Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation

Abstract From autolysed cultures of Mucor rouxii , two chitosanases, A and B, were purified to electrophoretic homogeneity. Apparent M _r values of 76 000 and 58 000 and p I values of 4.9 and 4.7 were determined for A and B, respectively. Both chitosanases showed a high specificity for chitosan and chitosan derivatives. They had optimum activities at pH 5.0 and at temperatures of 55°C and 50°C for A and B, respectively. Enzyme A was inhibited by acetate ions and enzyme B by high substrate concentration. Both enzymes showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of dimer, trimer and higher molecular mass oligomers of glucosamine. Glucosamine oligosaccharides were poorly hydrolysed by these enzymes. Both enzymes extensively degraded the chitosan extracted from M. rouxii cell walls.     

The arrangement of F-actin and microtubules during germination of Mucor rouxii sporangiospores

The distribution of F-actin microfilaments and microtubules was analyzed in germinating sporangiospores of Mucor rouxii by labeling with rhodamine-tagged phalloidin and by immunofluorescence microscopy. The transition from isodiametrical to apical growth was accompanied by a switch from uniform distribution of F-actin patches to a polarized accumulation of F-actin material at the germ tube tips. Immunoblotting of cell-free extracts of M. rouxii with a monoclonal anti-porcine -tubulin antibody (TU-01) disclosed two discrete bands of -tubulin suggesting the existence of two -tubulin genes in this fungus. Immunofluorescence microscopy of germinating cells stained with the same antibody revealed an elaborate network of cytoplasmic microtubules that persisted during the entire germination process and extended into the apex of the germ tube. Although their precise roles remain undetermined, the observed arrangement of cytoskeletal elements during germination is consistent with their presumed involvement in cell wall morphogenesis: the long axial microtubules serving as long-distance conveyors of wall-building vesicles to the apical region while the concentrated F-actin patches mark the participation of microfilaments in the zone of intense vesicle exocytosis at the hyphal apex.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DTT dithiothreitol - EGTA Ethylene glycol-bis (beta-aminoethyl ether) - N,N,N,N tetraacetic acid - F-actin Filamentous actin - MES 2-(N0morpholino)-ethanesulfonic acid - PIPES Piperazine-N,N-bis-2-ethanesulfonic acid - PMSF Phenyl-methylsulphonyl fluoride - TBS Tris-buffered saline     

Actin in Mucor rouxii

Abstract Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii . The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.     

Actin in Mucor rouxii

Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii. The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.     

Role of membranes of mycelial Mucor rouxii in synthesis and secretion of cell wall matrix polymers.

Membrane fractions of the mycelial form of Mucor rouxii contained endogenous polyuronides and glycoproteins with sugar compositions similar to the cell wall polyuronide (mucoran) and extracellular (wall and filtrate) glycoproteins, respectively. The polyuronide pool was rapidly labeled with D-[U-14C]glucose and on pulse-chase experiments appeared to turn over, suggesting that these polymers were precursors of the wall mucoran. In contrast, the glycoproteins appeared to accumulate. Although the membrane-associated glycoproteins had molecular weights similar to those of the extracellular glycoproteins, the bulk of the membrane-associated polyuronides were of a molecular weight lower than that of the wall polyuronide.     

Calmodulin-like activity in Mucor rouxii

Abstract A modulator factor with properties similar to those of calmodulin was found and partially purified from the soluble fraction of Mucor rouxii . These properties include: heat-stability, stimulation in a Ca²⁺-dependent fashion of cyclic nucleotide phosphodiesterase from bovine brain, and inhibition of this process by trifluoperazine. This calmodulin-like activity was detected in the soluble fraction of both mycelium and yeast-like cells of the fungus.     

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.     

Electron microscopy of Staphylococcus aureus cell wall lysis

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Mu?oz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.     

Electron microscopy of the cotton fibre: new observations on cell wall formation.

The ultrastructure of the cotton fibres was examined after developing successful fixation methods. Fibre cells were fixed at different stages of development. In cells which were elongating and producing primary cell walls, the Golgi apparatus appeared to be directly involved in secretion and synthesis of primary wall components. In cells which were synthesizing thick secondary cell walls, evidence suggested a major role for the endoplasmic reticulum and plasma memebrane in the synthesis and secretion of secondary wall materials. The possibility of a shift from a Golgi apparatus pathway for primary wall synthesis to an endoplasmic reticulum pathway for secondary wall synthesis is discussed. Plasma membrane micro-invaginations are present only during secondary wall synthesis and may represent sites of cellulose assembly. A model for primary wall biogenesis via the Golgi apparatus is presented, and the potential of the cotton fibre as a model system for studying cellulose biogenesis in higher plants is discussed.     

