Deletion of BGL2 results in an increased chitin level in the cell wall of Saccharomyces cerevisiae

It is shown that the deletion of BGL2 gene leads to increase in chitin content in the cell wall of Saccharomyces cerevisiae. A part of the additional chitin can be removed from the bgl2Δ cell wall by alkali or trypsin treatment. Chitin synthase 1 (Chs1) activity was increased by 60 % in bgl2Δ mutant. No increase in chitin synthase 3 (Chs3) activity in bgl2Δ cells was observed, while they became more sensitive to Nikkomycin Z. The chitin level in the cell walls of a strain lacking both BGL2 and CHS3 genes was higher than that in chs3Δ and lower than that in bgl2Δ strains. Together these data indicate that the deletion of BGL2 results in the accumulation and abnormal incorporation of chitin into the cell wall of S. cerevisiae, and both Chs1 and Chs3 take part in a response to BGL2 deletion in S. cerevisiae cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nikkomycin Z counteracts rylux BSU and Congo red inhibition ofSaccharomyces cerevisiae growth but does not prevent formation of aberrant cell walls

Rylux BSU and Congo red bind to chitin, interfere with proper cell-wall assembly, and stimulate chitin synthesis by increasing, most probably, chitin synthase 3 (ChS3) levels inSaccharomyces cerevisiae. On the other hand, the antibiotic nikkomycin Z inhibits chitin synthesis competitively. As ChS3 is the critical target of nikkomycin Z, its effect was tested in cells inhibited in growth by Rylux BSU or Congo red. Nikkomycin Z counteracted this inhibition but did not counteract aberrant cell-wall formation. These results indicate that chitin synthesis stimulation is the key step in Rylux BSU and Congo red inhibition and support the idea that increase in chitin synthesis represents a compensatory response to damaged cell-wall structure. As Rylux BSU and Congo red bind to newly synthesized chitin, further damage is caused in the wall and the response works in this case contrariwise. Nikkomycin Z breaks this vicious circle by counteracting the chitin synthesis stimulation.     

Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.     

Hyperpolarized growth of Saccharomyces cerevisiae cak1P212S and cla4 mutants weakens cell walls and renders cells dependent on chitin synthase 3.

In a screen for cell wall defects in Saccharomyces cerevisiae, we isolated a strain carrying a mutation in the Cdc28-activating kinase CAK1. The cak1P212S mutant cells exhibit multiple, elongated and branched buds, beta(1,3)glucan-poor regions of the cell periphery and lysed upon osmotic shock after treatment with the chitin synthase III inhibitor Nikkomycin Z. Ultrastructural examination of cak1P212S mutants revealed a thin, uneven cell wall and marked abnormalities in septum formation. In all of the above aspects, the cak1P212S mutants are similar to previously described cla4 mutants, suggesting that the cell wall defects are common to mutants with hyperpolarized growth. In cak1P212S mutants, chitin accumulates all over the surface of the cells and glucan synthase activity is located preferentially to the tips of elongated buds. We conclude that the cell wall weakness in cak1P212S mutants is caused by hyperpolarized secretion of glucan synthase and lack of reinforcement of the lateral cell walls. Showing that the defect depends at least in part on Cdc28, the cak1P212S hyperpolarized growth phenotype can be suppressed by a Cak1-independent Cdc28-allele. The results underline the importance of a minor cell wall component, the chitin of lateral walls, for the integrity of the cell in a stress situation.     

Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo.

Nikkomycin Z inhibits chitin synthase in vitro but does not exhibit antifungal activity against many pathogens. Assays of chitin synthase isozymes and growth assays with isozyme mutants were used to demonstrate that nikkomycin Z is a selective inhibitor of chitin synthase 3. The resistance of chitin synthase 2 to nikkomycin Z in vitro is likely responsible for the poor activity of this antibiotic against Saccharomyces cerevisiae.     

