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PubMed1996

Cloning and sequence analysis of a putative transcription factor (MTF1) gene from Mucor circinelloides.

We describe here the fortuitous cloning of a putative transcription factor gene (MTF1) from the dimorphic fungus Mucor circinelloides. Sequence analysis of MTF1 revealed an open reading frame (ORF) of 1059 nucleotides encoding a protein of M(r) 39601. The deduced amino acid sequence from the ORF imparts two glutamine-rich stretches which are homologous to a number of transcription factors characterized previously from various organisms. A Southern blot analysis of Mucor genomic DNA digested with different restriction endonucleases and probed with the 1.9 kb EcoR1 fragment of the putative transcription factor gene shows a single copy number of the the gene. Northern analysis during morphogenetic changes in Mucor suggested constitutive expression of the gene.

Amino Acid Sequence↗
PubMed1996

Cutaneous mucor infection treated with wide excision in two children who underwent marrow transplantation.

Cutaneous mucor infection developed in two children who had undergone bone marrow transplantation for treatment of leukemia. One infection occurred before transplantation, and the other occurred during the period of profound neutropenia after transplantation. Both children were treated with an extensive wide excision of the infected area, and there was no evidence of mucor along the resected edges of tissue. Both patients received extensive treatment with either amphotericin (case 1) or amphotericin and itraconazole (case 2). These two cases represent aggressive management of cutaneous mucor infections, which is believed to be required for the successful completion of a marrow transplantation procedure.

Adolescent↗
PubMed2003

Mucor mycosis infection presenting as a non-healing ulcer in an immunocompromised patient.

Mucor mycosis is an uncommon saprophytic opportunistic fungus causing localized cutaneous infection associated with high morbidity and, on dissemination, high mortality. We report the case of an immunocompromised patient with an aggressively progressing, painful non-traumatic ulceration, unresponsive to standard treatment. Deep biopsies for haematoxylin and eosin staining and fungal culture revealed the characteristic broad non-septate irregular hyphae of mucor allowing introduction of the appropriate treatment. Infection with mucor mycosis must be considered in today's medical environment as the number of immunocompromised patients increases.

Humans↗
PubMed1962

Nutrition, growth, and morphogenesis of Mucor rouxii.

Bartnicki-Garcia, S. (Rutgers, The State University, New Brunswick, N.J.) and Walter J. Nickerson. Nutrition, growth, and morphogenesis of Mucor rouxii. J. Bacteriol. 84:841-858. 1962.-Mucor rouxii was grown under three different atmospheres of incubation: air, N(2), and CO(2) in parallel cultures. The atmosphere of incubation markedly affected nutritional requirements, growth, and morphogenesis. Absence of oxygen greatly reduced growth and increased the nutritional demands of the fungus. Presence of a high tension of CO(2) resulted in a change from filamentous to yeastlike morphogenesis. Aerobically, a large variety of carbon sources was utilized; anaerobically, only hexoses served to meet requirements for carbon and energy. Aerobically, various amino acids supported abundant growth; anaerobically, they were poorly utilized. Ammonium and nitrate ions were better sources of nitrogen for anaerobic growth. In general, incubation under either air or N(2) resulted in development of coenocytic filamentous mycelium, whereas incubation under CO(2) resulted in development of budding yeastlike cells. Variations in temperature and time of incubation, inoculum size, type and concentration of carbon source, type of nitrogen source, and presence of various substances with known action on fungal morphogenesis altered growth in many cases, but did not significantly affect the patterns of vegetative morphogenesis conditioned by each atmosphere of incubation. However, vegetative morphogenesis was strongly affected by addition of certain chelating agents. Yeastlike development of M. rouxii was prevented by ethylene-diaminetetraacetic acid (EDTA) in concentrations which were also partially inhibitory for growth; under these conditions, development was filamentous. Chemically related chelating agents were similarly active. The growth-inhibitory and morphogenetic effects of EDTA were reversed by transition-group metal ions. Yeastlike development of M. subtilissimus, which does not require CO(2) for its induction, was also inhibited by EDTA.

