Search

The TU25 mutant strain of the basidiomycete Coprinus cinereus grows well at 28 degrees C but not at 37 degrees C. Microscopic examination revealed that TU25 exhibited swelling at hyphal apices after a shift-up from 28 degrees C to 37 degrees C. The temperature sensitivity and hyphal swelling co-segregated through meiosis as expected for a single Mendelian factor, designated hyt1 (hyphal tip). Both defects could be suppressed by the presence of osmotic stabilizers in the medium, suggesting that the hyt1 gene product is required for proper cell-wall function. Chemical analysis of the cell walls, however, failed to detect any clear difference in the composition of the cell-wall polysaccharides between the wild-type and TU25. A DNA fragment which complements the hyt1-1 mutation was cloned and sequenced. The predicted protein in the ORF essential for complementation of hyt1-1 is a novel protein.

We first obtained a cytochalasin-E supersensitive strain (CES14) of the basidiomycete Coprinus cinereus and then isolated 1000 revertants of CES14, expecting that at least some of the revertants have defects in the functions of actin filaments. Microscopic examination revealed that three of the revertants lack septa in their hyphae. Of the 1000 revertants, 18 including the 3 septumless strains were genetically analyzed. In 11 of the 18 revertants, reversion was due to extragenic suppressor mutations of ces14. Ten of the 11 mutations were in the same locus designated cer1, while the locus of the remaining mutation, which inhibits septum formation, could not be determined because of its failure to mate with any other cer1 strains. In one (CES14R42) of the cer1 mutants, actin appeared to be altered in the affinity to actin antibody in Western blotting after isoelectric focusing, suggesting that cer1 is a gene encoding actin. Phenotypic examination revealed that the 10 cer1 mutations all confer resistance to cytochalasin-E and that two of the cer1 mutations, both of which block septation, inhibit the formation of the actin ring.

Summary: To help understand the subcellular machinery responsible for cell wall formation in a fungus, we determined the abundance and subcellular distribution of chitin synthetase (chitin synthase, EC 2.4.1.16) and chitosomes in the asexual life cycle of Mucor rouxii. Cell-free extracts of ungerminated sporangiospores, hyphae/mycelium in exponential and stationary phase, and yeast cells were fractionated by isopycnic centrifugation in sucrose density gradients. The total amount of chitin synthetase per cell increased exponentially during aerobic germination of spores. In all developmental stages, the profile of chitin synthetase activity encompassed a broad range of sucrose density (d = 1.12-1.22) with two distinct zones: a low-density chitosome zone (d=approx. 1.12-1.16) and a highdensity, mixed-membrane zone (d = approx. 1.16-1.22). Chitosomes were a major reservoir of chitin synthetase in all stages of the life cycle, including ungerminated spores. Two kinds of chitin synthetase profiles were recognized and correlated with the growth state. In nongrowing cells (ungerminated sporangiospores and stationary-phase mycelium), the profile was skewed toward lower densities with a sharp chitosome peak at d = 1.12-1.13. In actively growing cultures (aerobic mycelium or anaerobic yeast cells), the entire profile of chitin synthetase was displaced toward higher densities; the average buoyant density of chitosomes was higher (d=1.14-1.16), and more chitin synthetase was associated with denser (d=1.16-1.23) membrane fractions. In all life cycle stages, chitosomal chitin synthetase was almost completely zymogenic. In contrast to the enzyme from spores or from growing cells, samples of chitosomal chitin synthetase from stationary-phase mycelium were unstable and contained a high proportion of larger vesicles in addition to the typical microvesicles. The presence of chitosomes in ungerminated spores indicates that these cells are poised to begin synthesizing somatic (= vegetative) cell walls at the onset of germination. The increased buoyant density of chitosomes in actively growing cultures suggests that the composition of these microvesicles changes significantly as they mobilize chitin synthetase to the cell surface.

