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PubMed2002

Identification and analysis of genes involved in the control of dimorphism in Mucor circinelloides (syn. racemosus).

Mucor circinelloides (syn. racemosus) is a non-pathogenic dimorphic fungus belonging to the class of zygomycetes. We are developing a novel system for heterologous protein production exploiting the dimorphic growth characteristics of M. circinelloides. In order to identify potential genetic regulators of morphology we have initiated a characterisation of key genes involved in signal transduction in Mucor. We have cloned and characterised pkaR and pkaC encoding the regulatory subunit (PKAR) and the catalytic subunit (PKAC), respectively, of the cAMP-dependent protein kinase A (PKA) of M. circinelloides. In anaerobically grown yeast cells, the levels of expression of both pkaR and pkaC were significantly higher than the levels of expression in aerobically grown mycelium. However, during the dimorphic shift, i.e. during the transition from anaerobic yeast growth to aerobic filamentous growth, the expression of pkaR was found to increase approximately two-fold. These results indicate that regulation of PKA activity is conferred at different levels according to growth and environmental conditions. Overexpression of pkaR resulted in a multi-branched colony phenotype on solid medium indicating that PKAR plays a role in filamentation and branching. Fragments of genes encoding factors of the mitogen-activated protein (MAP) kinase (MAPK) pathway have also been cloned: mpk1 (mitogen-activated protein kinase 1) encoding a MAPK homologue and ste12 encoding a transcription factor.

Amino Acid Sequence↗
PubMed2006

Comparative fatty acid profiling of Mucor rouxii under different stress conditions.

To understand the relationship between fatty acid metabolism and the growth morphology of Mucor rouxii, fatty acid profiling was studied comparatively in cells grown under conditions which included different atmospheric conditions or the addition of phenethyl alcohol (PEA). The significant difference in fatty acid profiles from M. rouxii grown under aerobic or anaerobic conditions was not found to be directly related to morphological growth. Oxygen limitation, which induced the formation of pure multipolar budding yeasts, led to a decrease in long-chain fatty acids-- particularly unsaturated fatty acids-- and an increase in medium-chain saturated fatty acids, a finding which contrasted with the aerobic cultures, including mycelia and PEA-induced bipolar budding cells. High levels of C18 : 1Delta(9) were found in aerobic yeast cultures with additional PEA when compared to that in the aerobically grown mycelia. The identification of unusual fatty acids in Mucor in response to alcoholic and hypoxic stresses - including odd-numbered fatty acids and 7-hydroxy dodecanoic acid (7-OH C12 : 0) in addition to the more common fatty acids - implied that an important role existed for these unusual fatty acids.

Aerobiosis↗
PubMed1986

Trichodermin esterase activity and trichodermin resistance in Mucor racemosus.

Mucor racemosus exhibited inducible phenotypic resistance toward the protein synthesis inhibitor trichodermin. Induction of resistance was elicited by exposure to trichodermin or to cycloheximide. Both adapted and nonadapted cells took up [14C]trichodermin from the medium. Trichodermin was found to be rapidly deacetylated to trichodermol upon entering the cell. Adapted cells deacetylated the drug more rapidly than nonadapted cells both in vivo and in vitro. The trichodermol resulting from deacetylation appeared in the medium, but the growth of adapting cells began well before the total conversion of trichodermin to trichodermol. Based on these data and the observation that trichodermol was a poor inhibitor of Mucor, adaptation appears to result from deacylation of the active antibiotic.

Biological Transport↗
PubMed1990

The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae.

An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.

Amino Acid Sequence↗
PubMed1974

Cyclic adenosine 3',5'-monophosphate and morphogenesis in Mucor racemosus.

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.

Aerobiosis↗
PubMed1976

Volatile factor involved in the dimorphism of Mucor racemosus.

Both hyphal and yeastlike development of Mucor racemosus and M. rouxii were demonstrated under 100% N2. Under standardized conditions in yeast extract-peptone-glucose medium, the morphology depended on the N2 flow rate and not on the glucose concentration. The effect was related to the rate of flushing of the atmosphere over the culture medium. The results indicate that a volatile compound produced by Mucor is involved in morphogenesis.

Anaerobiosis↗
PubMed1977

Control of beta-glucosidase synthesis in Mucor racemosus.

The beta-glucosidase of Mucor racemosus was shown to be synthesized when the organism was grown in the presence of such diverse carbon sources as glycerol, lactate, xylose, ribose, alpha-methylglucoside, alpha-phenylglucoside, maltose, and cellobiose. Enzyme synthesis was strongly repressed in the presence of hexoses. In addition, exogenous cyclic adenosine 3',5'-monophosphate (cAMP) resulted in enzyme repression. When cAMP was added exogenously after enzyme activity had accumulated, a reversible enzyme inactivation occurred. Growth on disaccharides (maltose or cellobiose) was severely retarded in the presence of cAMP, whereas that on glucose remained unaffected. The results indicate a probable role for cAMP in control of glucosidase synthesis in Mucor.

