Morphology and growth kinetics of hyphae of differentiated and undifferentiated mycelia of Neurospora crassa.

A comparison was made of the morphology and growth kinetics of hyphae of differentiated and undifferentiated mycelia of Neurospora crassa. Undifferentiated mycelia were formed during exponential growth on solid media or submerged culture. Hyphae at the margin of differentiated mycelia (colonies) differed from undifferentiated mycelia in diameter, extension rate, extension zone length, and intercalary and apical compartment length. The mean hyphal extension rate (E) of an undifferentiated mycelium was a function of the length of the mycelium's hyphal growth unit (G) and the organism's specific growth rate (alpha). Thus, E=Galpha.     

Effect of temperature and temperature shifts on growth and branching of a wild type and a temperature sensitive colonial mutant (Cot 1) of Neurospora crassa

Growth of a temperature sensitive colonial mutant (cot 1) of Neurospora crassa was compared with a wild type strain. The hyphal growth unit (the ratio between mycelial length and number of branches) of the wild type was not appreciably altered by temperature and there was a direct relationship between the specific growth rate () of these mycelia and their mean hyphal extension rate (E). The specific growth rate of cot 1 increased by about the same relative amount as the wild type between 15° and 30°C. Cot 1 grew and branched normally at 15° and 25°C but at 30°C the hyphal growth unit and mean hyphal extension rate of the mutant mycelia were reduced. Thus, between 15–30°C the ratio, E/ was constant for the wild type but not for cot 1.The effect of temperature and temperature shifts on extension zone length (Z), extension zone expansion time (Z _i ) and branching of leading hyphae of mature colonies were also studies.It is suggested that branching is governed by a mechanism which regulates the linear growth rate of hyphae; the cot 1 mutation may have a direct effect on wall extension or affect linear growth rate indirectly due to an influence on the transport of precursors to the tip.     

Effect of choline on the morphology, growth and phospholipid composition of Fusarium graminearum

Studies were made of the growth kinetics, morphology and phospholipid composition of two strains of Fusarium graminearum, a wild-type strain (A3/5) and a highly branched variant (C106) which arose spontaneously during cultivation of A3/5. No significant difference was observed between the hyphal diameters of the two strains and therefore increased branching of C106 could not be explained in the terms of an increase in hyphal radius in the absence of a change in hyphal growth unit volume. The two strains had the same specific growth rate in batch culture and this was not affected by the addition of up to 1.5 mM-choline to the medium. However, choline increased the mean hyphal extension rate and colony radial growth rate of both strains and this response was correlated with the formation of mycelia which were more sparsely branched than mycelia grown on medium lacking choline. Addition of betaine, choline, ethanolamine, monomethylethanolamine or dimethylethanolamine (but not serine, glycine, dimethylglycine, methylamine, hydroxylamine or beta-hydroxyethylhydrazine) to the medium also resulted in appreciable increases in the colony radial growth rates of A3/5 (increased by about 130% for choline) and C106 (increased by about 25% for choline). No significant difference was observed between the phospholipid compositions of the two strains, and the addition of 100 microM-choline to the medium had no significant effect on the phospholipid composition of either strain.     

Evidence for the independent regulation of hyphal extension and branch initiation in Fusarium graminearum A3 5

Abstract Addition of 100 μM choline chloride to the medium increased (by approx. 36%) both the length of the hyphal growth unit ( G a measure of mycelial branching) and the mean hyphal extension rate ( E ) of Fusarium graminearum but did not increase the maximum rate of extension of the hyphae ( E _max). The paramorphogen, edifenphos (Hinosan) reduced G and E without affecting specific growth rate (μ). However, when mycelia were treated with edifenphos plus choline, μ was reduced, G was increased by approx. 35%, but E and E _max were not affected. The results suggest that the primary effect of edifenphos is inhibition of hyphal extension, whilst the primary effect of choline is inhibition of branch initiation.     