Amino Acids↗
PubMed1963

EFFECTS OF THE ANTIFUNGAL PEPTIDE RO 2-7758 ON MORPHOLOGY OF MUCOR CORYMBIFERA.

Gale, Glen R. (Veterans Administration Hospital and Duke University Medical Center, Durham, N.C.). Effects of the antifungal peptide Ro 2-7758 on morphology of Mucor corymbifera. J. Bacteriol. 85:833-837. 1963.-Hyphae of Mucor corymbifera grown in the presence of the antifungal peptide, Ro 2-7758, were examined in an electron microscope. The predominant change, compared with control hyphae, was a marked thickening of the cell wall, with apparent maintenance of normal structure. Cell walls of control hyphae were from 0.03 to 0.11 mu in thickness; drug-treated walls varied from 0.22 to over 0.75 mu in thickness. The possible sites and mechanisms of action, as well as the significance of a thickened cell wall in antibiosis, are discussed.

Antifungal Agents↗
PubMed1964

ACCUMULATION OF PHOSPHORUS COMPOUNDS BY MUCOR RACEMOSUS.

James, A. W. (The Pennsylvania State University, University Park), and L. E. Casida, Jr. Accumulation of phosphorus compounds by Mucor racemosus. J. Bacteriol. 87:150-155. 1964.-In glucose-inorganic salts media containing excess KH(2)PO(4), Mucor racemosus accumulated 5 to 6% phosphorus on a dry-weight basis in its mycelium. Fractionation of phosphorus compounds in young, rapidly growing mycelium revealed that approximately 58% of this phosphorus was in the form of inorganic polyphosphate; nucleic acids accounted for an additional 17% of the total phosphorus. Upon further growth, morphological changes were observed in many of the hyphae, and polyphosphate appeared in the culture media. In media containing limited amounts of KH(2)PO(4), exogenous polyphosphate was utilized by M. racemosus, and the phosphorus content of the mycelium decreased to approximately 0.3%.

Culture Media↗
PubMed2000

Mechanism of activation of cAMP-dependent protein kinase: in Mucor rouxii the apparent specific activity of the cAMP-activated holoenzyme is different than that of its free catalytic subunit.

Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with a greater Km, the order of preference of the peptides by the M. rouxii catalytic subunit was similar to the one displayed by mammalian protein kinase A. Particularly significant is the replacement of serine by threonine in the basic peptide RRATVA, which impaired its role as a substrate of M. rouxii catalytic subunit. Mucor rouxii protein kinase A is a good model in which to study the mechanism of activation since cAMP alone is not enough to promote activation and dissociation. Four peptides were selected for the study of holoenzyme activation under conditions in which the enzymatic activity was not proportional to the holoenzyme concentration: RRASVA, RRRRASVA, KRRRLSSRA (S6 peptide), and LRRASLG (kemptide); protamine was used as reference. Differential activation degree was observed depending on the peptide used and on cAMP concentration. Ratios of activity between different substrates displayed by the holoenzyme under the above conditions did not reflect the one expected for the free catalytic subunit. The degree of inhibition of the holoenzyme activity by an active peptide derived from the thermostable protein kinase inhibitor was dependent on the substrate used and on the holoenzyme concentration, while it was found to be independent of these two parameters for free catalytic subunit. Polycation modulation of holoenzyme activation by cAMP was also dependent on the polycation itself and on the peptide used as substrate. The observed kinetic differences between holoenzyme and free catalytic subunit were decreased or almost abolished when working at low enzyme or at high cAMP concentrations. Two hypotheses compatible with the results are discussed: substrate participation in the dissociation process and/or holoenzyme activation without dissociation.

Amino Acid Sequence↗
PubMed1999

Evidence that MRas1 and MRas3 proteins are associated with distinct cellular functions during growth and morphogenesis in the fungus Mucor racemosus.