The myosin ATPase inhibitor 2,3-butanedione-2-monoxime disorganizes microtubules as well as F-actin in Saccharomyces cerevisiae

Interactions between microtubules and filamentous actin (F-actin) are essential to many cellular processes, but their mechanisms are poorly understood. We investigated possible roles of the myosin family of proteins in the interactions between filamentous actin (F-actin) and microtubules of budding yeast Saccharomyces cerevisiae with the general myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM). The growth of S. cerevisiae was completely inhibited by BDM at 20 mmol/L and the effect of BDM on cell growth was reversible. In more than 80% of BDM-treated budding yeast cells, the polarized distribution of F-actin was lost and fewer F-actin dots were observed. When cells were synchronized in G₁ with α-factor and released in the presence of BDM, cell number did not increase and cells were mainly arrested in G₁ DNA content without any bud, suggesting that myosin activity is required for new bud formation and the start of a new cell cycle. More than 10% of the BDM-treated cells also revealed defects in nuclear migration to the bud neck as well as in nuclear shape. Consistent with these defects, the orientation of mitotic spindles was random in the 57% of cells treated with 20 mmol/L BDM and immunostained with anti-tubulin antibody. Furthermore, microtubule structures were completely disorganized in most of the cells incubated in 50 mmol/L BDM, while similar amounts of tubulin proteins were present in both BDM-treated and untreated cells. These results show that the general myosin inhibitor BDM disorganizes microtubule structures as well as F-actin, and suggest that BDM-sensitive myosin activities are necessary for the interaction of F-actin and microtubules to coordinate polarized bud growth and the shape and migration of the nucleus in S. cerevisiae. This revised version was published online in July 2006 with corrections to the Cover Date.     

chs-4, a class IV chitin synthase gene fromNeurospora crassa

InSaccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by theCSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized aCSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomyceteNeurospora crassa and have used a reverse genetics approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of aN. crassa gene (designatedchs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those ofS. cerevisiae andCandida albicans. N. crassa strains in whichchs-4 had been inactivated by the Repeat-Induced Point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in thechs-4 ^RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme inN. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.A. Beth Din and C. A. Specht contributed equally to this work     

Stoffwechselprodukte von Mikroorganismen

Zusammenfassung Der Streptomycet Tü 901, Streptomyces tendae, bildet ein antifungisch wirkendes Nukleosid-Antibioticum, Nikkomycin. Als Angriffsort kommt die Chitinsynthese in Frage.Mit Hilfe der Massenspektrometrie und des chemischen Abbaus konnten Uracil, eine Aminohexuronsäure und eine neue, einen Pyridinring enthaltende Aminosäure nachgewiesen werden.

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Metabolic products of microorganisms154. Nikkomycin, a new inhibitor of fungal chitin synthesis

From the fermentation broth of Streptomyces tendae Tü 901 a substance was isolated, which inhibits the growth of several fungi. The new antibiotic affects the chitchin biosynthesis. Its structure was identified by mass spectrometry of the products obtained after chemical degradation. Nikkomycin is a nucleoside-peptide antibiotic consisting of uracil, an amino hexuronic acid and a new amino acid containing a pyridin ring.

153. Mitteilung: G. Wolf, J. Wörth, H. Achenbach: Untersuchungen der Pigmente aus Streptomyces michiganesis. Arch. Microbiol. 106, 245–249 (1975)     

WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis

The chitin synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II chitin synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37°C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Δ mutants showed that yeast cells often failed to separate, tended to be enriched with chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell–daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2Δ strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25°C and was not viable at 37°C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.     

The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37 degrees C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a beta-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.     

AsSlt2基因对茄链格孢细胞壁完整性的调控

【背景】由茄链格孢(Alternaria solani)引起的马铃薯早疫病被普遍认为是马铃薯生产上的第二大叶部病害,在马铃薯各产区普遍发生,给马铃薯生产造成了巨大的经济损失。【目的】明确AsSlt2基因对茄链格孢细胞壁完整性的影响。【方法】在含有刚果红、细胞壁降解酶和十二烷基硫酸钠(sodiumdodecylsulfate,SDS)等细胞壁胁迫的培养基上观察ΔAsSlt2缺失突变株的生长情况,计算相对生长抑制率;通过实时荧光定量PCR (RT-qPCR)方法检测ΔAsSlt2菌株中细胞壁合成相关基因的表达情况;进一步检测ΔAsSlt2细胞壁中几丁质的含量及胞外酶活性。【结果】ΔAsSlt2缺失突变株对SDS、刚果红、细胞壁降解酶等细胞壁胁迫的敏感性增强,在加入细胞壁降解酶后突变株原生质体释放量显著增多;ΔAsSlt2对外源氧胁迫更敏感,突变株胞外过氧化物酶和漆酶活性均显著降低;进一步研究发现,ΔAsSlt2细胞壁中几丁质含量减少,几丁质合成相关基因与漆酶合成相关基因的表达量均明显降低。【结论】AsSlt2基因在茄链格孢细胞壁的完整性及抵御外界胁迫方面发挥重要作用。     

