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Abstract
Mucor racemosus is a member of the order Mucorales of the Zygomyceta. Although Mucor exhibits multiple morphologies, interest has focused on vegetative yeast-hyphal dimorphism. Part of the interest in Mucor dimorphism concerns their potential as etiological agents of disease. In healthy humans, Mucor rarely causes disease, but in the host compromised by immune deficiency, immune suppression, or a serious underlying disease, the incidence of Mucor infections is much higher. Although the yeast form of Mucor has been observed in human and animal tissue, invariably it is the hyphal form that is associated with disease and pathology (Rippon, 1982). Thus, there may be a very practical reason for understanding the process of dimorphism among Mucor, since from such knowledge may emerge improved chemotherapeutic procedures, not only for infections involving Mucor, but also for infections involving the other pathogenic dimorphic fungi. In addition to the clinical implication of Mucor dimorphism, in the past several years there has been growing interest in Mucor as a model system to investigate fundamental questions of the molecular biology of cellular morphogenesis. In particular, these studies have focused on differential gene expression during Mucor development and on the mechanisms that may serve to regulate such expression.
Bats (Chiroptera), the second largest group of mammals, are known for their unique immune system and their ability to act as vectors for various zoonoses. Bats also act as important carriers of fungi, which include plant, animal, and human pathogens. Their roosting areas, foraging behaviors, and even migration routes make bats ideal vectors for fungi. We isolated 75 culturable fungal species from bats in Yunnan Province, China, with 36 species representing known pathogens of plants, animals, and humans, while 39 species are non-pathogenic fungi. Among these species, 77% (58 species) belonged to Ascomycota, 9% (seven species) belonged to Basidiomycota, and 13% (10 species) belonged to Mucoromycota. Even though several taxonomic studies on fungi associated with bats have been published, studies exploring the role of bats as fungal vectors are lacking. This study discusses the fungi host-specific traits and pathogenicity and the impact and ecological significance of bats as fungal vectors.
Mucor dimorphism has interested microbiologists since the time of Pasteur. When deprived of oxygen, these fungi grow as spherical, multipolar budding yeasts. In the presence of oxygen, they propagate as branching coenocytic hyphae. The ease with which these morphologies can be manipulated in the laboratory, the diverse array of morphopoietic agents available, and the alternative developmental fates that can be elicited from a single cell type (the sporangiospore) make Mucor spp. a highly propitious system in which to study eukaryotic cellular morphogenesis. The composition and organization of the cell wall differ greatly in Mucor yeasts and hyphae. The deposition of new wall polymers is isodiametric in yeasts and apically polarized in hyphae. Current research has focused on the identity and control of enzymes participating in wall synthesis. An understanding of how the chitosome interacts with appropriate effectors, specific enzymes, and the plasma membrane to assemble chitin-chitosan microfibrils and to deposit them at the proper sites on the cell exterior will be critical to elucidating dimorphism. Several biochemical and physiological parameters have been reported to fluctuate in a manner that correlates with Mucor morphogenesis. The literature describing these has been reviewed critically with the intent of distinguishing between causal and casual connections. The advancement of molecular genetics has afforded powerful new tools that researchers have begun to exploit in the study of Mucor dimorphism. Several genes, some encoding products known to correlate with development in Mucor spp. or other fungi, have been cloned, sequenced, and examined for transcriptional activity during morphogenesis. Most have appeared in multiple copies displaying independent transcriptional control. Selective translation of stored mRNA molecules occurs during sporangiospore germination. Many other correlates of Mucor morphogenesis, presently described but not yet explained, should prove amenable to analysis by the emerging molecular technology.
All classes of the Eumycota contain members exhibiting yeast/mycelial dimorphism. In certain environments the fungus grows as spherical (Fig. 1A) or ellipsoidal (Fig. IB) budding yeasts. Under other conditions it grows by apical extension and branching of multicellular (Fig. ID) or coenocytic (Fig. 1C) hyphae. Dimorphism is common in several important plant pathogens (e.g., Cerato-cystis ulmi, Ustilago may dis, and Rhodosporidium sphaerocarpum) and numerous human pathogens (e.g., Candida albicans, Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidioides brasiliensis). Pathogenicity is generally limited to only one of the alternative morphologies. This correlation between morphology and pathogenicity has served as a major incentive to elucidate the molecular basis of dimorphism.
This report reviewed the specific characteristics of Mucor circinelloides in solid cultures and its capacity for multiple anamorphic expressions in synthetic broth, including holoblastic, holothallic, enterothallic conidia, and vesicular conidia head group as well as yeast cells. Attempt was made to show that the sequence of events in the conversion process, with inherent cytosolic nucleation and protoplast formation, to terminal budding yeast cell would serve as a model for studying morphogenetic transformation in the fungi.
In attempting to review the metabolism of fungi in a concise manner one is confronted with both a plethora and a lack of information.
Fungi are a very diverse kingdom of organisms, members of which have been evolving independently for over a billion years
with often short generation times. Although their morphology is rather similar, one might expect a metabolic diversity that
is at least as significant as the diversity in the animal kingdom. This diversity is reflected in the variety of substrates
fungi can metabolize and use as carbon sources as well as in the chemicals they can produce as fermentation products or so-called
secondary metabolites. These properties have been exploited in a large number of industrial applications. Growth requirements
of others such as VAM-forming fungi or some biotrophic pathogens can be so intricate that it has proven impossible to grow
them in culture. A discussion of fungal metabolism therefore has to be restricted to the most important or most general pathways.
Sporangiospores from Mucor rouxii or M. bacilliformis placed in an appropriate culture medium enter into a developmental growth program with three discrete phases: Ia, characterized by acquisition of a spherical shape by the spore; Ib, where a maximal spherical volume is obtained by the spore; and II, after germ tube or bud emergence. During spore germination protein and RNA content increased exponentially without a noticeable lag phase and with no change in rate during all the growth phases. Their inhibition stopped spore germination at discrete steps, suggesting that specific mRNAs and proteins were required at each phase. DNA biosynthesis started after a long lag phase, about 30 min before the onset of growth phase II. Its inhibition prevented the cells from entering phase II, but did not prevent nuclear division. It is suggested that Mucor spores are in the G2 phase of the cell cycle, with their genetic information “programmed” for spherical growth, and that DNA replication is required for the synthesis of specific mRNAs necessary for the onset of polarized growth.
Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.
An extraordinary feature of the majority of human pathogenic fungi is that they exhibit dimorphism and in many cases pleomorphism. In addition it is common to find that the morphology of the fungus in the infected tissue is different from that of the propagule that initiated the infection. It is generally assumed, but not well established experimentally, that the morphogenesis accompanying the parasitic infection in some way confers a survival advantage on the fungus. Basic studies on the phenomenon of fungal morphogenesis are important for at least two reasons: firstly, to expand our understanding of the mechanism of mycotic pathogenicity, and, secondly, to use these fungi as relatively simple systems for studying eukaryotic differentiation. For a more detailed treatment of fungal dimorphism the reader is referred to the excellent monograph of Szaniszlo (133). This review summarizes the salient features of the different morphogenic forms of a number of the human pathogenic fungi and where possible relates these to the infection process.
A case is presented in which yeastlike cells resembling the yeast phase of Paracoccidioides brasiliensis were observed by microscopic examination of a urine sample. A mold identified as Mucor circinelloides was isolated from the specimen. It was converted to the yeast form by cultivation on Sabouraud agar incubated in a GasPak jar at 37 degrees C. The isolate was eventually shown not to be related to the patient's illness; however, the superficial resemblance of the yeast phase to P. brasiliensis caused some confusion in making a correct diagnosis.
Three allyl-alcohol-resistant mutants were isolated in the dimorphic fungus Mucor rouxii and characterized with regard to their alcohol dehydrogenase (ADH) activity in vitro and in vivo as well as their ability to execute the morphological alternatives of dimorphism under different environmental stimuli, either in the absence or in the presence of oxygen. These studies indicated that fermentation and yeast-cell development are independent events and that ADH activity is essential for growth of the fungus in the absence of oxygen. Heterokaryon construction and analysis indicated that in the three mutant strains the corresponding genetic alterations are recessive nuclear mutations which behave as allelic in complementation tests.
Mucorales constitute a group of fungi that, because of their growth characteristics, have been used extensively in the study of cell differentiation, cell morphogenesis, and stimuli perception. We have studied the role of polyamine metabolism in the development of different Mucorales, with emphasis on Mucor and Phycomyces species. It has been observed that previous to each differentiative step, the cellular levels of the most regulated enzyme of the pathway, ornithine decarboxylase (ODC), and polyamines suffer a noticeable increase. Addition of diaminobutanone (DAB), a competitive inhibitor of ODC, blocks all the corresponding differentiative phenomena. In its presence, germinating spores fail to produce germ tubes and keep growing isodiametrically; mycelia do not sporulate but continue their vegetative growth, and yeast cells are unable to engage in a dimorphic transition without alterations in their growth rate. This differential effect of the ODC inhibitor in growth and development is apparently due to the location of ODC in at least two different cell compartments, one of which is impermeable to the drug. Inhibition of development is counteracted by putrescine and more noticeably by 5-azacytidine (5AC), a strong inhibitor of DNA methylation. Methylation levels of DNA are high in spores, and they become reduced after germination. Demethylation is inhibited by hydroxyurea, which blocks DNA replication, and by DAB. The effect of the latter is reversed by 5AC. These results suggest a relationship between polyamines and DNA methylation. Analysis of metallothioneine gene (CUP) behavior and expression during spore germination has confirmed this hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)
A soluble NAD-dependent alcohol dehydrogenase (ADH) activity was detected in mycelium and yeast cells of wild-type Mucor rouxii. In the mycelium of cells grown in the absence of oxygen, the enzyme activity was high, whereas in yeast cells, ADH activity was high regardless of the presence or absence of oxygen. The enzyme from aerobically or anaerobically grown mycelium or yeast cells exhibited a similar optimum pH for the oxidation of ethanol to acetaldehyde (∼pH 8.5) and for the reduction of acetaldehyde to ethanol (∼pH 7.5). Zymogram analysis conducted with cell-free extracts of the wild-type and an alcohol-dehydrogenase-deficient mutant strain indicated the existence of a single ADH enzyme that was independent of the developmental stage of dimorphism, the growth atmosphere, or the carbon source in the growth medium. Purified ADH from aerobically grown mycelium was found to be a tetramer consisting of subunits of 43 kDa. The enzyme oxidized primary and secondary alcohols, although much higher activity was displayed with primary alcohols. K
m values obtained for acetaldehyde, ethanol, NADH2, and NAD+ indicated that physiologically the enzyme works mainly in the reduction of acetaldehyde to ethanol.
The differential display technique was used to identify genes from Mucor circinelloides involved in the yeast-to-hypha transition. Using a limited set of primer combinations, cDNA fragments corresponding to mRNAs differentially expressed during the dimorphic transition were isolated. Northern analyses showed that the accumulation of the transcript detected by hybridisation with one of the cDNA fragments increased during the transition and was undetectable at the mycelial stage. Sequence analysis and database searches of this fragment revealed high similarity to ornithine decarboxylase (ODC) encoding genes. The odcA gene of M. circinelloides was isolated from genomic and cDNA libraries and characterised. Electrophoretic karyotyping and hybridisations showed that the odcA gene is single-copy and linked to the leuA and rDNA genes. The single transcript detected (2.1 kb), was considerably longer than the deduced ORF. Through non-radioactive primer extension analysis four transcription initiation sites were mapped to positions -61, -167, -239 and -436 from the start codon. The ODC mRNA levels increased during the yeast-to-hypha transition, reaching a maximum at 120 min, which was accompanied by a rise in ODC enzymatic activity. The expression pattern of the odcA gene showed that in M. circinelloides the ODC levels are transcriptionally regulated, in contrast with other dimorphic fungi in which a post-transcriptional regulation has been proposed.
We report a histologically and mycologically proven sinonasal mucormycosis case causing palatal necrosis in a nondiabetic patient with renal failure. Mycological examination of Giemsa stained imprinted tissue preparations revealed abundant yeast-like cells besides the typical mucoraceous hyphae. The fungus was isolated from surgical specimens and identified as Rhizopus oryzae by phenotypic and genotypic tests. Laboratory studies were performed to investigate the association of the yeast-like cells observed in tissue specimens and the fungus recovered in culture. In vitro induced yeast-like cell development of the case isolate was found under certain growth conditions and documented by photomicrographs.
