSC322 and A. tubingensis NRRL593. Extracellular glucose oxidase (GOX) activity levels were clearly higher in colonies developed in solid medium and in concentrated extracts of the spent medium of liquid cultures of the Ed8 strain in comparison with the reference strains. In addition, concentrated extracts of the spent medium of A. tubingensis Ed8, but not those of the reference strains, exhibited the ability to reduce Cr(VI). In line with this observation, it was found that A. niger purified GOX is capable of mediating the conversion of Cr(VI) to Cr(III) in a reaction dependent on the presence of glucose that is stimulated by organic acids. Furthermore, it was found that a decrease in Cr(VI) may occur in the absence of the GOX enzyme, as long as the reaction products gluconolactone and hydrogen peroxide are present; this conversion of Cr(VI) is stimulated by organic acids in a reaction that generates hydroxyl radicals, which may involve the formation of an intermediate peroxichromate(V) complex. These findings indicated that fungal glucose oxidase acts an indirect chromate reductase through the formation of Cr(VI) reducing molecules, which interact cooperatively with other fungal metabolites in the biotransformation of Cr(VI).

Role of Bacillus subtilis error prevention oxidized guanine system in counteracting hexavalent chromium-promoted oxidative DNA damage.

Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular reduction of hexavalent chromium [Cr(VI)] to trivalent chromium [Cr(III)] may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system.

Bacterial biodiversity from anthropogenic extreme environments: a hyper-alkaline and hyper-saline industrial residue contaminated by chromium and iron.

Anthropogenic extreme environments are among the most interesting sites for the bioprospection of extremophiles since the selection pressures may favor the presence of microorganisms of great interest for taxonomical and astrobiological research as well as for bioremediation technologies and industrial applications. In this work, T-RFLP and 16S rRNA gene library analyses were carried out to describe the autochthonous bacterial populations from an industrial waste characterized as hyper-alkaline (pH between 9 and 14), hyper-saline (around 100 PSU) and highly contaminated with metals, mainly chromium (from 5 to 18 g kg(-1)) and iron (from 2 to 108 g kg(-1)). Due to matrix interference with DNA extraction, a protocol optimization step was required in order to carry out molecular analyses. The most abundant populations, as evaluated by both T-RFLP and 16S rRNA gene library analyses, were affiliated to Bacillus and Lysobacter genera. Lysobacter related sequences were present in the three samples: solid residue and lixiviate sediments from both dry and wet seasons. Sequences related to Thiobacillus were also found; although strains affiliated to this genus are known to have tolerance to metals, they have not previously been detected in alkaline environments. Together with Bacillus (already described as a metal reducer), such organisms could be of use in bioremediation technologies for reducing chromium, as well as for the prospection of enzymes of biotechnological interest.

Fusarium oxysporum Adh1 has dual fermentative and oxidative functions and is involved in fungal virulence in tomato plants.

An alcohol dehydrogenase gene, adh1, has been identified in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that adh1 is highly expressed in mycelia grown in potato dextrose liquid medium (PDB) under hypoxic conditions, as compared to mycelia grown under aerobic conditions. One spontaneous allyl alcohol-resistant (Ally(R)) mutant exhibited insertion of an incomplete F.oxysporum transposable element, while another mutant contained a short (13 nucleotide) deletion, in both cases interrupting the coding region of the adh1 gene. These mutations caused deficiency in Adh activity due to loss of the main constitutive isoform of Adh1, as well as alteration of different physiological parameters related to carbon and energy metabolism, including the ability to use ethanol as a carbon source under aerobic conditions; impaired growth under hypoxic conditions with glucose as the carbon source; and diminished production of ethanol in glucose-containing medium. Interestingly, the adh1 mutations resulted in a significant delay in fungal disease development in tomato plants. Complementation with the wild-type adh1 allele repaired all defects caused by mutation, indicating that the product of the adh1 gene has dual enzymatic functions (fermentative and oxidative), depending on culture conditions, and is also required for full fungal virulence.