The Aspergillus nidulans phsB4 mutation alters colonial growth and development of the mould at acidic pH

The phsB4 mutant of the mould Aspergillus nidulans, identified as showing increased sensitivity to acid pH, is mitotically unstable and its conidia swell and lyse, forming protoplasts during germination and early development in shaken liquid cultures. On solid medium, we observed balloon-shaped hyphal swellings, a phenotype also exhibited by the chitin synthase gene (chsD) disruptants. We also observed that lysis was osmotically remediable with 0.5 M NaCl, but the balloon-shaped hyphal swelling was remedied in a pH-dependent way i.e., this phenotype was remedied only at pH values above 6.5. Based on the nature of our mutant selection, the pH sensitive phenotype of the selected strains, the known occurrence of hyphal swelling in cell wall mutants of A. nidulans, and the transformation with cosmids that hybridize to chsD gene, the phsB and chsD genes are possibly alleles.     

Evaluation of different yeast cell wall mutants and microalgae strains as feed for gnotobiotically grown brine shrimp Artemia franciscana

The nutritional value of isogenic yeast strains and two microalgal species for gnotobiotically grown Artemia was examined. Yeast cell wall mutants were always better feed for Artemia than their respective wild type. Yeast cells harbouring null mutants for enzymes involved early in the biochemical pathway for cell wall mannoproteins synthesis performed best as feed for Artemia. Yeast cells defective in chitin or β-glucan production were scored in second order. The mnn6 isogenic yeast mutant, harbouring a null mutation for mannoprotein phosphorylation, performed poorly as feed for Artemia, although with good growth. These results suggest that any mutation affecting the yeast cell wall scaffolding by reducing the amount of covalent links between the major components of yeast cell wall, namely mannoproteins, β-glucans and chitin, is sufficient to improve the digestibility for Artemia. The results with microalgae indicated that within one species, strains can have different nutritional value under gnotobiotic conditions. The growth phase was another parameter influencing feed quality, although here it was not possible to reveal the exact cause. It is anticipated that the standard Artemia gnotobiotic growth test is an excellent tool to study the mode of action of bacteria, with a probiotic as well as with a pathogenic character.     

A Highlights from MBoC Selection: Distinct roles of cell wall biogenesis in yeast morphogenesis as revealed by multivariate analysis of high-dimensional morphometric data

The cell wall of budding yeast is a rigid structure composed of multiple components. To thoroughly understand its involvement in morphogenesis, we used the image analysis software CalMorph to quantitatively analyze cell morphology after treatment with drugs that inhibit different processes during cell wall synthesis. Cells treated with cell wall–affecting drugs exhibited broader necks and increased morphological variation. Tunicamycin, which inhibits the initial step of N-glycosylation of cell wall mannoproteins, induced morphologies similar to those of strains defective in α-mannosylation. The chitin synthase inhibitor nikkomycin Z induced morphological changes similar to those of mutants defective in chitin transglycosylase, possibly due to the critical role of chitin in anchoring the β-glucan network. To define the mode of action of echinocandin B, a 1,3-β-glucan synthase inhibitor, we compared the morphology it induced with mutants of Fks1 that contains the catalytic domain for 1,3-β-glucan synthesis. Echinocandin B exerted morphological effects similar to those observed in some fks1 mutants, with defects in cell polarity and reduced glucan synthesis activity, suggesting that echinocandin B affects not only 1,3-β-glucan synthesis, but also another functional domain. Thus our multivariate analyses reveal discrete functions of cell wall components and increase our understanding of the pharmacology of antifungal drugs.     