SUMMARY: Modifications of cell-wall polysaccharides during stipe elongation in Coprinus cinereus were investigated by gel-filtration and Smith-degradation analyses. During stipe elongation two polysaccharide fractions (I and IVa) decreased in average molecular weight, whereas two other fractions (II and III) increased marginally in molecular weight. The decreases in molecular weight of the former fractions were accompanied by changes in glycosidic linkage composition. Glucans comprised the bulk of all fractions; additionally there was an appreciable contribution from mannose and xylose in fraction II. The results are discussed in relation to the mechanisms of stipe elongation.

In total, 404 variants resistant to the antimicrotubule agent benomyl were isolated from UV-irradiated oidia of the basidiomyceteCoprinus cinereus. Part of the variants showed, in addition to benomyl resistance, heat sensitivity or heat dependence. Fifteen variants selected on the basis of different phenotypes were subjected to further analyses. All of the 15 variations were due to single-gene mutations, and the mutations comprised four groups (benA, benB, benC, andbenD) in terms of genetic linkage. Some of the 15 mutations affected nuclear migration in dikaryosis and/or fruiting processes.

Summary: Cell walls isolated from rapidly elongating stipes of Coprinus macrorhizus basidiocarps lost 55% of their dry weight during 24 h incubation at 37 °C. Neutral sugars accounted for 73% of the solubilized products, glucose being the major sugar component. In the carbohydrate fraction solubilized after relatively short periods of autolysis, polymeric (degree of polymerization > 10), dimeric and monomeric components were detected, and the larger components were converted to monosaccharide as the reaction proceeded. Three polysaccharide fractions of the cell wall were highly susceptible to autolytic enzymes, whereas another fraction was not. Chitin appeared to be relatively resistant. Analyses of the cell walls from various stages during stipe elongation revealed a positive relation between the rate of stipe elongation and the initial rate of autolysis, suggesting that the autolytic enzymes are involved in the mechanisms of stipe elongation.

甜菜の貯蔵中に微生物により生成する多糖類,すなわちデキストランおよびレバンの濾過作業に対する影響を検討した. 両者とも分子量は106～109ダルトンと類似した高分子構造を有していたが,炭酸飽充に対する影響には顕著な差異があり,デキストランが粗汁中に200ppm以上,第2炭酸飽充で100ppm以上存在すると濾過障害を発生するのに対し,レバンは全く影響を示さなかった.高分子のデキストラン(MW 5×105以上)には,第2炭酸飽充で生成する炭酸カルシウムの結晶に吸着し,凝集体の生成を阻止する作用があり,このため見掛けの粒径が小さくなり,濾布の目詰りを起こして濾過障害を発生すると結論された.このデキストランの作用は,低分子化すると減少し,標準デキストランで検討した結果,分子量104ダルトンでは実用上無視できる結果を得た.一方,シュガーエンドの濾過障害に対しても高分子デキストランが主因と考えられ,この場合も分子量104ダルトン程度に分解すると櫨過障害を解消できる結果が得られた.レバンはシュガーエンドの濾過障害には無視できない影響を有していた. 次に,デキストランの低分子化の目的で, endo型デキストラナーゼの応用を検討した.デキストラナーゼによるデキストランの分解反応はきわめて容易に進行し,粗汁中のデキストラン当り1/100の添加量で60°C,5分の反応により104ダルトンまで分解した. 引続き現場試験を行った結果,粗汁中のデキストラン当り1/50～1/100のデキストラナーゼの添加により,第2炭酸飽充の濾過障害は完全に解消する結果が得られ,その実用性を確認することができた.