Aspergillus nidulans class V and VI chitin synthases CsmA and CsmB, each with a myosin motor-like domain, perform compensatory functions that are essential for hyphal tip growth

The polarized synthesis of cell wall components such as chitin is essential for the hyphal tip growth of filamentous fungi. The actin cytoskeleton is known to play important roles in the determination of hyphal polarity in Aspergillus nidulans. Previously, we suggested that CsmA, a chitin synthase with a myosin motor-like domain (MMD), was involved in polarized chitin synthesis in a manner dependent on the interaction between the MMD and the actin cytoskeleton. The genome database indicates that A. nidulans possesses another gene encoding another chitin synthase with an MMD. In this study, we characterized this gene, which we designated csmB. The csmB null mutants examined were viable, although they exhibited defective phenotypes, including the formation of balloons and intrahyphal hyphae and the lysis of subapical regions, which were similar to those obtained with csmA null mutants. Moreover, csmA csmB double null mutants were not viable. Mutants in which csmB was deleted and the expression of csmA was under the control of the alcA promoter were viable but severely impaired in terms of hyphal growth under alcA-repressing conditions. We revealed that CsmB with three copies of a FLAG epitope tag localized at the hyphal tips and forming septa, and that the MMD of CsmB was able to bind to actin filaments in vitro. These results suggest that CsmA and CsmB perform compensatory functions that are essential for hyphal tip growth.     

Chitin biosynthesis in protoplasts and subcellular fractions of Aspergillus fumigatus.

The biosynthesis of chitin has been obtained in broken mycelia and protoplasts of the fungus Aspergillus fumigatus. The specific activity of chitin synthase (EC 2.4.1.16) in a membrane preparation from protoplasts derived from the hyphal tips of A. fumigatus was 26.8-fold greater than that of the chitin synthase in broken mycelia, indicating that the active chitin synthase is located primarily in a membrane-bound site at the hyphal tip. Polyoxin D was a potent competitive inhibitor of the enzyme, having Ki 5.2 +/- 0.8 micron with respect to the natural substrate UDP-N-acetyl-D-glucosamine, which has Km 1.58 mM.     

Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.     

CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8 Delta null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2 Delta chs8 Delta double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1 Delta mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity.     

Effects of validamycin A on the morphology, growth and sporulation of Rhizoctonia cerealis, Fusarium culmorum and other fungi

All Basidiomycotina screened were sensitive to validamycin A, whereas most Ascomycotina and all Mucorales and Oomycetes were insensitive. Studies with Rhizoctonia cerealis and Fusarium culmorum showed that, in semi-solid culture, the antibiotic caused a decrease in colony radial growth rate and that this was associated with a decrease in mean hyphal extension rate and an increase in hyphal branching. However, the antibiotic did not alter the morphology of R. cerealis grown in liquid culture (shaken or stationary). Validamycin A caused a reduction in the number and viability of conidia produced by F. culmorum.     

Botrytis cinerea virulence is drastically reduced after disruption of chitin synthase class III gene (Bcchs3a)

Botrytis cinerea is an important phytopathogenic fungus requiring new methods of control. Chitin biosynthesis, which involves seven classes of chitin synthases, could be an attractive target. A fragment encoding one of the class III enzymes was used to disrupt the corresponding Bcchs3a gene in the B. cinerea genome. The resulting mutant exhibited a 39% reduction in its chitin content and an 89% reduction in its in vitro chitin synthase activity, compared with the wild-type strain. Bcchs3a mutant was not affected in its growth in liquid medium, neither in its production of sclerotia, micro- and macroconidia. In contrast, the mutant Bcchs3a was severely impaired in its growth on solid medium. Counterbalancing this defect in radial growth, Bcchs3a mutant presented a large increase in hyphal ramification, resulting in an enhanced aerial growth. Observations by different techniques of microscopy revealed a thick extracellular matrix around the hyphal tips. Moreover, Bcchs3a mutant had a largely reduced virulence on Vitis vinifera and Arabidopsis thaliana leaves.     

The influence of supplemental components in nutrient medium on chitosan formation by the fungus Absidia orchidis

Chitosan, a derivative of chitin, is a natural component of some fungus cell walls. It is formed by the complex action of chitin synthase and chitin deacetylase. The in vitro activity of these two enzymes is known to be influenced by several factors. We investigated the influence of ferrous ions, manganese ions, cobalt ions, trypsin, and chitin, as individual supplements to the nutrient medium, on the in vivo activity of chitin synthase and chitin deacetylase to form chitosan in the fungus Absidia orchidis. Manganese and ferrous ions gave the most significant results. These ions increase chitosan yields through an increase in biomass production rather than an increase of chitosan content in cell walls. Manganese and ferrous ions lowered the activity of chitin deacetylase; however, their influence on the activity of chitin synthase was more complex. The effects of trypsin and chitin on biomass and cell wall chitosan content were negligible, while cobalt ions completely inhibited the growth of fungi.     