Effect of Fusarium oxysporum f. sp. lycopersici on the soil-to-root translocation of heavy metals in tomato plants susceptible and resistant to the fungus.

The purpose of this work was to gain an insight on the potential role of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici in the translocation of metals and metalloids from soil to plant roots in tomato (Lycopersicum esculentum). Two varieties of tomato (one susceptible and another resistant to infection by Fusarium oxysporum f. sp. lycopersici) were challenged with the fungus for different periods of time, and several elements (V, Cr, Mn, Co, Cu, Zn, As, Se, Mo, Ag, Cd, Pb) were determined in roots and in soil substrate. Additionally, phenolic plant products were also analyzed for the evaluation of the plant response to biotic stress. In order to obtain representative results for plants cultivated in noncontaminated environments, the infected and control plants were grown in commercial soil with natural, relatively low metal concentrations, partly associated with humic substances. Using such an experimental design, a specific role of the fungus could be observed, while possible effects of plant exposure to elevated concentrations of heavy metals were avoided. In the infected plants of two varieties, the root concentrations of several metals/metalloids were increased compared to control plants; however, the results obtained for elements and for phenolic compounds were significantly different in the two plant varieties. It is proposed that both Lycopersicum esculentum colonization by Fusarium oxysporum f. sp. lycopersici and the increase of metal bioavailability due to fungus-assisted solubilization of soil humic substances contribute to element traffic from soil to roots in tomato plant.

Effect of Fusarium oxysporum f. sp. lycopersici on the degradation of humic acid associated with Cu, Pb, and Ni: an in vitro study.

The intent of this work was to gain further insight on the fungus-assisted degradation/solubilization of humic acid and the related changes in metal-binding profiles. In the experimental design, Aldrich reagent humic acid (HA) or HA enriched with Cu, Pb, and Ni (HA(Me)) was added to Fusarium oxysporum f. sp. lycopersici cultures in vitro. The cultures were supplied by different carbon- and nitrogen-containing nutrients (glucose, Glc, or glutamate, Glu and ammonium, NH4+, or nitrate, NO3-, ions, respectively) in order to examine their possible effect on HA and HA(Me) decomposition. During the first 48 h of fungus growth, gradual acidification to pH 2 was observed in medium containing Glc + NH4+, while for other cultures, alkalinization to pH 9 occurred and then, the above conditions were stable up to at least 200 h. Size exclusion chromatography (SEC) with UV/Vis detection showed progressive degradation and solubilization of both HA and HA(Me) with the increasing time of fungus growth. However, the molecular mass distributions of HA-related soluble species were different in the presence of metals (HA(Me)) as referred to HA and were also influenced by the composition of growth medium. The solubilization of Pb, Cu, and Ni and their association with HA molecular mass fractions were studied using inductively coupled plasma mass spectrometry (ICP-MS) detection. Under acidic conditions, relatively high concentrations of low-molecular-mass metallic species were found in culture supernatants, while in alkaline media, metal solubilization was generally poorer. In contrast to low pH culture, SEC-ICP-MS results obtained in alkaline supernatants indicated metal binding to degradation products of humic substances of MM > 5 kDa. In summary, the results of this study suggest that fungus-assisted degradation of HA and HA(Me) might be controlled using appropriate N- and C- sources required for fungus growth, which in turn would affect molecular mass distribution of soluble metallic species thus potentially influencing their actual bioaccessibility.

Cr(VI) reduction by an Aspergillus tubingensis strain: role of carboxylic acids and implications for natural attenuation and biotreatment of Cr(VI) contamination.