Protoplast formation during spore germination inStreptomyces

Germ tubes from spores ofStreptomyces were very sensitive to lysozyme attack. A good yield of stable protoplasts was obtained 30 min after addition of the enzyme, making the growth of the microorganism in a high-glycine-content medium unnecessary. Physiologically unaltered, stable protoplasts, which are formed from cells in the same stage of their developmental cycle, may be obtained in the presence of lysozyme. After protoplast release, abundant membranous structures were observed inside the empty walls.     

Regulation of ornithine decarboxylase activity in Mucor bacilliformis and Mucor rouxii

Ornithine decarboxylase (ODC) from Mucor bacilliformis and Mucor rouxii was studied. Enzymatic activity was maximal at pH 7.2–7.4 and at 30°C. The K_m was 0.17 mM for the M. bacilliformis enzyme. Putrescine was a competitive inhibitor of ODC with a K_i of 2–3 mM. Enzymatic activity was undetectable in sporangiospores but increased rapidly during the first stages of spore swelling, reaching the highest levels during germ tube or bud emergence, and then decreased. Incubation at 30°C inhibited spore germination in M. bacilliformis and prevented development of ODC activity. More ODC activity was present in mycelial than in yeast cells. Morphological transition of yeast cells into hyphae by an anaerobic-aerobic shift induced a rapid and transient increase in ODC activity. Similar results were obtained when the morphogenetic transformation of M. rouxii was induced by CO₂ elimination in an anaerobic environment. Transfer of mycelial cells to anaerobiosis resulted in a rapid decrease in enzyme activity. Changes in ODC activity were accompanied by a change in the pool of polyamines. The possible role of ODC in growth and cell differentiation in Mucor is discussed.     

Inhibition and activation of chitin synthesis by Mucor rouxii cell extracts

The chitin synthetase activity of a “microsomal” fraction from Mucor rouxii increased during storage under the appropriate conditions. This increase in activity was blocked by an inhibitor present in the soluble cytoplasm. The inhibitor was thermostable, nondialysable, and its action was pH dependent. Sensitivity to the inhibitor was lost on prolonged storage. These observations agree with the mechanism of control of chitin synthetase in Saccharomyces proposed by Cabib and Farkas in which the enzyme is produced in a zymogen state and then converted to its active form by proteolysis. The inhibitor blocks this conversion.     

Relationship between spore germination kinetics and lag time during growth of Mucor racemosus

AIMS: Spore germination requires microscopic observation whereas fungal growth results in a macroscopic examination. This paper aims at establishing a relationship between the percentage of germinated spores and parameters easily available from visible development. METHODS AND RESULTS: About 225 spores of Mucor racemosus were inoculated on PDA medium and incubated at 15 degrees and 25 degrees C. Germination kinetics were modelled by a logistic function. Fungal development provided two parameters, a growth rate, micro, and a lag period, lambda, defined as the slope of the straight line of the graph radius (mm) vs time (h) and the intercept of this line with the X-axis, respectively. CONCLUSIONS: It was found that the lag period coincided with the completion of the germination process, although the number of spores inoculated should be controlled carefully. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing that this result can be generalized, this procedure would constitute a significant breakthrough for predicting food spoilage by moulds.     

Processing of asparagine-linked saccharides in Mucor rouxii

Asparagine-linked Glc₁Man₉GlcNAc₂, Glc₁Man₈GlcNAc₂ and Glc₁Man₇GlcNAc₂ were detected in mycelial-form cells of the dimorphic fungus Mucor rouxii inculbated with [U-¹⁴C]glucose for 3 min. The oligosaccharides were absent from glycoproteins isolated from cells chased for 15 min with the unlabed monosaccharide. This was due to deglucosylation of the oligosaccharides and not to further addition of mannose residues to them. The half-lives of the glucosylated compounds were much shorter, therefore, in M. rouxii than in other eucaryotic cells. Further processing of N-linked saccharides led to the synthesis of mannan-like glycoproteins, some of whch contained methyl groups in position 3 or the mannose residues. Methylation occurred only at the non-reducing ends and prevented further elongation of the branches.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

A pathway of chitosan formation in Mucor rouxii: enzymatic deacetylation of chitin

An enzyme which hydrolyzes the acetamido groups of N-acetylglucosamine residues in chitin was partially purified from $\text{Mucor rouxii}$. The enzyme deacetylates also N-acetylchitooligoses, whereas it is inactive toward bacterial cell wall peptidoglycan, N-acetylated heparin, a polymer of N-acetylgalactosamine, di-N-acetylchitobiose, or N-acetylglucosamine. The enzyme shows a pH optimum of 5.5 and is markedly inhibited by acetate. The occurrence of this enzyme accounts for the formation of chitosan in fungi.     

Electron microscopy of the cell wall complex ofMethylomonas albus

In surface view, the cell wall complex ofMethylomonas albus possesses a hexagonal pattern of ridges. Thin sections reveal a continuous layer of goblet-shaped elements attached to the outermost surface of the lipopolysaccharide membrane. A possible interpretation of the cell wall complex ofM. albus, based on the fine-structural data is presented.