Carbon↗
PubMed1978

Glucose metabolism and dimorphism in Mucor.

Mucor racemosus fermented glucose to ethanol, carbon dioxide, and glycerol. When this fungus was grown anaerobically in either the yeast or mycelial form, the catabolism of glucose was very similar. Yeast cells shifted to aerobic conditions maintained a high flux of glucose carbon through the glycolytic and pentose phosphate pathways. Mycelial cells grown aerobically catabolized glucose in a manner consistent with a respiratory metabolism. Although there was no consistent pattern of glucose metabolism in the mycelial form of Mucor, growth in the yeast form consistently was correlated with a high flux of glucose carbon through the catabolic pathways.

Fermentation↗
PubMed1978

Regulation of macromolecular synthesis during hyphal germ tube emergence from Mucor racemosus sporangiospores.

Protein and RNA syntheses were examined during hyphal germ tube emergence from sporangiospores of a dimorphic phycomycete, Mucor racemosus. Both classes of macromolecules were synthesized immediately upon introduction of the dormant sporangiospores into nutrient medium. The specific rates of synthesis of both protein and RNA accelerated during initial germ tube emergence and reached a maximum when the emergence of new germ tubes ended. The specific rates of synthesis later decreased during further hyphal elongation. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by sucrose density gradient centrifugation, and the rate of amino acid addition to nascent polypeptide chains was calculated throughout the developmental sequence. The results showed that both the percentage of ribosomes active in protein synthesis and the velocity of ribosome movement along the mRNA were continuously adjusted throughout hyphal germ tube development. The free intracellular amino acid pools were measured throughout development. Alanine, glutamate, and aspartate were present at very high concentrations in the dormant spores but were rapidly depleted during hyphal germ tube emergence. The results of these studies are discussed in relation to hyphal germ tube development from yeast cells of Mucor and dormant spores of other fungal species.

Amino Acids↗
PubMed1982

Lipid synthesis during morphogenesis of Mucor racemosus.

Lipid synthesis increases coordinately with protein and RNA synthesis during morphogenesis of Mucor racemosus. The lipid synthesis inhibitor cerulenin can completely block morphogenesis under conditions in which cell growth continues. An increase in phospholipid turnover may be an important correlate to morphogenesis of Mucor spp., especially the turnover of phosphotidyl inositol and phosphatidyl ethanolamine. The increase in ornithine decarboxylase, which occurs during morphogenesis, is inhibited by the addition of cerulenin.

Acetates↗
PubMed1985

Dispersed repetitive DNA sequence of Mucor racemosus.

A dispersed repetitive DNA sequence has been identified within the genome of the fungus Mucor racemosus. Recombinant phage clones, as well as a plasmid harboring the sequence, have been isolated. Examination of cloned fragments comprising part of the repetitive sequence has led to a partial characterization of the element. The sequence has been detected in other Mucor species, and although the apparent number and chromosomal position of the repetitive sequence vary from strain to strain, it is clear that at least portions of the element have been conserved.

DNA, Fungal↗
PubMed1992

Developmental regulation of CUP gene expression through DNA methylation in Mucor spp.

Inserts which carried the CUP gene from Saccharomyces cerevisiae or Mucor racemosus were used as hybridization probes to measure the methylation state and expression of the CUP gene from Mucor rouxii at different stages of growth. It was observed that the fungus contains a CUP multigene family. All the CUP genes were present in a hypermethylated DNA region in nongrowing and isodiametrically growing spores and were not transcribed at these stages. After germ tube emergence, CUP genes became demethylated and transcriptionally active. Development, demethylation, and transcription of CUP genes were blocked by the ornithine decarboxylase inhibitor 1,4-diaminobutanone. These results suggest that genes that are activated during development became demethylated in this fungus.

Carrier Proteins↗
PubMed1992

Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus.

A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined.

Amino Acid Sequence↗
PubMed1966

Control of dimorphism in Mucor rouxii.

Haidle, C. W. (The University of Texas, Austin), and R. Storck. Control of dimorphism in Mucor rouxii. J. Bacteriol. 92:1236-1244. 1966.-Yeastlike cells of Mucor rouxii NRRL 1894 were converted to filaments in a medium containing glucose, mineral salts, casein hydrolysate, nicotinic acid, and thiamine when the gas phase was changed from CO(2)-N(2) or N(2) alone to air. Germ tubes began to appear 3 to 4 hr after exposure to air. Ribonucleic acid (RNA) precursors were incorporated into RNA in a discontinuous fashion during this conversion, but the incorporation was continuous during the anaerobic growth of yeastlike cells and during the aerobic germination of sporangiospores. The incorporation of labeled amino acids during the conversion was exponential. Labeling of ribosomal RNA occurred as shortly as 5 min after replacement of CO(2)-N(2) with air. However, P(32)-labeled RNA isolated 20 min after exposure to air had a guanine plus cytosine (GC) content of 41% (mole%) as compared with the 47% found for labeled and unlabeled RNA isolated at other stages of the life cycle of this organism or later during the conversion. In addition, the overall base composition of this 20-min pulse-labeled RNA resembled that of deoxyribonucleic acid (GC = 39%), suggesting that a significant proportion of this RNA is of the messenger type. Furthermore, the synthesis of cytochrome oxidase was induced upon exposure of yeastlike cells to air. Cyanide, acriflavine, and cycloheximide, which inhibited the action or synthesis of cytochrome oxidase, also inhibited the yeast to filament transition.