Pyruvate kinase has been purified from bovine skeletal muscle by a procedure that includes only heat, ammonium sulfate fractionation, and chromatography on carboxymethyl Sephadex. Crystallization was accomplished by dialysis at 4° against 47% saturated ammonium sulfate. Since this simple purification scheme, with only slight modifications, has also been used to isolate turkey and rabbit muscle pyruvate kinases, it may be generally applicable to the isolation of muscle pyruvate kinase from birds and mammals.
Bovine muscle pyruvate kinase prepared by the procedure described here was found to be homogeneous, as determined by disc gel electrophoresis at pH 9.5, gel electrophoresis in the presence of sodium dodecyl sulfate, sedimentation velocity and sedimentation equilibrium, isoelectric focusing, and immunodiffusion. It sediments at 9.9 S (s⁰20,w) and has a maximum velocity of 400 µmoles per min per mg or more at 25°, or 2.4 times that value at 37°. The enzyme has maximal activity at pH 7.1, with an apparent Michaelis constant for phosphoenolpyruvate of 0.04 mm or less, and for ADP of 0.4 mm. Its isoelectric pH during electrofocusing is 8.9.
Sedimentation equilibrium yielded a molecular weight of 230,000 with a range of ±3,000 in dilute phosphate buffer, and 57,000 ± 1,500 in 3.5 m guanidine hydrochloride, using a partial specific volume of 0.740 ml per g calculated from the amino acid composition. The molecular weight in guanidine hydrochloride, confirmed by gel electrophoresis in sodium dodecyl sulfate, indicates the presence of four polypeptide chains in the native enzyme. Binding studies suggest a total of four phosphoenolpyruvate binding sites, or one per subunit. Ion sensitivities and other chemical and physical parameters are very similar to those reported for rabbit muscle pyruvate kinase.
Polyamines were examined in several yeasts and filamentous fungi. Whereas putrescine, spermidine, and spermine were present in the yeasts, spermine was not detected in any of the filamentous fungi.
The sequence of 72 base pairs of the rightward operator (O-R) of bacteriophage lambda is presented as determined with simple and rapid methods for direct DNA sequencing. The sequence of an operator mutant is also described. The methods are of general use in sequencing DNA fragments with unique 5' ends up to 50 base pairs in length. Previous experiments have shown that this operator contains multiple sites recognized by the lambda phage repressor. We believe we have identified three of these sites.
Mucor genevensis was grown in both glucose-limited and glucose-excess continuous cultures over a range of dissolved oxygen concentrations (<0.1 to 25 muM) to determine the effects of glucose and the influence of metabolic mode (fermentative versus oxidative) on dimorphic transformations in this organism. The extent of differentiation between yeast and mycelial phases has been correlated with physiological and biochemical parameters of the cultures. Under glucose limitation, oxidative metabolism increased as the dissolved oxygen concentration increased, and this paralleled the increase in the proportion of the mycelial phase in the cultures. Filamentous growth and oxidative metabolism were both inhibited by glucose even though mitochondrial development was only slightly repressed. However, the presence of chloramphenicol in glucose-limited aerobic cultures inhibited mitochondrial respiratory development but did not induce yeast-like growth, indicating that oxidative metabolism is not essential for mycelial development. Once mycelial cultures had been established under aerobic, glucose-limited conditions, subsequent reversal to anaerobic conditions or treatment with chloramphenicol caused only a limited reversal (<35%) to the yeast-like form. Glucose, however, induced a complete reversion to yeast-like form. It is concluded that glucose is the most important single culture factor determining the morphological status of M. genevensis; mitochondrial development and the functional oxidative capacities of the cell appear to be less important factors in the differentiation process.
Fatty acid and sterol content and composition were determined for the dimorphic mold, Mucor genevensis, grown under a variety of experimental conditions. Fatty acids account for 6 to 9% of the dry weight of aerobically grown mycelium, and 70 to 80% of these are unsaturated. The organism contains gamma-linolenic acid which is characteristic for Phycomycetes, and in sporangiospores this compound represents 40% of the total fatty acids. Of the sterols found in mycelium, 80% is ergosterol, and stigmasterol was positively identified as one of the minor components. In anaerobically grown yeastlike cells, sterol content is less than 10% of the level found in aerobically grown cells, and fatty acids amount to less than 2% of the dry weight. These fatty acids are predominantly short chain and less than 10% are unsaturated. Yeastlike cells obtained under aerobic conditions by growth in the presence of phenethyl alcohol have fatty acid and sterol compositions characteristic of aerobically grown mycelium. It is concluded that the dimorphology of the organism is not directly related to lipid composition.
The present study demonstrates the importance of mitochondrial activities in controlling Mucor rouxii morphogenesis. The respiratory capacity of the spores of this facultatively anaerobic, dimorphic fungus becomes repressed if germination and growth take place in the absence of oxygen. The level of activity of mitochondrial enzymes such as cytochrome oxidase and malate dehydrogenase is lower in the anaerobic yeastlike cells than it is in ungerminated spores and in aerobic hyphae, but the reverse is true for glycolytic enzymes such as pyruvate kinase and alcohol dehydrogenase. Following exposure to air, yeastlike cells convert into hyphae after a lag period corresponding to aerobic adaptation. Anaerobic cultures grown in the presence of ethylenediaminetetraacetate (EDTA) at a concentration of 10(-4) M exhibit hyphal morphology. These cells, which are fully adapted to anaerobic fermentation, nevertheless have potentially active mitochondria with the same levels of respiratory enzymes as ungerminated spores. These cells are able to grow immediately after aeration, without an adaptation lag. Evidence is presented which indicates that the morphogenetic effect of EDTA is not the result of elimination of free metals. Additional evidence proving mitochondrial control of morphogenesis in M. rouxii is that chloramphenicol (4 mg/ml) induced the formation of respiratory-deficient, yeastlike cells in aerobic cultures.