The filamentous fungus Mucor racemosus provides a simple and unique model system for defining the function of individual ras genes in a gene family which is closely related to mammalian ras genes. The current study was designed to investigate the role of Mras1 and Mras3 in different stages of fungal morphogenesis, including sporangiospore germination, sporulation, and dimorphic transitions. The overall patterns of Mras1 and Mras3 transcript and protein accumulation were markedly different but, in general, transcripts and proteins were present at low levels during spherical growth and their accumulated level increased severalfold during polar growth (germ tube emergence and elongation). In contrast to Mras1, relatively high levels of Mras3 transcript accumulated during sporulation and MRas3 protein accumulated in sporangiospores. Transformation of M. racemosus with an activated allele of Mras3 reduced growth rate during aerobic sporangiospore germination, while a dominant-negative allele of Mras3 caused a 40% decrease in viable asexual spores. An activated allele of Mras1 increased growth rate during sporangiospore germination but neither activated nor dominant-negative alleles of Mras1 affected total number of asexual spores. Expression of MRas3 and MRas1 proteins appear to be subject to different regulatory mechanisms: exogenous dibutyryl-cAMP and fusidienol caused a strong repression of the level of MRas3 protein (but not MRas1) concurrent with the inhibition of polar growth. Differential posttranslational modification and intracellular localization of MRas1 and MRas3 proteins were also observed. The data strongly suggest that Mras3 and Mras1 play different roles in regulation of cell growth and morphogenesis in Mucor.

Alkyl and Aryl Transferases↗
PubMed1985

Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit.

The polymeric structure of the cyclic AMP-dependent protein kinase (E.C.2.7.1.37) from the dimorphic fungus Mucor rouxii was analyzed through studies of gel filtration and sucrose gradient centrifugation of the holoenzyme and its subunits and by photoaffinity labeling of the regulatory subunit. It was demonstrated that it is a tetramer composed by two regulatory subunits (R) of mol. wt. 75 000 and two catalytic subunits (C) of mol. wt. 41 000 forming a holoenzyme R2C2 of mol. wt. 242 000. Frictional coefficients of 1.55 and 1.62 for the holoenzyme and for the regulatory dimer, respectively, indicate a significant degree of dimensional asymmetry in both molecules. A procedure for the purification of the catalytic subunit of the kinase is presented. The holoenzyme could be bound to a cyclic AMP-agarose column and the catalytic subunit could be eluted by 0.5 M NaCl, well resolved from the bulk of protein. This particular behaviour of the holoenzyme in cyclic AMP-agarose chromatography allowed the inclusion of this step in the purification of the catalytic subunit and corroborated that the holoenzyme was not dissociated by cyclic AMP alone. The isolated catalytic subunit displays Michaelis-Menten behavior towards kemptide, protamine and histone and is inhibited by sulfhydryl reagents, indicating that the molecule has at least one cysteine residue essential for enzyme activity. The catalytic activity of the isolated C subunit is inactivated by the mammalian protein kinase inhibitor, and is inhibited by the regulatory subunit from homologous and heterologous sources. In general, the properties of the catalytic subunit suggest a structural similarity between Mucor and mammalian C subunits.

Catalysis↗
PubMed1991

Integrative transformation by homologous recombination in the zygomycete Mucor circinelloides.

Transformation of a Mucor circinelloides Leu- strain with the plasmid pAD45, harbouring the wild-type allele (leuA+) and a chymosin gene, led to the identification of mitotically stable transformants after one to three vegetative growth cycles on non-selective medium. Southern analysis of the stable transformed strains demonstrated that the vector is integrated, as an intact molecule, into the resident Mucor leuA locus. Retransformation of Escherichia coli with genomic DNA restricted with enzymes having no or only a single recognition site within the inserted sequence did not permit isolation of plasmids or fragments carrying the leuA or chymosin gene.

Alleles↗
PubMed1991

Transformation of a methionine auxotrophic mutant of Mucor circinelloides by direct cloning of the corresponding wild type gene.

