Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid β-oxidation, is usually lethal in both humans and animals, precluding the development of animal models of the disease. In contrast, CACT deficiency is conditionally lethal in the fungus Aspergillus nidulans, since loss-of-function mutations in acuH, the translocase structural gene, do not prevent growth on carbon sources other than ketogenic compounds, such as fatty acids. Here, we describe the molecular characterization of extant acuH alleles and the development of a fungal model for CACT deficiency based on the ability of human CACT to fully complement, when expressed at physiological levels, the growth defect of an A. nidulans ΔacuH strain on acetate and long-chain fatty acids. By using growth tests and in vitro assays this model enabled us to carry out a functional characterization of human CACT mutations showing that it may be useful for distinguishing potentially pathogenic human CACT missense mutations from neutral, single residue substitution-causing polymorphisms.

The role of actin in apical growth and enzyme secretion in the filamentous fungus Aspergillus nidulans was studied by treating the hyphae with cytochalasin A (CA), which inhibits actin polymerization. Indirect immunofluorescence microscopy revealed actin at the tips of main hyphae and branches, and at the sites of developing septa. CA inhibited the growth of the fungus and changed the growth pattern of hyphal tips from cylindrical tubes to spherical beads. The regions with swellings showed no actin fluorescence, and neither was actin seen in association with septa. After 4 h exposure, hyphae were able to resume the normal tip growth pattern in the presence of CA for a short period of time and new cylindrical hyphae, with actin fluorescence at the apex, emerged from the swollen tips. Later, the tips of the hyphae swelled again. which led to a beaded apperance. We also studied the effect of CA on the secretion of alpha-and beta-galactosidase. alpha-Galactosidase is secreted into the culture medium, whereas beta-galactosidase remains in the mycelium, with part of its activity bound to the cell wall. When A. nidulans mycelium was incubated in the presence of CA, a reduction in the secretion of alpha-galactosidase into the culture medium and a decrease in the alpha-and beta-galactosidase activities bound to the cell wall was detected. However, the CA dose used for the hyphae did not modify the secretion of the enzymes from protoplasts. Results described here provide evidence that a polymerized actin cytoskeleton is required for normal apical growth, hyphal tip shape and polarized enzyme secretion in A. nidulans. Cytochalasin-induced disruptions of the actin cytoskeleton could result in the alterations of apical growth and inhibition of enzyme secretion observed by blocking secretory vesicle transport to the apex.

The effects of methyl benzimidazole-2-yl carbamate (MBC) on microtubule and actin cytoskeleton were analyzed by indirect immunofluorescence and transmission electron microscopy in a wild-type strain and a benomyl-resistant mutant (benA10) ofAspergillus nidulans. The treatment of the wild-type strain with sublethal doses of MBC not only caused depolymerization of cytoplasmic microtubules (MTs), but also changed the pattern of actin at the hyphal tips. In the MBC-treated hyphae, the actin fluorescence was concentrated at the very tip region of the hypha, whereas in the control hyphae, the actin fluorescence was weak at the very tip and strong below the tip. The dose of MBC used for the wild-type strain did not depolymerize the MTs or modify the actin organization at the apex in the mutant strain, which confirmed that the change in actin distribution in the wild-type strain was due to the disruption of MTs. In the mutant strain, a seven times higher concentration of MBC than in the wild-type strain was required to depolymerize MTs and to alter the actin organization at the apex. The ultrastructural study of the MBC-treated hyphae revealed that the area containing apical vesicles was larger and the number of microvesicles was higher than in control hyphae. These changes probably resulted from the disassembly of MTs and the reorientation of actin cytoskeleton in MBC-treated apexes and suggested that MTs would organize the actin at the apex, which in turn would restrict the vesicle fusion to a narrow area at the hyphal tip. In treated hyphae of both strains without cytoplasmic MTs, mitotic spindles were detected although in lower number and with slightly modified morphology.

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced beta-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the beta-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The beta-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans.