Growth of Mucor genevensis, a facultatively anaerobic dimorphic mold, in high concentrations of chloramphenicol (4 mg/ml) leads to increased numbers of yeast-like cells and small club-like mycelial forms. This change in morphology is accompanied by a threefold increase in the mass doubling time, the loss of cyanide-sensitive respiration, and the development of cyanide-insensitive respiration. Associated with these changes is the absence of cytochromes aa(3) and b and the inability of the organism to utilize ethanol; in addition, mitochondria appear more numerous and have less internal membrane. A further inhibitory action of the antibiotic, other than eliminating functional mitochondria, appears likely since microaerobic cultures which lack respiratory ability have twice the mass doubling time in the presence of the drug. Although a small inhibition of amino acid incorporation by cytoplasmic ribosomes is found with a high chloramphenicol concentration, it is insufficient to account for the effect on growth of the microaerobic culture. The nature of this additional effect of chloramphenicol remains to be determined, but it has been shown that increasing the glucose concentration can partially reverse this action of the antibiotic. The effect of the drug on the morphology of the organism is not as dramatic as that of phenethyl alcohol in producing yeast-like forms. However, in view of the action of chloramphenicol in eliminating functional mitochondria in M. genevensis the suggestion that phenethyl alcohol exerts its effect in promoting yeast-like morphology by uncoupling oxidative phosphorylation should be re-examined.
Tetrahymena is able to adapt to the presence of sublethal concentrations of many drugs which inhibit a wide variety of cellular functions. In spite of the generality of this phenomenon in Tetrahymena, the mechanism of adaptation at the cellular and molecular levels is unknown. This study deals mainly with adaptation to the protein synthesis inhibitors, cycloheximide and emetine. The physiological response of Tetrahymena to sublethal concentrations of these drugs is an immediate cessation of cell division for a period of time dependent on the drug concentration, followed by an abrupt resumption of exponential growth at a constant rate. By measuring the length of the growth lags under a variety of experimental conditions, we have confirmed several observations made by Frankel and coworkers, and provide evidence for two new phenomena associated with adaptation to cycloheximide: (a) adaptation to cycloheximide also results in adaptation of cells to emetine, another protein synthesis inhibitor not closely related structurally to cycloheximide. We have termed this phenomenon cross adaptation, (b) exposure to concentrations of cycloheximide too low to cause any growth lags or inhibition of protein synthesis significantly shortens the time required by cells to adapt to higher concentrations of cycloheximide. We have termed this phenomenon facilitation. Facilitation shows some degree of specificity in that facilitation with cycloheximide has no effect on adaptation to emetine. From this, we infer the existence of two distinct systems involved in adaptation to cycloheximide, one of which shows a higher degree of specificity towards cycloheximide than the other. We also show that transfer of adapted or facilitated cells to drug-free medium results in a gradual but complete resensitization. The kinetics of resensitization suggest that the cellular machinery responsible for adaptation and facilitation does not leave the cell, but is simply diluted out during cell division.
The sporangiospores of Mucor bacilliformis (NRRL 2346) were compared to those of Mucor fragilis (ATCC 10777), Mucor hiemalis (ATCC 1205), Mucor mucedo (ATCC 7941), Mucor racemosus (ATCC 1216a) and Mucor rouxii (NRRL 1894) with respect to number of nuclei, weight, amount of DNA, RNA and protein and rate of germination. They were found to be mostly uninucleate like those of the other five organisms. They have, however, a much smaller weight and contain significantly less DNA, RNA and protein. The relative proportions of these macromolecules are also characteristically different for M. bacilliformis. The ratio of RNA to DNA is about two, whereas for the other Mucor species it varies from eight to thirteen. These results suggest that the sporangiospores of M. bacilliformis are deficient in RNA.
Some properties of the inducible α-glucosidase system of Mucor rouxii were investigated. This enzymatic activity was induced after resuspending glucose-grown cells in a maltose-supplemented medium. The wall-bound activity of α-glucosidase was determined by using intact cells in the enzymatic assay; this activity represented from 80 to 90% of the total activity present in the induced cells. The addition of glucose before, or during, the induction period repressed α-glucosidase synthesis. α-Glucosidase induction was tested under aerobic and anaerobic conditions. It was found that the enzyme synthesis and the appearance of wall-bound activity were not affected by changing the gaseous environment. On the other hand, it was observed that anaerobically grown yeast-like cells were much less efficient than aerobic mycelia to develop wall-bound α-glucosidase activity. This could explain earlier observations about the incapacity of M. rouxii to utilize maltose as a substrate for anaerobic growth. This idea was strengthened by the fact that, if an anaerobic culture was induced to develop under a mycelial morphology by adding to the medium the chemical agent EDTA, these cells also acquired the capacity to grow on maltose and concomitantly possessed wall-bound α-glucosidase activity. The relevance of the structure of the cell wall on the capacity of M. rouxii to metabolize maltose is discussed.
The sporangiospores of Mucor bacilliformis (NRRL 2346) were compared to those of Mucor fragilis (ATCC 10777), Mucor hiemalis (ATCC 1205), Mucor mucedo (ATCC 7941), Mucor racemosus (ATCC 1216a) and Mucor rouxii (NRRL 1894) with respect to number of nuclei, weight, amount of DNA, RNA and protein and rate of germination. They were found to be mostly uninucleate like those of the other five organisms. They have, however, a much smaller weight and contain significantly less DNA, RNA and protein. The relative proportions of these macromolecules are also characteristically different for M. bacilliformis. The ratio of RNA to DNA is about two, whereas for the other Mucor species it varies from eight to thirteen. These results suggest that the sporangiospores of M. bacilliformis are deficient in RNA.
An efficient technique for the enrichment of mutants of M. racemosus by differential freeze-killing is described. Ungerminated spores were resistant to free-killing. During germination susceptibility increased up to a maximum coinciding with the appearance of germ tubes. Under optimum conditions a thousandfold preferential survival of growth-limited spores over actively germinating spores was obtained. Using this differential killing, cultures treated with mutagen were enriched with respect to auxotrophic and temperature-sensitive mutants, with the desired mutants constituting a large fraction of the survivors of the freeze-thaw treatments. This technique may be applicable to mutant enrichment in other filamentous fungi.