Experiments conducted in shake flask cultures, in minimal medium of pH 5.3 containing 50 microg mL(-1)Cr(VI) with glucose as a carbon source, indicated that the biomass of Aspergillus sp. strain Ed8, a chromate-tolerant fungal strain previously isolated from a chromium-polluted soil, responds to the presence of citrate in the medium by increasing the rate of Cr(VI) reduction; this effect required the use of live biomass and was not observed in medium with lactate. Other natural carboxylic acids or non-natural metal chelating agents showed a stimulatory effect of Cr(VI) reduction by Ed8 biomass; salicylate, tartrate and citrate were the stronger stimulators of the specific rate of Cr(VI) reduction, with about 12, 8 and 7-fold stimulatory effects, respectively, as compared to control cultures without additions. A procedure for Cr(VI) removal from a diluted electroplating effluent was devised, based on the use of growth medium amended with citrate or a mixture of salycilate-tartrate and cycles of recharge of growth medium-diluted effluent. In addition, conditions were adjusted in a 2-L bioreactor to reach a 20-fold increase in the volume of the reduction system with no loss of efficiency. Strain Ed8 was identified as an Aspergillus tubingensis isolate (included in Aspergillus niger species complex) on the basis of the ITS1-5.8s rDNA-ITS2 sequence similarity.

Analytical speciation of chromium in in-vitro cultures of chromate-resistant filamentous fungi.

In this work, different analytical speciation schemes have been used to study the reduction of Cr(VI) by a chromate-resistant strain of filamentous fungi Ed8 (Aspergillus sp), indigenous to contaminated industrial wastes. As demonstrated previously, this strain has the capability to reduce chromate present in the growth medium without its accumulation in the biomass, yet the reduced chromium end-products have not been characterized. Liquid growth medium, initially containing 50 mg L(-1) Cr(VI), was analyzed for Cr(III)/Cr(VI) and for total Cr at different time intervals (0-24 h) after inoculation with fungi. Three hyphenated procedures, based on the Cr(III)-EDTA formation and species separation by anion-exchange or ion-pairing reversed-phase chromatography with ICP-MS or DAD detection were used. The results obtained for Cr(VI) in each case were consistent, demonstrating efficient reduction of chromate during 24 h of Ed8 growth. However, pre-column complexation with EDTA did not ensure complete recovery of the reduced forms of chromium in the above procedures. An alternative speciation scheme, based on extraction of Cr(VI)-benzyltributylammonium bromide (BTAB) ion pairs into chloroform and subsequent determination of residual chromium by ICP-MS has provided evidence on the effective conversion of chromate into reduced chromium species in the growth medium. The results indicate the feasibility of using Ed8 strain for chromate bioremediation purposes. Analytically it can be concluded that speciation of chromium in biological systems should not be limited to its two most common oxidation states, because the actual reduced chromium species are not converted quantitatively to Cr(III)-EDTA.

The protective effect of selenium inorganic forms against cadmium and silver toxicity in mycelia of Pleurotus ostreatus.

The effect of two inorganic selenium forms has been investigated in the mycelia of Pleurotus ostreatus exposed to cadmium and silver salts in the shaken cultures. The degree of toxicity was assessed by the determination of malondialdehyde (MDA; a common biomarker of lipid peroxidation). The mycelia were exposed to one element form (up to 5 mg l(-1)) and also to the following combinations: cadmium(II) + selenium(IV); cadmium(II) + selenium(VI); silver(I) + selenium(IV); silver(I) + selenium(VI). The concentrations of cadmium, silver, selenium, and MDA were assessed in the mixed cytosol and cell membrane fractions (CCM). A positive correlation between MDA and cadmium was found in the CCM (beta=0.7775, P=0.0001), whereas the effect of silver was less significant (beta=0.4642, P=0.039). These results indicate that silver(I) and cadmium(II) have different capacities to induce lipid peroxidation in P. ostreatus. The protective role of selenium against metal-induced oxidative damage was found to be dependent on the oxidation state of the element form in the growth medium. The strongest beneficial effect was observed in mycelia exposed to cadmium(II) + selenium(IV) (inverse correlation between MDA and selenium in the CCM: beta=-0.7129, P=0.009) and it has been ascribed to a lower incorporation of the toxic metal and/or to possible intracellular interaction between selenium and cadmium. Under exposure to silver(I), the protective effect of selenium(IV) was less noticeable (correlation between MDA and selenium in the CCM; beta=-0.6068, P=0.036); in the presence of selenium(VI), no beneficial effect was observed.