Acridines↗
PubMed1968

Control of dimorphism in Mucor by hexoses: inhibition of hyphal morphogenesis.

In anaerobic cultures of Mucor rouxii, morphogenesis was strongly dependent on hexose concentration as well as pCO(2). At low levels of hexose or CO(2), or both, hyphal development occurred; at high levels, the fungus developed as yeast cells. Other dimorphic strains of Mucor responded similarly to hexose and CO(2) but differred in their relative sensitivity to these agents. Glucose was the most effective hexose in eliciting yeast development of M. rouxii; fructose and mannose were next; and galactose was last. The fungus may be grown into shapes covering its entire dimorphic spectrum simply by manipulating the hexose concentration of the medium. Thus, at 0.01% glucose, hyphae were exceedingly long and narrow; at higher sugar concentrations, the hyphae became progressively shorter and wider; finally, at about 8% glucose, almost all cells and their progeny were isodiametric (spherical budding cells). Such yeast development occurred without a manifested requirement for exogenous CO(2). The stimulation of yeast development by hexose is not an artifact due to increased production of metabolic CO(2) (hyphae or yeast cells released metabolic CO(2) at similar rates). Presumably, the effect was caused by some other hexose catabolite which interfered with hyphal morphogenesis (apical growth); deprived of its polarity, the fungus grew into spherical yeastlike shapes. Although 10% glucose inhibited the development of hyphae from germinating spores, it did not prevent the elongation of preformed hyphae. This suggests that hexose inhibits hyphal morphogenesis not by blocking the operation of the enzyme complex responsible for apical growth but by preventing its initiation; such inhibition may be regarded as a repression of hyphal morphogenesis.

Carbon Dioxide↗
PubMed1975

Yeast-like growth of Mucor alternans (van Tieghem) in tissue-culture medium 199.

Mucor alternans(van Tieghem) was found to be a diphasic species in that it grew exclusively in the yeast-like budding phase under anaerobic conditions in the complex medium yeast extract-peptone-glucose broth and in tissue-culture medium 199. In the latter medium this growth form occurred also at 37C, at an initial pH of 7.2, and at glucose concentrations of 0.1 and 1.0%. The authors suggest that because of its synthetic nature the tissue-culture medium could be used with advantage in the study of nutritional requirements of dimorphic mucors.

Aerobiosis↗
PubMed1975

Acid proteases from species of Mucormii. partial characterization of the acid protease produced by a strain of Mucor miehei isolated in Cuba.

The acid protease produced by a strain of Mucor miehei isolated in Cuba was purified by column electrofocusing and partially characterized as to amino-acid composition, molecular weight, helical content, total carbohydrate content, and approximate isoelectric point; A detailed comparison of these results was reported previously for Mucor miehei protease (Ottesen, M. & Rickert, W;S. (1970) C.R. Trav. Labmcarlsberg 37, 301) suggested that the two enzymes are similar but not identicalmthis conclusion was reinforced by an analysis of circular-dichroism spectra.

Amino Acids↗
PubMedDate not supplied

Mucor pyelonephritis. Report of a case diagnosed by urine cytology, with diagnostic considerations in the workup of funguria.

BACKGROUND: Isolated renal mucormycosis is an uncommon kidney infection affecting patients with underlying systemic diseases and intravenous (IV) drug abuse. We report a unique case in the cytologic literature in which urine cytology provided insight into the diagnosis, renal mucormycosis. CASE: The patient, a diabetic and IV drug abuser, presented with complaints of left flank pain, fever and dysuria. All urine cultures were negative. A computed tomography (CT) scan showed changes consistent with left acute pyelonephritis, and the patient was treated for a presumed diagnosis of bacterial pyelonephritis. Late in the hospital stay, the cytology laboratory diagnosed Mucor in a single urine specimen, but the patient had already been discharged. The patient was never treated for funguria, only to present again with left flank pain 13 months later. An abdominopelvic CT scan showed progression to left chronic pyelonephritis. The patient, however, left the hospital against medical advice before any further workup could be completed. CONCLUSION: Renal mucormycosis should be considered part of the differential diagnosis in patients with underlying diseases or IV drug abuse who present with symptoms of acute pyelonephritis. The differential diagnosis of Mucor funguria should also include fungal ball in the renal pelvis or urinary bladder and fungal cystitis.

Adult↗