Contents, Introduction. Fungi as representative eukaryotes. Physical characteristics of monomers and subunits.Ribosomal RNA (rRNA). 17 S and 25 S rRNA. 5 S rRNA. 5.8 S rRNA. Ribosomal RNA modifications: Methylation and pseudouridylation.Ribosomal proteins (r-proteins). Number of r-proteins. Structural characteristics. Ribosomal protein modifications: Phosphorylation and methylation. Tertiary distribution of rRNA and protein in the ribosome.Ribosomal structural genes. Ribosomal DNA (rDNA). 5 S rRNA genes. Distribution of rRNA genes among yeast chromosomes. rRNA gene inheritance. Ribosomal protein genes.Ribosome synthesis. RNA polymerase. Processing of precursor rRNA. Precursor rRNA modification: Methylation and pseudouridylation. Ribosomal protein synthesis. Association of r-protein with pre-rRNA.Ribosome synthesis control. Potential control points. Relationship between rRNA and r-protein synthesis. Relationship between rRNA and protein synthesis. Negative control of transcription. Balanced positive and negative control. Ribosomal protein synthesis control.Conclusion. Acknowledgments. References.
Wild-typeMucor racemosus yeasts grown under CO2 treated with dibutyryl cyclic adenosine 3′,5′-monophosphate (dbcAMP) and glucose do not convert to hyphae during a CO2-to-air shift. By using procedures which select for hyphal cells, we have isolated a class of mutants ofM. racemosus which do convert to hyphae from CO2 yeast in the presence of dbcAMP and glucose but otherwise respond like wild-type cells to dbcAMP. One of these mutants (MLT-3) has been analyzed and appears to be wild type with respect to two other dbcAMP effects: (1) dbcAMP prevented the appearance of an NAD-dependent glutamate dehydrogenase during a CO2-to-air shift; (2) dbcAMP inhibited sporangiospore germination. It is probable that the altered gene product in not involved in cAMP metabolism nor does it alter permeability of this organism to dbcAMP, but rather represents an alteration of one of the systems under cAMP control.
When cycloheximide (0.2 μg per ml) was added to synchronized cultures of Tetrahymena pyriformis GL-C, the initial rate of incorporation of 14C-leucine was reduced to about 20% of the rate observed in control cells. After one hour, the rate increased fairly abruptly to about 60% of the control rate. The cells in cycloheximide underwent synchronous division about three hours after addition of cycloheximide. A second addition of cycloheximide had little effect on either the rate of incorporation or on the time of cell division in the drug. The medium in which cells had recovered brought about full inhibition of 14C-leucine incorporation in fresh cells, indicating that recovery was not accompanied by appreciable degradation of the cycloheximide. It was therefore concluded that during recovery the cells were either adapting to the cycloheximide or excluding it. The recovery process shows some specificity, since cells which had recovered from cycloheximide, and had become insensitive to a second dose of this drug, still retained full sensitivity to another drug, colchicine. Conversely, cells recovering in colchicine became insensitive to fresh colchicine but remained sensitive to cycloheximide.
The intracellular concentration of S-adenosylmethionine (SAM) and the specific activity of S-adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, EC 2.5.1.6) were examined in wild-typeMucor racemosus, as well as a morphological mutant termedcoy, under conditions designed to prevent the morphogenesis of yeasts to hyphae. When the mutant was grown in a defined medium supplemented with methionine and induced to shift by exposure to air, there was an increase in intracellular SAM analogous to that previously reported with the wild type. However, when thecoy mutant was grown in the absence of methionine, the intracellular concentration decreased dramatically and the mutant failed to undergo the yeast to hypha transition. An inhibitor of SAM synthetase activity, cycloserine, was used to lower the intracellular concentration of SAM in the wild-type organisms. Under these conditions, wild-typeM. racemosus failed to undergo the transition from yeasts to hyphae when exposed to air.
Some properties of the inducible α-glucosidase system of Mucor rouxii were investigated. This enzymatic activity was induced after resuspending glucose-grown cells in a maltose-supplemented medium. The wall-bound activity of α-glucosidase was determined by using intact cells in the enzymatic assay; this activity represented from 80 to 90% of the total activity present in the induced cells. The addition of glucose before, or during, the induction period repressed α-glucosidase synthesis. α-Glucosidase induction was tested under aerobic and anaerobic conditions. It was found that the enzyme synthesis and the appearance of wall-bound activity were not affected by changing the gaseous environment. On the other hand, it was observed that anaerobically grown yeast-like cells were much less efficient than aerobic mycelia to develop wall-bound α-glucosidase activity. This could explain earlier observations about the incapacity of M. rouxii to utilize maltose as a substrate for anaerobic growth. This idea was strengthened by the fact that, if an anaerobic culture was induced to develop under a mycelial morphology by adding to the medium the chemical agent EDTA, these cells also acquired the capacity to grow on maltose and concomitantly possessed wall-bound α-glucosidase activity. The relevance of the structure of the cell wall on the capacity of M. rouxii to metabolize maltose is discussed.
The germination of spores of Mucor rouxii into hyphae was inhibited by 2 mm dibutyryl cyclic adenosine 3′,5′-monophosphate or 7 mm cyclic adenosine 3′,5′-monophosphate; under these conditions spores developed into budding spherical cells instead of filaments, provided that glucose was present in the culture medium. Removal of the cyclic nucleotides resulted in the conversion of yeast cells into hyphae. Dibutyryl cyclic adenosine 3′,5′-monophosphate (2 mm) also inhibited the transformation of yeast to mycelia after exposure of yeast culture to air.Since in all living systems so far studied adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase are involved in maintaining the intracellular cyclic adenosine monophosphate level, the activity of both enzymes and the intracellular concentration of cyclic adenosine monophosphate were investigated in yeast and mycelium extracts. Cyclic adenosine monophosphate phosphodiesterase and adenylate cyclase activities could be demonstrated in extracts of M. rouxii. The specific activity of adenylate cyclase did not vary appreciably with the fungus morphology. On the contrary, cyclic adenosine monophosphate phosphodiesterase activity was four- to sixfold higher in mycelial extracts than in yeast extracts and reflected quite accurately the observed changes in intracellular cyclic adenosine monophosphate levels; these were three to four times higher in yeast cells than in mycelium.