A bacterial strain was isolated from a sample of contaminated heating oil and identified as a strain of Acinetobacter calcoaceticus, named MM5. The bacterial isolate was able to grow on petroleum derivatives and brought about an emulsification of those compounds. A bioemulsifier was extracted from the culture medium of MM5 strain and partially characterized. This compound was able to emulsify petroleum fuels and both aliphatic and aromatic pure hydrocarbons and was stable over a wide range of temperatures. Studies developed by light, scanning electron and transmission electron microscopy showed that, during the growth on petroleum derivatives, the microorganisms were orientated on the surface of drops enclosed in a skin or membranous polymer produced by the bacteria. These droplets may represent the hydrocarbon/water emulsion of the liquid culture. The growth of A. calcoaceticus MM5 on media containing both hydrocarbon and water-soluble substrates as carbon sources also results in the formation of a film, consisting of amorphous and membranous layers. The bacteria were connected to the biofilm and showed intercellular contacts through cell-surface appendages, forming a complex network. The importance of the biofilms for bacterial adhesion to oil droplets and for its nourishment is discussed.

Several microorganisms (bacteria, moulds and yeasts) were isolated from contaminated heating oil, some of which were able to degrade hydrocarbons. The filamentous fungi Hormoconis resinae H7, and the bacterial strain, Acinetobacter calcoaceticus MM5, were able to degrade hydrocarbons in sea sand artificially contaminated with petroleum derivatives. Furthermore, we analyzed the effect of different factors on the degradation of hydrocarbons in sea sand by the selected strain A. calcoaceticus MM5 regarding its possible use in bioremediation processes.

The ability of Acinetobacter calcoaceticus MM5 to degrade heating oil was studied. Certain culture conditions relevant to the degradation of this petroleum derivative were also analyzed. Degradation of heating oil by MM5 was followed by gas chromatography during a 30-day incubation period. A. calcoaceticus MM5 degraded essentially the n-alkane components of the fuel, and left the branched hydrocarbons pristane and phytane undegraded. The degradation of heating oil was favoured by aeration of the cultures. There was an absolute nutritional requirement for nitrogen and phosphorus sources for hydrocarbon degradation. Strain MM5 degraded heating and diesel oil to a similar extent. The presence of commercial additives in the heating oil did not modify the degradation of this fuel. Infrared spectroscopy of remaining heating oil after incubation with strain MM5 showed the appearance of two new bands, corresponding to compounds with carbonyl groups derived from the metabolism of hydrocarbons.

Beta-Galactosidase from mycelial extract of Aspergillus nidulans has been purified by substrate affinity chromatography and used to obtain anti-beta-galactosidase polyclonal antibodies. A. nidulans growing in lactose as carbon source synthesizes one active form of beta-galactosidase which seems to be a multimeric enzyme of 450 kDa composed of monomers with 120 and 97 kDa. Although the enzyme was not released to the culture medium, some enzymatic activity was detected in a cell-wall extract, thus suggesting that it can be an extracellular enzyme. Beta-Galactosidase of A. nidulans is a very unstable enzyme with an optimum pH value of 7.5 and an optimum temperature of 30 degrees C. It was only active against beta-galactoside substrates like lactose and p-nitrophenyl-beta-D-galactoside (PNPG).

The effect of the antimicrotubular drug methyl benzimidazol-2-yl carbamate (MBC) on the production and secretion of acid phosphatase, alpha-galactosidase and beta-galactosidase in Aspergillus nidulans was studied. A wild type and two benomyl resistant mutant (benA10 and benC28) strains were used. All the strains secreted acid phosphatase and alpha-galactosidase into the culture medium, whereas beta-galactosidase remained in the mycelium with a portion of its activity bound to the cell wall.When the wild type strain was incubated in the presence of a sublethal dose of MBC, a decrease of the activity of the enzymes studied was found. A reduction in the secretion of acid phosphatase and alpha-galactosidase into the culture medium was also observed. In addition, a decrease in the percentage of alpha- and beta-galactosidase activities bound to the cell wall was detected.The MBC dose used for the wild type strain did not modify either the total enzyme activities or the secretion of the enzymes studied in the benomyl resistant mutant benA and benC strains. However, when those strains were grown in the presence of a sublethal dose of MBC, a decrease in the total enzyme activities, as well as a reduction in acid phosphatase and alpha-galactosidase secretion was found. In addition, alterations in the percentage of enzyme activities bound to the cell wall were observed in both mutant strains. Results described in this work clearly suggest that microtubules are involved in the polarized secretion of enzymes in A. nidulans.

