Chimeric pheromone receptors in the basidiomycete Schizophyllum commune

The pheromone receptor system of the basidiomycete Schizophyllum commune is capable of ligand discrimination to confer mating specificity. The pheromone receptors of the B alpha locus were investigated for ligand discrimination in a strategy of domain swapping experiments. Several altered phenotypes of chimeric receptors have been found. These include constitutive pheromone receptors which need no ligand for activation of the downstream cascade of events. In addition, receptors still dependent on ligand were identified that had altered pheromone activation profiles, including promiscuous receptors that are activated by pheromones of all nine specificities, including the former self. In addition, highly discriminative receptors were created which are activated by only two of the eight non-self-specificities. The chimeric receptors identify the last third of the receptor as the determinant for B alpha 1 specificity, whereas B alpha 2 specificity resides in noncontiguous domains covering the first and middle parts of the receptor molecule.     

Allelic divergence at Bα1 pheromone receptor genes of Schizophyllum commune

Abstract The multiallelic mating type locus Bal of Schizophyllum commune encodes a pheromone receptor and putative pheromone genes. A comparison of two alleles encoding receptors that share the same specificity Bα1 was performed using strains of different geographic origin. The amino acid sequence alignment revealed strong conservation of the largest part of the receptor. Only in the distal C-terminus major amino acid was divergence encountered. This C-terminal region of 117 of the total of 639 amino acids was shown to be unnecessary for function in vivo by transformation experiments.     

Heterokaryon formation in the basidiomycete Schizophyllum commune by electrofusion of protoplasts

Summary Conditions for high frequency electrofusion of protoplasts from the basidiomycete Schizophyllum commune are described. Visual inspection revealed up to 30% of the protoplasts engaged in fusion. Using complementing nutritional mutations, nearly 7% of the regenerated protoplasts could be recovered as heterokaryotic mycelia. The method is probably equally applicable to other basidiomycetes such as Agaricus bisporus, permitting the recovery of fusion products in the absence of selection markers.     

普通裂褶菌真菌学及实验室研究进展

普通裂褶菌是一种条件致病菌,可侵犯人体多个器官多种组织,导致各种表现形式的疾病,如脑脓肿、脑膜炎、鼻窦炎、肺部真菌球、蜂窝肺、肺结节、甲真菌病等.由于普通裂褶菌感染引起的真菌病病例少见报道,临床医生对其认识不足,再加上某些实验室不具备鉴定条件,所以感染该菌的病例常被漏诊、误诊.现将近年来临床分离的普通裂褶菌株真菌学及实...     

裂褶菌及裂褶菌多糖研究进展

裂褶菌是一种十分重要的食药兼用真菌,含有丰富的生理活性物质,裂褶菌多糖作为一种极有开发前景的生物活性物质已得到国内外的普遍重视。综述了裂褶菌的生物学特性、营养成分、药用价值、栽培现状以及裂褶菌多糖的化学分析和药理作用的研究进展,并讨论了裂褶菌和裂褶菌多糖的研究前景。     

裂褶菌营养菌丝蛋白质成分的分析

对裂褶菌菌丝中蛋白质的氨基酸组成及含量进行了测定。结果表明 ,裂褶菌中所含粗蛋白的质量分数为 2 8.76 % ,所测定的 17种氨基酸总质量分数为 12 0 .13g·kg- 1 。其中 7种必需氨基酸的质量分数为 4 5 .36g·kg- 1 ,占氨基酸总量的 37.76 % ;10种非必需氨基酸的质量分数为 79.5 0g·kg- 1 ,占氨基酸总量的 6 2 .2 4 %。     

A bacteriolytic muramidase from the basidiomycete Schizophyllum commune

The basidiomycete Schizophyllum commune produces an extracellular bacteriolytic enzyme when grown on heat-killed cells of Bacillus subtilis as sole C, N and P source. The enzyme catalyses the dissolution of isolated B. subtilis cell walls at an optimum pH of 3.2-3.4, releasing muramyl reducing groups, which indicates that it is a muramidase. Although low levels of enzyme activity are present when the fungus is grown in the absence of bacteria, full enzyme production appears to be induced by bacterial cells and repressed by glucose. Whole bacteria are not lysed by the enzyme at pH 3.3, but are rendered osmotically fragile, and lyse when the pH is raised to 7 or higher. The muramidase is effective against several Gram-positive bacteria but did not lyse any of the Gram-negative species tested.     

Developmentally regulated proteases from the basidiomycete Schizophyllum commune

The basidiomycete Schizophyllum commune produces three chromatographically distinguishable proteases which are capable of attack on a variety of other enzymes from S. commune and other sources. These proteases, which are produced during a specific phase of the development cycle, exhibit typical enzyme kinetic patterns, are active in the neutral to weakly alkaline pH range and are inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and ovomucoid. No pattern of specificity toward the test enzymes could be discerned. The proteases co-purify with the activity which causes the increase in cold lability of S. commune phosphoglucomutase reported previously. In addition, one of the protease enzymes could be purified to the point where it had no significant ability to release trichloroacetic acid products from denatured substrates at pH 3 or pH 7. When undenatured hemoglobin was used as a substrate, the purified protease releases a relatively large molecular weight nonheme peptide. Relatively large peptides are also formed after proteolysis of rabbit muscle phosphoglucomutase. These results suggest that the protease carries out only limited proteolysis.     