Protein kinase and cyclic adenosine 3′,5′-monophosphate (cAMP) binding activities have been detected in cell extracts of the dimorphic fungus Mucor rouxii. The subcellular distribution of both activities indicates that most of the binding protein is in the high-speed supernatant (S100), while about 70% of the total protein kinase activity remains in particulate fractions. S100 preparations have been analyzed by diethylaminoethyl cellulose column chromatography. Binding activity can be resolved in two peaks (A and B) and protein kinase in three peaks (I, II, and III). Peaks I and II are casein dependent and insensitive to cAMP. Peak III utilizes histone as substrate and is activated (two- to fourfold) by cAMP. Theophylline strongly inhibits cAMP binding activity and mimics the effect of cAMP on cAMP-dependent protein kinase. The possible relationship between cAMP binding activity and cAMP-dependent protein kinase is suggested.
The pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) isoenzymes from kidney cortex have been studied further. Pyruvate kinase type I which is the major component, shows homotropic cooperativity towards the substrate phosphoenol pyruvate, the effect is independent of pH. This isoenzyme is markedly inhibited by alanine and other amino acids but its activity is not modified by ATP or fructose 1,6-diphosphate. The kinetic behavior of this isoenzyme is then clearly different from that of the other pyruvate kinases from rat tissues.Pyruvate kinase type II exhibits sigmoid kinetics with respect to phosphoenolpyruvate. The enzyme is activated by fructose 1,6-diphosphate and inhibited by ATP, alanine and cysteine. The allosteric properties of the enzyme are strongly affected by changes in the pH. It is concluded that this kidney cortex isoenzyme is very similar to the pyruvate kinase type L from liver.
beta-Glucosidase activity in crude extracts of Mucor racemosus exists in a soluble form and in a wall-bound form which sediments at 3,500 x g. The soluble form and a wall-bound form were purified to homogeneity by ammonium sulfate fractionation. DEAE-Sephadex chromatography, and SP-Sephadex chromatography. Both forms were identical in all parameters measured. Each enzyme is a glycoprotein of 91,000 daltons, with an identical amino acid composition and N-terminal amino acid of lysine; both contain about 10% carbohydrate. Both forms catalyze the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside with identical kinetic constants.
The in vivo regulation of glutamate dehydrogenase (GDH) was studied in Mucor racemosus as a function of nutritional conditions and morphological state. Both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP)-dependent GDH activities were found. The effect of carbon and nitrogen source on the specific activity of the NAD-dependent GDH suggests that its role is primarily catabolic. The NAD-dependent activity was generally an order of magnitude greater in mycelial cells than in yeast-phase cells grown on the same medium. During yeast-to-hyphal morphogenesis the increase in NAD-dependent activity preceded the appearance of hyphal cells both under aerobic and anaerobic conditions. Exogenous dibutyryl-cyclic AMP prevented the increase in NAD-dependent GDH concomitantly with the suppression of morphological differentiation. The NADP-dependent activity did not change appreciably during morphogenesis.
A variety of cultural conditions were examined to determine the relationship between respiratory capacity and the growth of Mucor racemosus in the yeast and mycelial form. The results show that both yeasts and hyphae can develop when the respiratory capacity is low (e.g., in N2). In addition, the yeast form of the fungus could be grown in air in the presence of cyclic adenosine 5'-monophosphate with high respiratory rates characteristic of air-grown mycelia. These results indicate that their is not an obligatory relationship between respiratory capacity and morphogenesis in M. racemosus. Low intracellular levels of cyclic adenosine 5'-monophosphate, however, were correlated with aerobic mycelial development, whereas yeast development under CO2 was characterized by higher cyclic adenosine 5'-monophosphate levels.
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.
A variety of cultural conditions were examined to determine the relationship between pyruvate kinase isozyme patterns and morphology in Mucor racemosus. The results indicate that M. racemosus has two isozymes of pyruvate kinase, form A and form B, which are clearly separable on ion-exchange columns (diethylaminoethyl-cellulose). Addition of glucose to cultures growing on amino acids in air resulted in the induction of form A and the termination of form B synthesis. Cycloheximide added at the same time as glucose blocked the formation of form A but did not interfere with the termination of form B synthesis. Removal of glucose resulted in termination of form A synthesis and the induction of form B. Cycloheximide blocked the induction of form B and did not interfere with the termination of form A synthesis. The data show that the isozyme type is not directly related to morphology, but depends only on the presence or absence of glucose.
Ribosomal proteins of the dimorphic fungus Mucor racemosus were isolated and characterized by 2-dimensional gel electrophoresis. Proteins from ribosomes of the yeast and mycelial phase were compared, and were found to be qualitatively indistinguishable. The only consistent difference in the patterns of proteins was in a protein of the 40S subunit, S-6. This protein was phosphorylated in yeast and hyphae forms, but not in asexual sporangiospores. Studies on protein S-6 showed that it contained 3 phosphate residues per molecule of protein when maximally phosphorylated. In this form 3 different tryptic peptides were shown to contain a single phosphoserine. The S-6 protein also existed in forms containing 1 or 2 phosphates per molecule, depending on growth conditions.
Saccharomyces cerevisiae cells contain a small internal pool of the secretory enzymes invertase and acid phosphatase. This pool increases up to 8-fold at 37 degrees C in a temperature-sensitive, secretion-defective mutant strain (sec 1-1). Cell division and incorporation of a sulfate permease activity stop abruptly at the restrictive temperature, while protein synthesis continues for several hours. Electron microscopy of mutant cells incubated at 37 degrees C reveals a large increase in the number of intracellular membrane-bound vesicles, which are shown by histochemical staining to contain the accumulated acid phosphatase. The vesicles are removed and the accumulated enzymes are secreted when cells are returned to a permissive temperature in the presence or absence of cycloheximide. These results are consistent with a vesicle intermediate in the yeast secretory pathway and suggest that exocytosis may contribute to cell-surface growth.
