A.M. Pedregosa

Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans.

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 site 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 appearance. 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.

Induction of β-oxidation enzymes and microbody proliferation in Aspergillus nidulans

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced β-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 β-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 β-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.

Emulsifier production and microscopical study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcoaceticus MM5

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.

Study of factors influencing the degradation of heating oil by Acinetobacter calcoaceticus MM5

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.

Biodegradation of diesel and heating oil by Acinetobacter calcoaceticus MM5: its possible applications on bioremediation

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.

Biosurfactant-mediated biodegradation of straight and methyl-branched alkanes by Pseudomonas aeruginosa ATCC 55925

Accidental oil spills and waste disposal are important sources for environmental pollution. We investigated the biodegradation of alkanes by Pseudomonas aeruginosa ATCC 55925 in relation to a rhamnolipid surfactant produced by the same bacterial strain. Results showed that the linear C11-C21 compounds in a heating oil sample degraded from 6% to 100%, whereas the iso-alkanes tended to be recalcitrant unless they were exposed to the biosurfactant; under such condition total biodegradation was achieved. Only the biodegradation of the commercial C12-C19 alkanes could be demonstrated, ranging from 23% to 100%, depending on the experimental conditions. Pristane (a C19 branched alkane) only biodegraded when present alone with the biosurfactant and when included in an artificial mixture even without the biosurfactant. In all cases the biosurfactant significantly enhanced biodegradation. The electron scanning microscopy showed that cells depicted several adaptations to growth on hydrocarbons, such as biopolymeric spheres with embedded cells distributed over different layers on the spherical surfaces and cells linked to each other by extracellular appendages. Electron transmission microscopy revealed transparent inclusions, which were associated with hydrocarbon based-culture cells. These patterns of hydrocarbon biodegradation and cell adaptations depended on the substrate bioavailability, type and length of hydrocarbon.

Effects of methyl benzimidazoIe-2-yl carbamate on microtubule and actin cytoskeleton inAspergillus nidulans

SummaryThe 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.

Effect of the microtubule inhibitor methyl benzimidazol-2-yl carbamate (MBC) on production and secretion of enzymes in Aspergillus nidulans

The effect of the antimicrotubular drug methyl benzimidazol-2-yl carbamate (MBC) on the production and secretion of acid phosphatase, α-galactosidase and β-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 α-galactosidase into the culture medium, whereas β-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 α-galactosidase into the culture medium was also observed. In addition, a decrease in the percentage of α- and β-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 α-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 .

Effect of the microtubule inhibitor methyl benzimidazol-2-yl carbamate (MBC) on protein secretion and microtubule distribution in Cladosporium cucumerinum

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 [35S]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.

Functional analysis of mutations in the human carnitine/acylcarnitine translocase in Aspergillus nidulans

Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid beta-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 DeltaacuH 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.