A transformation system has been developed for Mucor circinelloides, by direct cloning of a wild-type methionine gene that complements the auxotrophic mutation. The marker gene isolated was associated with an autonomous replication sequence (ARS) functional in this zygomycete. Southern hybridisation analyses of transformants showed sequence homology both with vector DNA and with Mucor wild-type DNA. The transformation frequency (up to 6000 per microgram DNA) and the mitotic instability of the transformed cells were studied. The hybridisation pattern of undigested DNA from the transformants suggests that the inserts contain a novel autonomous replication element for this filamentous fungus.

Blotting, Southern↗
PubMed1990

Primary and secondary structure of the 25S rRNA from the dimorphic fungus Mucor racemosus.

A 9.76 Kb ribosomal DNA repeat unit from the nuclear genome of the dimorphic fungus Mucor racemosus (Zygomycetes) was identified using a hybridization probe from the yeast Saccharomyces cerevisiae (Ascomycetes). This material was cloned in Escherichia coli plasmids as four overlapping pieces and mapped with respect to cleavage sites for 12 restriction endonucleases. The nucleotide sequence of the complete 25S rRNA gene and flanking regions was determined. The 5' and 3' ends of the structural gene were identified by comparison with the published sequence for the S. cerevisiae gene. The Mucor gene was found to possess 3469 bp and have a GC content of 42.8%. It was compared with the homologous gene from several other eukaryotes and found to be most similar to that from Saccharomyces. A potential secondary structure of the putative RNA transcript consistent with the structures proposed for the E. coli and Saccharomyces molecules was constructed by computer modelling.

Base Sequence↗
PubMed1993

Mitochondrial genome of the dimorphic zygomycete Mucor racemosus.

Mitochondria were isolated from the dimorphic zygomycete Mucor racemosus by differential centrifugation. DNA from the organelles was purified by cesium chloride-ethidium bromide isopycnic centrifugation. Examination of the mitochondrial DNA by electron microscopy revealed a circular chromosome approximately 63.8 kbp in circumference. The chromosome was digested with restriction endonucleases and the resulting DNA fragments were separated by agarose-gel electrophoresis. Electophoretic mobilities and stoichiometry of the fragments indicated a mixed population of mtDNA molecules each with a size of about 63.4 kbp. Physical maps were constructed from analyses of fragments generated in single and double restriction digests and from the hybridization of fragments to probes for the large and small mitochondrial rRNA genes from Saccharomyces cerevisiae. The Mucor mitochondrial chromosome was found to exist in the form of two flip-flop isomers with inverted repeat sequences encoding both rRNA genes.

Cloning, Molecular↗
PubMed1980

Cyclic adenosine 3',5'-monophosphate and germination of sporangiospores from the fungus Mucor.

Cyclic adenosine 3',5'-monophosphate (cAMP) metabolism was examined in germinating sporangiospores of Mucor genevensis and Mucor mucedo. Exogenous cAMP prevented normal hyphal development from sporangiospores. Internal pools of cAMP fluctuated profoundly during development. Spherical growth of the spores was characterized by large pools of cAMP whereas germ tube emergence and hyphal elongation were characterized by small pools of cAMP. These observations suggest a possible role for cAMP in sporangiospore germination. Adenylate cyclase activities fluctuated significantly during germination with maximum values attained during spherical growth. In contrast, cAMP phosphodiesterase activities remained constant throughout germination. Internal cAMP levels may therefore be regulated by adjustment of adenylate cyclase activities. The binding of cAMP by soluble cell proteins was measured. cAMP-binding activity changed greatly during germination. Dormant and spherically growing spores possessed the highest activities. Developing hyphae contained the lowest activities. Use of the photoaffinity label, 8-azido-[32P]cAMP, in conjunction with sodium dodecyl sulfate-polyacrylamide-gel electrophoresis allowed the identification of a small population of morphogenetic-stage-specific proteins which bind cAMP and may be of regulatory significance to development.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Formation of antigenic extracellular polysaccharides by selected strains of Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum.