Large contrasts are observed between the messenger RNA populations of different tissues and of embryos at different stages of development. Nevertheless, coding sequences for genes not expressed in a cell appear to be present in its nuclear RNA. Though many nuclear RNA transcripts of single copy DNA sequences are held in common between tissues, an additional set, probably consisting of non-message sequences, is not shared. Nuclear RNA also contains transcripts of repetitive DNA sequences. Certain repeat families are represented at high levels in the nuclear RNA of particular tissues and much lower levels in others. It is surprising that both complements of most repeat sequences are present in nuclear RNA. These observations lead to model for regulation of gene expression in which the formation of repetitive RNA-RNA duplexes controls the production of messenger RNA.
The effects of the protein synthesis inhibitors trichodermin and anisomycin on the growth of the eucaryotic myxomycete Physarum polycephalum have been examined. When either of these drugs is added to log phase monoxenic cultures of myxamoebae, cell division is immediately arrested, but on continued incubation, growth resumes at a rate only slightly lower than that of drug free cultures. The length of the drug induced growth lag is roughly proportional to drug concentration. When adapted cells are transferred to fresh drug containing medium, growth is not inhibited. However, if the drug concentration is increased, transient inhibition is again exhibited. Measurement of the antibiotic concentration in used media demonstrates no significant external inactivation of either drug during adaptation. The resumption of growth cannot be attributed to the selection of stable drug-resistant mutants: single amoebal colonies arising on drug plates are found to be as drug-sensitive as control colonies when retested after subculture. In addition, when adapted cells are transferred to drug free medium, the phenotypic drug-resistance is completely lost after several generations of growth. As recovery occurs in the continuous presence of drug and is not due to the accumulation of drug-resistant mutants, this response appears to be an example of drug adaptation. Cross adaptation between anisomycin and trichodermin is also demonstrated, suggesting a common system is involved in adaptation to these structurally dissimilar, but functionally similar, drugs.
The intracellular location of active protein synthesis was examined during the emergence of germ tubes from both sporangiospores and yeast-phase cells of Mucor racemosus. It was determined that protein synthesis occurs in all regions of the cell and not preferentially at the growing tip.
The present study was undertaken in order to elucidate the molecular mechanisms responsible for regulating changes in the specific rate of protein synthesis during the yeast-to-hyphae morphogenesis in the fungus, Mucor racemosus. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by means of pulse-labeling and density gradient fractionation techniques. The percentage of ribosomes active in protein synthesis was observed to decrease throughout the morphological transition. The rate of amino acid addition to nascent polypeptide chains was calculated and the transit time of messenger RNA translation was measured. The results showed a significant increase in the velocity of ribosome movement along the message which was continuously adjusted throughout hyphal development.
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.
As is evident from the above summary of the recent literature, plus many other papers not cited here, there is an extensive literature indicating the physiological significance of these amines. The most important studies can be summarized as follows. (a) Polyamines and their biosynthetic enzymes are ubiquitous. (b) Microbiological mutants have been described in which there is a definite requirement of polyamines for growth. (c) The concentration of polyamines and their biosynthesis enzymes increase when the growth rate increases. These increases usually precede or are simultaneous with increases in RNA, DNA, and protein levels. (d) Ornithine decarboxylase has a remarkably fast turnover rate in animal cells, and the level of this enzyme rapidly changes after a variety of growth stimuli. (e) Polyamines have a high affinity for nucleic acids and stabilize their secondary structure. They are found associated with DNA in bacteriophages and have a variety of stimulatory effects on DNA and RNA biosynthesis in vitro. (f) Polyamines stimulate protein synthesis in vivo and in vitro. (g) Polyamines protect spheroplasts and halophilic organisms for lysis, indicating their ability to stabilize membranes. Despite these observations, no specific mechanism has been firmly established for the action of the polyamines in vivo. It is clear that these compounds are physiologically important, however, and further work is necessary to establish the mechanism of their action.
Cells of Mucor racemosus were labeled with l-[(14)C]leucine during the yeast-to-hyphae morphogenesis that follows a change of atmosphere from CO(2) to air. Pulse-labeling kinetics and the steady-state accumulation of incorporated l-[(14)C]leucine were determined throughout the period of cellular differentiation. We determined that the l-[(14)C]leucine was taken up by all forms of the organism, was not altered from the form of l-leucine, and was incorporated exclusively into protein. The intracellular pool of free l-leucine was small in comparison with those of the other l-amino acids, remained relatively constant in size during morphogenesis, and was rapidly equilibrated with exogenous leucine. Approximately the same internal radiospecific activities were attained throughout development shortly after addition of l-[(14)C]leucine to a culture. Experiments performed with leucine auxotrophs suggested that endogenous synthesis of leucine in prototrophs does not affect the measured rates of incorporation. Experiments performed with (14)C-labeled l-isoleucine, l-proline, l-lysine, and l-arginine produced results qualitatively the same as with l-leucine. The accumulation of incorporated l-[(14)C]leucine in a culture of M. racemosus undergoing the air-induced yeast-to-hyphae transition reflected the change in growth rate that accompanied the morphogenesis. However, the specific rate of protein synthesis measured throughout the developmental process displayed a characteristic acceleration during the emergence of germ tubes which was followed by a decline when all further growth took the form of hyphal elongation. Data are presented suggesting that this response is a correlate of morphogenesis rather than a consequence of the atmospheric change per se.
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.
The distribution of chloroform-methanol and alkali-extractable lipids in the cell walls of aerobically grown filamentous cells from Mucor rouxii has been determined. The results have been compared with the corresponding lipid composition of yeast-like cells from M. rouxii, which can be produced in two ways: by growth under anaerobic conditions and by aerobic growth in the presence of 0.22% phenethyl alcohol (PEA). It was observed that in most cases the crude cytoplasmic fraction contained higher levels of several lipids (i.e., squalene, sterols, triterpenes, and fatty acids) than did the corresponding cell walls. The cell walls did, however, contain both "free" (chloroform-methanol extractable) and "bound" (alkali extractable) lipids although the relative amounts were markedly dependent on the cell growth environment. The aerobically grown filamentous cell walls contained higher levels of squalene, sterols, triterpenes, and fatty acids than did aerobically grown yeast-like PEA-induced cell walls and there was also considerable variation in the "free"/"bound" ratios of the various lipid components. The lipid levels in both the cell walls and cytoplasm of the anaerobically grown cells were considerably lower than those of the cells grown under aerobic conditions. In addition, the differences in the growth environment were also reflected in the compositions of the individual lipid fractions from both the cell wall and the cytoplasm fraction.