Early death at medium acidification and survival after low pH adaptation inCryptococcus neoformans

WhenCryptococcus neoformans was grown in yeast nitrogen base (YNB) supplemented with 0.5% glucose, the medium was acidified to below pH 3 during the exponential growth phase, which caused early growth-phase death in susceptible strains. Even in resistant strains, 30–70% cells died if incubated for 2 d in YNB supplemented with 1.5% glucose, whereas the remaining cells survived long. Two types of fatal alterations have been observed in dead cells. In the first type, release of cytoplasm occurred through weakened parts of the cell wall; structures attached to cell walls of dead cells were shown to be rich in proteins by FITC staining, indicating their cytoplasmic origin. In the second type, cells shrank distinctly with no sign of wall rupture. The shrinkage may be due to dysfunction of the plasma membrane at low pH. The mechanism of cell survival in medium below pH 3 was also examined. Aniline blue alone, or calcofluor together with methylene blue, allowed cell wall glucan or chitin and dead cell cytoplasm to be stained simultaneously. In the later stages of incubation, cells showing bright staining for cell wall glucan and chitin emerged. These changes in cell wall synthesis could be considered as an adaptation mechanism to acidification of the medium, because such cells survived longer than cells showing no change in the cell wall staining pattern.     

Inactivation of chitin synthase in Saccharomyces cerevisiae

The activity of chitin synthase extracted from whole cells of Saccharomyces cerevisiae shows reproducible changes during the course of batch cultivation. During exponential growth 5–10% of the enzyme occurs in the active form, whereas in the stationary phase no active enzyme can be detected. Of three yeast proteinases, A, B and C, only B is able to activate pre-chitin synthase and inactivate chitin synthase. A new model of the regulation is presented which accounts for the specific location as well as for termination of chitin synthesis during the budding cycle.These results were reported at the 4th International Symposium on Yeasts in Vienna, July 1974, and are part of doctoral thesis by A.H., University Freiburg (1974).     

Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg²⁺ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg²⁺ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca²⁺), while Cu⁺² completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I₅₀'s of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V_max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone.     

Umchs5,a Gene Coding for a Class IV Chitin Synthase inUstilago maydis

A fragment corresponding to a conserved region of a fifth gene coding for chitin synthase in the plant pathogenic fungusUstilago maydiswas amplified by means of the polymerase chain reaction (PCR). The amplified fragment was utilized as a probe for the identification of the whole gene in a genomic library of the fungus. The predicted gene product ofUmchs5has highest similarity with class IV chitin synthases encoded by theCHS3genes fromSaccharomyces cerevisiaeandCandida albicans, chs-4fromNeurospora crassa,andchsEfromAspergillus nidulans. Umchs5null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed significant reduction in growth rate, chitin content, and chitin synthase activity, specially in the mycelial form. Virulence to corn plantules was also reduced in the mutants. PCR was also used to obtain a fragment of a sixth chitin synthase,Umchs6.It is suggested that multigenic control of chitin synthesis inU. maydisoperates as a protection mechanism for fungal viability in which the loss of one activity is partially compensated by the remaining enzymes.     

Differential Expression of Chitin Synthase III and IV mRNAs in Ascomata of Tuber borchii Vittad

A full-length genomic clone encoding a class III chitin synthase (CHS) and one DNA fragment corresponding to a class IV CHS were isolated from the mycorrhizal fungus Tuber borchii and used for an extensive expression analysis, together with a previously identified DNA fragment corresponding to a class II CHS. All three Chs mRNAs are constitutively expressed in vegetative mycelia, regardless of the age, mode of growth, and proliferation capacity of the hyphae. A strikingly different situation was observed in ascomata, where class III and IV, but not class II, mRNAs are differentially expressed in a maturation stage-dependent manner and accumulate, respectively, in sporogenic and vegetative hyphae. These data, the first on the expression of distinct Chs mRNAs during fruitbody development, point to the different cellular roles that can be played by distinct chitin synthases in the differentiation of spores of sexual origin (CHS III) or in ascoma enlargement promoted by the growth of vegetative hyphae (CHS IV).