Twenty aerobic bacterial strains were isolated from altered heating oil. Among them the strain catalogued as MM5 and identified as Acinetobacter calcoaceticus is able to grow on hydrocarbon substrates. When strain MM5 was grown on heating oil, crude oil and tetradecane, increases of protein concentration and of caprilate-lipase and acetate-esterase enzymatic activities were observed in the culture filtrate, with a simultaneous pH drop. A strong emulsification of petroleum by-products was also noticed. Degradation of heating oil was followed by gas chromatography and infrared spectroscopy. Presence of available nitrogen and phosphorus sources were essential for hydrocarbon biodegradation. Intracellular electron transparent inclusions were observed by transmission electron microscopy when strain MM5 cells were grown on hydrocarbons. Light and scanning electron microscopy showed bacteria interconnected by an extracellular polymer and attached to hydrocarbon droplets and to sheets of polymeric material. A bioemulsifier was extracted from the cell-free culture supernatants of strain MM5 grown on tetradecane. The emulsifier is a high molecular weight product that comprises proteins, sugars and fatty acids and which is resistant to high temperature. Strain MM5 should be helpful for the design of strategies for the bioremediation of hydrocarbon contaminated sites.

The role of microtubules (MTs) in the protein secretion process in Cladosporium cucumerinum has been studied using the antimicrotubular drug methyl benzimidazol-2-yl carbamate (MBC). Sublethal doses of this compound increased the level of acid and alkaline phosphatase enzymes in the culture filtrate, although fungal growth was reduced. The incorporation of [S-35]methionine into the extracellular proteins was not modified by MBC. However, alterations in the electrophoretic pattern of proteins were observed in the presence of the fungicide. Distribution of MTs in hyphae was studied by indirect immunofluorescence microscopy; in untreated control samples, the fluorescence was especially abundant in the apical dome of the hyphae; following incubation of the fungus in MBC, a decrease in tubulin-positive staining in this zone was observed.

Twenty bacterial strains were isolated from a sample of contaminated heating oil and screened for their ability to use petroleum and several common fuels as the sole source of carbon and energy. One of the isolates, named MM5, was able to grow on petroleum derivatives and brought about an emulsification of those compounds. Gas chromatography studies showed that strain MM5 was able to degrade hydrocarbons of heating oil. MM5 has been tentatively identified as a strain of Acinetobacter calcoaceticus. The fine structure of MM5 was examined by transmission electron microscopy. Incubation in the presence of hydrocarbon substrates resulted in the development of intracellular electron-transparent inclusions. These structures were absent in the non-hydrocarbon cultures studied.

alpha-Galactosidases from mycelial extract and culture filtrate of Aspergillus nidulans have been purified to homogeneity and utilised to obtain polyclonal antibodies anti-alpha-galactosidase. The enzymatic characteristics and the cross reactivity of the antibodies suggest that alpha-galactosidases isolated from the two sources were the same enzyme. Thus, A. nidulans synthesized and secreted only one enzymatic form of alpha-galactosidase which is a multimeric enzyme of 370 kDa composed of four monomers of 87 kDa and a pI of 6.3. The optimum temperature of activity was 50 degrees C and the optimum pH 4-5. The enzyme was stable over a wide range of pH but quite unstable to temperature. alpha-Galactosidase of A. nidulans is a very specific enzyme, it is active only on p-nitrophenyl-alpha-D-galactoside (PNPG), melibiose and raffinose. When PNPG was utilised as substrate melibiose, raffinose, galactose and glucose were competitive inhibitors of the activity.

Cladosporium cucumerinum produced multiple isozymes of extracellular acid phosphatase, in response to phosphate concentration in the medium and related to growth phase. Production of the largest, a 174 kDa molecular form, was not affected by culture conditions and this isozyme could be a constitutive enzyme. Another form, with a mol. wt of 59 kDa, was subject to phosphate repression and was secreted to the culture medium in the early phase of fungal growth. Two minor forms, which exhibited mol. wt of 389 and 562 kDa, were detected in culture filtrates at the end of exponential growth. A single 174 kDa molecular form of acid phosphatase was detected in mycelial extracts. Furthermore, two forms of alkaline phosphatase were determined in culture filtrates and mycelial extracts in response to the phosphate level in the medium. A repressible alkaline phosphatase, with a mol. wt of 174 kDa and a form which was promoted by phosphate, with a mol. wt of 98 kDa were also found.