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.
The protein synthesis elongation factor EF-1 alpha of Mucor racemosus hyphae contained eight or nine methylated amino acids
per molecule, whereas the factor from sporangiospores was nonmethylated. During the course of spore germination, the specific
activity of the factor in crude extracts increased sixfold. This increase in activity was accompanied by a constant level
of EF-1 alpha-specific mRNA and a constant level of EF-1 alpha protein. Methylation of the protein, however, accelerated during
the germination process, in parallel with the increase in specific activity of the factor. We propose that the activity of
EF-1 alpha is regulated during germination through methylation of the protein and does not involve transcriptional regulation.
Pyruvate kinase has been purified from bovine skeletal muscle by a procedure that includes only heat, ammonium sulfate fractionation,
and chromatography on carboxymethyl Sephadex. Crystallization was accomplished by dialysis at 4° against 47% saturated ammonium
sulfate. Since this simple purification scheme, with only slight modifications, has also been used to isolate turkey and rabbit
muscle pyruvate kinases, it may be generally applicable to the isolation of muscle pyruvate kinase from birds and mammals.
Bovine muscle pyruvate kinase prepared by the procedure described here was found to be homogeneous, as determined by disc
gel electrophoresis at pH 9.5, gel electrophoresis in the presence of sodium dodecyl sulfate, sedimentation velocity and sedimentation
equilibrium, isoelectric focusing, and immunodiffusion. It sediments at 9.9 S (s020,w) and has a maximum velocity of 400 µmoles per min per mg or more at 25°, or 2.4 times that value at 37°. The enzyme has maximal
activity at pH 7.1, with an apparent Michaelis constant for phosphoenolpyruvate of 0.04 mm or less, and for ADP of 0.4 mm. Its isoelectric pH during electrofocusing is 8.9.
Sedimentation equilibrium yielded a molecular weight of 230,000 with a range of ±3,000 in dilute phosphate buffer, and 57,000
± 1,500 in 3.5 m guanidine hydrochloride, using a partial specific volume of 0.740 ml per g calculated from the amino acid composition. The
molecular weight in guanidine hydrochloride, confirmed by gel electrophoresis in sodium dodecyl sulfate, indicates the presence
of four polypeptide chains in the native enzyme. Binding studies suggest a total of four phosphoenolpyruvate binding sites,
or one per subunit. Ion sensitivities and other chemical and physical parameters are very similar to those reported for rabbit
muscle pyruvate kinase.
The induction of beta-glucosidases (EC 3.2.1.21) was studied in Neurospora crassa. Cellobiase was induced by cellobiose, but other inducers had little effect on this enzyme. Cellobiase activity was very low in all stages of the vegetative life cycle in the absence of di-beta-glucoside inducer. Aryl-beta-glucosidase was semiconstitutive at late stages of culture growth prior to conidiation. At early stages, aryl-beta-glucosidase was induced by cellobiose, laminaribiose, and gentiobiose, and weakly induced by galactose, amino sugars, and aryl-beta-glucosides. The induction properties of the beta-glucosidases are compared with those of the other disaccharidases of Neurospora. The induction of beta-glucosidases was inhibited by glucose, 2-deoxy-d-glucose, and sodium acetate. Sodium phosphate concentrations between 0.01 and 0.1 M stimulated induction of both enzymes, while concentrations above 0.1 M were inhibitory. The optimal condition for induction of both beta-glucosidases was pH 6.0. Cellobiase induction was relatively more inhibited than aryl-beta-glucosidase in the range of pH 6.0 to 8.0.
Stable mutants of Mucor bacilliformis having lost the ability to grow filamentously and to sporulate occur spontaneously with a frequency of about one in every 3000 colonies. On solid and in liquid medium these mutants have a typical yeastlike morphology and reproduce by budding. The detailed study of one of these mutants shows that the inability to form filaments and spores is accompanied by the loss of cytochrome oxidase activity. This mutant is unable to take up oxygen but has a high level of alcoholic fermentation, which appears to be the major if not the sole source of energy.
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.
1.1. Maltose is utilized by Mucorrouxii by means of an inducible system. Induction is brought about by growth in the presence of maltose, and it is prevented by addition of cycloheximide.2.2. Maltose is not fermented by glucose-grown spores nor oxidized or fermented by spheroplasts unless cell walls isolated from maltose-grown mycelium are incorporated into the incubation medium. On the other hand, glucose is rapidly utilized by glucose-grown spores and spheroplasts.3.3. There is no uptake of radioactive material when spheroplasts are incubated with [I-14C]glucose is rapidly taken up by spheroplasts.4.4. The fungus contains an inducible cell wall-bound α-glucosidase. Isolated cell walls from the maltose-grown mycelium hydrolyse maltose, giving glucose as the only product.5.5. From these evidences it is concluded that maltose does not penetrate readily into the fungus, and that it must be hydrolysed outside the permiability barrier by the inducible cell wall-bound α-glucosidase in order to be utilized.
A Saccharomyces strain (1403-7A) has been described which carries the maltose gene, MAL4, and produces 15–500 fold more enzyme when grown in glucose than any other strain tested. The factor responsible for this high basal level is genetically dominant and closely linked to or identical with the structural gene. Maltase produced by this strain has a different heat stability, specific activity, and michaelis constant from the maltase produced by another strain (1394-9B) carrying the MAL4 allele. It has been suggested that hyper production of enzyme is a function of the MAL4 locus and that structural alteration of the enzyme can occur in some strains at a post transcriptional level.
Unlike five other strains of Mucor rouxii previously studied, certain nutritional factors must be present for rapid growth and completely yeastlike development of M. rouxii (National Regional Research Laboratory 1894) under CO(2); high CO(2) tensions markedly inhibit growth of this strain. Addition of yeast extract, peptone, or enzymatically hydrolyzed casein in substrate amounts to a basal medium (containing acid-hydrolyzed casein) completely relieved CO(2) inhibition of growth and permitted yeastlike development. The "CO(2) growth factor" activity of these supplements proved to be dialyzable and acid labile. These findings, together with the results of gel filtration and amino acid analysis, suggested that CO(2) growth factor activity can be attributed to small peptides.