Hexavalent chromium removal in vitro and from industrial wastes, using chromate-resistant strains of filamentous fungi indigenous to contaminated wastes.

Two chromate-resistant filamentous fungi, strains H13 and Ed8, were selected from seven independent fungal isolates indigenous to Cr(VI)-contaminated soil because of their ability to decrease hexavalent chromium levels in the growth medium. Morphophysiological studies identified strain H13 as a Penicillium sp. isolate and Ed8 as an Aspergillus sp. isolate. When incubated in minimal medium with glucose as a carbon source and in the presence of 50 microg/mL Cr(VI), these strains caused complete disappearance of Cr(VI) in the growth medium after about 72 h of incubation. Total chromium concentration in growth medium was constant during culture growth, and no accumulation of chromium in fungal biomass was observed. Quantitative determinations of oxidized and reduced chromium species during the reduction process revealed stoichiometric conversion of Cr(VI) to Cr(III). A decrease in Cr(VI) levels from industrial wastes was also induced by Ed8 or H13 biomass. These results indicate that chromate-resistant filamentous fungi with Cr(VI)-reducing capability could be useful for the removal of Cr(VI) contamination.

Se-enriched mycelia of Pleurotus ostreatus: distribution of selenium in cell walls and cell membranes/cytosol.

The incorporation of Se to fungi has been studied, focusing on element distribution among different cellular compartments and, in particular, polysaccharide structures contained in cell walls. Se-enriched mycelia of Pleurotus ostreatus were obtained in submerged cultures. The incorporation of selenium from the growth medium to mycelia was observed with the relative distribution between cytosol plus cell membranes fraction (CCM) and cell walls fraction (CW) of about 44 and 56%, respectively. CCM fractions were analyzed by size exclusion chromatography with on-line UV (280 nm) and ICP-MS detection (80Se). The results obtained showed selenium binding to components of different molecular masses (about 24% of total selenium coeluted with the compounds of molecular mass > 10 kDa). A polysaccharide-containing fraction of mycelia was treated alternatively with Tris-HCl at pH 7.5 or with chitinase. Better solubility and increased contribution of low molecular mass compounds were observed in chitinase extracts (UV detection), confirming the degradation of polysacharides by the enzyme. The total area under the ICP-MS chromatogram of chitinase extract was 2 times higher with respect to the area for Tris-HCl extract. Furthermore, the relative contribution of selenium in the low molecular mass fraction (molecular mass < 1 kDa) in chitinase extract was 72% as compared to 45% in Tris-HCl extract (based on peak area measurements with respect to total area under the chromatogram). The results obtained suggest selenium binding to chitin-containing polysaccharide structures in fungi cell walls.

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry.

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.

Presence and physiologic regulation of alcohol oxidase activity in an indigenous fungus isolated from petroleum-contaminated soils.

A soluble alcohol oxidase (AO) activity was detected in the mycelium of a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde. Zymogram analysis conducted with purified fractions from aerobic mycelium of YR-1 strain extracts indicated the existence of two AO enzymes (AO-1 and AO-2). Purified samples of both enzymes analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated the presence of three protein bands with molecular sizes 20,38, and 46 kDa that could be part of the native enzyme. In samples of both enzymes, the 46-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against AO from Hansenula polymorpha. The purified AO-2 enzyme oxidized different alcohols, although higher activity was displayed with hexadecanol. Km values obtained for methanol and hexa-decanol indicated a higher affinity for the latter. Analysis of the aminoter-minal sequence of the 46-kDa protein of AO-2 enzyme indicated significant similarity to enzymes involved in the metabolism of biphenyl polychloride compounds.