In this study, polyclonal IgG antibodies raised against extracellular polysaccharides (EPS) of Mucor racemosus were characterised as almost specific for moulds belonging to the order of Mucorales. Cross-reactivity in the ELISA could be observed only towards the yeast Pichia membranaefaciens. EPS were isolated from various cultures of M. hiemalis growing on six different carbon sources and two nitrogen sources, with ratios varying from 0.13 to 0.44 relative to the amount of biomass. Other strains including Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum also excreted EPS, with ratios varying from 0.05 to 0.23. In all cases, the excreted EPS had similar antigenic properties as determined by ELISA. No enzymatic degradation of the antigenic parts of the polysaccharides could be observed upon prolonged incubation. Considering that all tested strains formed similar amounts of antigenic EPS there might be scope for the specific detection of biomass of Mucoralean moulds using ELISA techniques for example in food.

Antibodies, Fungal↗
PubMedDate not supplied

An amphotericin B-resistant case of rhinocerebral mucor mycosis.

A 51-year-old male patient with diabetes mellitus complicated by ketoacidotic imbalance developed a rhinocerebral mucor mycosis that advanced despite early amphotericin B therapy and extensive surgical intervention. The MIC of amphotericin B for the isolated mucor species was 64 mg/l, meaning that in vitro resistance also existed. Only long-term treatment with ketoconazole (600 mg/day, perorally) was successful in curing the disease.

Amphotericin B↗
PubMed2005

Agrobacterium tumefaciens-mediated transformation of Mucor circinelloides.

The Agrobacterium tumefaciens-mediated transformation of the zygomycetous fungus Mucor circinelloides is described. A method was also developed for the hygromycin B-based selection of Mucor transformants. Transformation with the hygromycin B phosphotransferase gene of Escherichia coli controlled by the heterologous Aspergillus nidulans trpC promoter resulted in hygromycin B-resistant clones. The presence of the hygromycin resistance gene in the genome of the transformants was verified by polymerase chain reaction and Southern hybridization: the latter analyses revealed integrations in the host genome at different sites in different transformants. The stability of transformants remained questionable during the latter analyses.

Agrobacterium tumefaciens↗
PubMed2003

Differential response to low temperature of two Delta6 fatty acid desaturases from Mucor circinelloides.

A recently identified Delta6 fatty acid desaturase in Mucor rouxii shows a low sequence homology (approximately 24% at the amino acid level) to that isolated from Mortierella alpina, but is phylogenetically closer to a plant enzyme, suggesting the occurrence of Delta6 desaturase isozymes in Mucorales molds. In the present study, two types of Delta6 desaturases, mcD6-1 ( Mo. alpina type) and mcD6-2 ( M. rouxii type), were cloned from Mucor circinelloides. When the cloned genes were expressed in the yeast Saccharomyces cerevisiae in the presence of a linoleic acid substrate (C18:2Delta9, 12), a newly generated gamma-linolenic acid (C18:3Delta6, 9, 12) was detected in the cells, which confirmed the suspected enzymatic function of the recombinant protein. This is the first report of Delta6 desaturase isozymes present in one organism. Northern analysis demonstrated that the amount of mcD6-2 mRNA was less than half of that of mcD6-1 mRNA in cells grown at 28 degrees C. However, upon cultivation of the cells at 15 degrees C for 0.5-1 h, mcD6-2 mRNA rapidly increased by up to 1.5-fold and then gradually decreased. By contrast, mcD6-1 transcripts levels did not fluctuate significantly for 1 h after the temperature shift, but declined by 75% over the next 2 h. The gamma-linolenic acid content in total fatty acid from M. circinelloides decreased at 28 degrees C, but was maintained at approximately 30% at 15 degrees C. These data suggest that Delta6 desaturase isozymes play physiologically distinct roles in the maintenance of cellular lipids and adaptation to low temperature.

Amino Acid Sequence↗