Relationship between the nitrogenase activity and dark respiration rate of Synechococcus RF-1

Growth of three different anaerobic rumen fungi Neocallimastix frontalis, Piromonas communis and Sphaeromonas communis was assessed in vitro at regular intervals by measurements of protein and chitin content and of chitin synthase activity of the cell free extracts. Similar trends and a comparable amount of protein and chitin were observed in the three species. However, chitin synthase activity was higher in S. communis and contrary to the activity of the other two strains did not decrease after maximum enzyme activity was reached. There were positive correlations between chitin content, protein content and chitin synthase activity during the active growth phase of the fungi indicating that they could be confidently used to determine in vitro growth phase and biomass concentration.     

Model for Branch Initiation in Aspergillus nidulans Based on Measurements of Growth Parameters

The rate of hyphal elongation and the number of branches per hypha were measured on short sporelings of Aspergillus nidulans growing at different rates. The rate of elongation was proportional to total length in unbranched and branched hyphae. At each growth rate, the number of branches per hypha increased with increasing length and gave approximately straight-line graphs when plotted against length. The average number of branches per unit of hyphal length was quite different for the various growth rates and increased in direct proportion to the growth rate. The results are interpreted to mean that (i) growing tips have a maximum rate at which they can elongate and which is reached at hyphal lengths characteristic of the particular growth rate and (ii) a new branch is formed when the capacity of the hypha to elongate exceeds that of the existing tips.     

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).     

Characterization of chitin and chitin synthase from the cellulosic cell wall fungusSaprolegnia monoi¨ca

The presence of chitin in hyphal cell walls and regenerating protoplast walls ofSaprolegnia monoi¨ca was demonstrated by biochemical and biophysical analyses. α-Chitin was characterized by X-ray diffraction, electron diffraction, and infrared spectroscopy. In hyphal cell walls, chitin appeared as small globular particles while cellulose, the other crystalline cell wall component, had a microfibrillar structure. Chitin synthesis was demonstrated in regenerating protoplasts by the incorporation of radioactiveN-acetylglucosamine into a KOH-insoluble product. Chitin synthase activity of cell-free extracts was particulate. This activity was stimulated by trypsin and inhibited by the competitive inhibitor polyoxin D (K_i 20 μM). The reaction product was insoluble in 1M KOH or 1M acetic acid and was hydrolyzed by chitinase into diacetylchitobiose. Fungal growth and cell wall chitin content were reduced when mycelia were grown in the presence of polyoxin D. However, hyphal morphology was not altered by the presence of the antibiotic indicating that chitin does not seem to play an important role in the morphogenesis ofSaprolegnia.     

Cell wall biogenesis in a double chitin synthase mutant (chsG-/chsE-) of Aspergillus fumigatus

Previous studies (Aufauvre-Brown et al., 1997; Mellado et al., 1996a,b ) have shown that only two genes of the Aspergillus fumigatus chitin synthase family, chsG and chsE, play a role in the morphogenesis of this fungal species. An A. fumigatus strain lacking both chsG (class III CHS) and chsE (class V CHS) genes was constructed by gene replacement of the chsE gene with a copy that has its conserved coding region interrupted by the hph resistance cassette in an A. fumigatus chsG- genetic background. Unexpectedly the double disruption was not lethal. The double mutant AfchsG-/chsE- strain (i) has reduced chitin synthase activity with or without trypsin stimulation, (ii) has a reduced colony radial growth rate, (iii) produces highly branched hyphae, (iv) exhibits aberrant features, such as periodic swellings along the length of the hyphae and a block in conidiation that can be partially restored by an osmotic stabilizer (v) shows alterations in the shape and germination capacity of the conidia, and (vi) has a cell wall that contains half the chitin of the parental strain and is, unexpectedly, highly enriched in alpha-(1-3) glucan.     

The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo

The chitin synthase of Saccharomyces is a plasma membrane-bound zymogen. Following proteolytic activation, the enzyme synthesizes insoluble chitin that has chain length and other physical properties similar to chitin found in bud scars. We isolated mutants lacking chitin synthase activity (chs1) and used these to clone CHS1. The gene has an open reading frame of 3400 bases and encodes a protein of 130 kd. The fission yeast S. pombe lacks chitin synthase and chitin. When a plasmid encoding a CHS1-lacZ fusion protein is introduced into S. pombe, both enzymatic activities are expressed in the same ratio as in S. cerevisiae, demonstrating that CHS1 encodes the structural gene of chitin synthase. Three CHS1 gene disruption experiments were performed. In all cases, strains with the disrupted gene have a recognizable phenotype, lack measurable chitin synthase activity in vitro but are viable, contain normal levels of chitin in vivo, and mate and sporulate efficiently.     

Two chitin synthases in Saccharomyces cerevisiae

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.