The dikaryon #5026 (A2B2)+#5132 (A7B7) of the basidiomyceteCoprinus macrorhizus was treated with ultraviolet light (UV),N-methyl-N′-nitro-N-nitrosoguanidine (NG), and 5-bromouracil (BU) in order to induced developmental variations in fruiting. Among a total of 11,383 isolates, 742 were homokaryons. Fruiting test was made with the remaining 10,641 dikaryotic isolates, of which 1,594 showed abnormality in basidiocarp development. Both UV and NG were very efficient in inducing such variations, but BU was not. The induced variations were classified into seven basic types as follows: 1) “knotless”, in which hyphal knots, the first sign of fruiting, do not differentiate; 2) “primordiumless,” in which hyphal knots are formed, but no further development occurs; 3) “maturationles”, in which primordia are formed, but the maturation of primordia into adult fruit bodies does not occur; 4) “elongationless”, in which the elongation of stipe is blocked, and a basidiocarp with a very short stipe is produced; 5) “expansionless,” in which pilei do not expand normally; 6) “sporeless,” in which the formation of basidiospores is blocked and albinic pilei bearing none or only a small amount of spores are produced; and 7) “autolysisless,” in which the autolysis of pilei does not appear to occur. It has been noticed that the four steps of maturation, i.e. stipe elongation, pileus expansion, spore formation, and perhaps pileus autolysis, can proceed independently. Compound types of variations such as “elongation-expansionless,” “elongation-sporeless,” “expansion-sporeless,” and “elongation-expansion-sporeless” were also induced. UV treatment induced maturationless at the highest rate, while NG treatment sporeless.

Temperature-sensitive variants defective in hyphal growth of the basidiomyceteCoprinus cinereus were isolated from oidia treated by UV orN-methyl-N′-nitro-N-nitrosoguanidine. In 23 out of 27 variant strains isolated, the temperature-sensitive variation was shown to be due to recessive single-gene mutation. All of the 23 mutants exhibited apparent interallelic complementation with one another, with the exception of one pair. The mutants were mated with their respective progeny to construct temperature-sensitive dikaryons carrying the mutant genes homozygously. Fruiting test of the respective dikaryons showed that eight mutations reduce stipe elongation during basidiocarp maturation at a restrictive temperature (37°C).

Cell-free extracts and cell wall autolysates prepared from the stipes of basidiocarp ofCoprinus cinereus were examined for (1→3)-β-glucanase activities. Gel filtration revealed two major peaks and a minor one of (1→3)-β-glucanases in both of the preparations, the former ones being designated as glucanase I and glucanase II. Glucanase I with a molecular weight of 300,000 had activity towardp-nitrophenyl-β-d-glucoside (pNPG) as well as laminarin, whereas glucanase II with a molecular weight of 70,000 had no activity toward pNPG. Both enzymes had only negligible activity toward pustulan. During stipe elongation, the level of glucanase-II activity remarkably increased with increasing rate of the elongation, whereas that of glucanase-I activity remained almost constant, in both the cell-free extract and the cell wall autolysate. Near the end of stipe elongation, both glucanase activities were lowered in the cell wall autolysate, but remained high in the cell-free extract.

A new bicyclogermacrene, capgermacrene C (1), along with a known compound, 1,4-peroxy-5-muurolene (2), were isolated from a population of Bornean soft coral Capnella sp. The structures of these metabolites were determined by extensive spectroscopic analysis, including NMR, and HRESIMS. Both compounds were subjected to antibacterial activity tests against antibiotic resistant clinical bacteria, but produced only negligible inhibition.

One new cembrane, ent -sinuflexibilin D (1), along with seven known compounds (2-8) were isolated from a population of Bornean soft coral Sinularia flexibilis. Their structures were elucidated on the basis of spectroscopic analyses. Cytotoxicity and antifungal activities of these compounds were evaluated in vitro. In addition, muurolene 7 was first isolates from a marine source.

Two new aromatic compounds, phillipsins A (1) and B (2) were isolated from the Bornean wild ginger Zingiber phillippsii Mood & Theilade, and were characterized based on spectroscopic data (HRESI-MS, FTIR as well as 1D and 2D NMR). These metabolites also exhibited strong antifungal activity against selected fungi.