裂褶菌培养基形态学观察和DNA序列分析

目的通过观察裂褶菌在5种培养基上的生长状态、扫描电镜及DNA序列分析,了解该菌形态学及分子生物学等方面的特征。方法菌落转种于沙氏培养基(SDA),麦芽浸膏琼脂(MEA),马铃薯葡萄糖琼脂(PDA),玉米粉琼脂(CMA)和察氏琼脂(CZA)平皿培养基,27℃和37℃培养2周,观察菌落生长情况,进行扫描电镜检测及DNA序列分析。结果菌落在SDA,MEA和PDA上生长状态较好,呈蓬松白色羊毛状;尿素酶试验阳性,放线菌酮耐受试验阴性。光镜下见分支分隔菌丝、侧生的钉状突起及类水母体变异子实体。扫描电镜见菌丝分隔处闭锁联合、侧生钉状突起和泪滴状球形分泌物。经26S rDNAD1/D2区序列分析证实该菌株为裂褶菌。结论裂褶菌只有丝状型一种菌落形态;分支分隔菌丝及分隔处闭锁联合,侧生钉状突起和泪滴状球形分泌为其形态学特征;孢子由类水母状子实体产生。     

Action spectrum for fruiting in the basidiomycete Schizophyllum commune

An action spectrum for fruit body formation was determined in the range 280–723 nm for a dikaryon of Schizophyllum commune Fr. Action maxima occurred at 280 and 340–360 nm (main peak), and there were minor peaks at 437 and 467 nm. The quantum effectiveness at 360 nm was ca seven-fold compared to that of 437 nm light. Wavelengths longer than 500 nm were ineffective. Light also induced formation of brown pigment in the area producing fruit bodies. Wavelengths ranging from 260 to 300 nm injured aerial hyphae at the border of the colony. The possibility that a flavin or a pteridine may be the photoreceptor is discussed.     

Cell wall metabolism during fruiting of the basidiomycete Schizophyllum commune

During the development of fruit bodies of the basidiomycete Schizophyllum commune, the alkali-insoluble (R glucan) and alkali-soluble (S glucan) cell wall fractions are synthesized during the entire course of morphogenesis. The water soluble glucan (WSG) is not synthesized after an early stage. There is also a relative increase in the proportion of S glucan during development which appears related to a change in the proportion of the components synthesized. Data are also presented to show that several fruiting mutants also have specific cell wall differences, and that there is a significant contribution to cell wall structure by genes which do not cause a macroscopically observable change in phenotype.     

Direct studies of dikaryotization in Schizophyllum commune

The growth, duplication and fate of multikaryotic hyphae bearing true clamp connections, as derived from compatible matings of Schizophyllum commune, were studied by phase contrast microscopy. The nuclei (N) of multikaryotic apices maintained a near central position during hyphal growth. True clamp connection formation occurred with near synchronous mitosis followed by septal synthesis across the clamp neck and main hyphal axis. Nuclear progeny after mitosis in a hexakaryon included 6 N in the apex, 1 N in the clamp and 5 N in the penultimate cell; the solitary nucleus in the clamp later entered the penultimate cell. Similar events occurred for clamp connection formation and mitosis in the trikaryon, quadrikaryon or pentakaryon, whether in the apex or primary branches. Nuclear content of the multikaryotic apex (2 N through 10 N) had no apparent effect on the rate of individual hyphal growth. Reduction of the nuclear number in a trikaryon occurred by long-term entrappment of a solitary nucleus in the clamp and subsequent outgrowth of the dikaryotic penultimate cell. Occasionally, more than one nucleus became entrapped in the clamp cell. The ephemeral nature of the multikaryon was indicated by the fact that older cultures appeared to be exclusively dikaryotic hyphae at the colony periphery.     

An efficient gene deletion procedure for the mushroom-forming basidiomycete Schizophyllum commune

Gene deletion in Schizophyllum commune is hampered by a low incidence of homologous integration. As a consequence, extensive screening is required to identify a transformant with the desired genotype. To alleviate this and to facilitate the assembly of deletion plasmids, vector pDelcas was constructed. This construct has a set of restriction sites, which allows for directional cloning of the flanking sequences at both sides of a nourseothricin resistance cassette. Moreover, it contains a phleomycin resistance cassette elsewhere in the plasmid, which is used to screen for transformants with an ectopic integration of the pDelcas derivative. The use of pDelcas derivatives in combination with an improved PCR screening protocol permitted the efficient identification of S. commune deletion strains. This procedure may also function in other basidiomycetes.     

Dikaryons of the basidiomycete fungus Schizophyllum commune: evolution in long-term culture

The impact of ploidy on adaptation is a central issue in evolutionary biology. While many eukaryotic organisms exist as diploids, with two sets of gametic genomes residing in the same nucleus, most basidiomycete fungi exist as dikaryons in which the two genomes exist in separate nuclei that are physically paired and that divide in a coordinated manner during hyphal extension. To determine if haploid monokaryotic and dikaryotic mycelia adapt to novel environments under natural selection, we serially transferred replicate populations of each ploidy state on minimal medium for 18 months (approximately 13,000 generations). Dikaryotic mycelia responded to selection with increases in growth rate, while haploid monokaryotic mycelia did not. To determine if the haploid components of the dikaryon adapt reciprocally to one another's presence over time, we recovered the intact haploid components of dikaryotic mycelia at different time points (without meiosis) and mated them with nuclei of different evolutionary histories. We found evidence for coadaptation between nuclei in one dikaryotic line, in which a dominant deleterious mutation in one nucleus was followed by a compensatory mutation in the other nucleus; the mutant nuclei that evolved together had the best overall fitness. In other lines, nuclei had equal or higher fitness when paired with nuclei of other histories, indicating a heterozygote advantage. To determine if genetic exchange occurs between the two nuclei of a dikaryon, we developed a 24-locus genotyping system based on single nucleotide polymorphisms to monitor somatic exchange. We observed genetic exchange and recombination between the nuclei of several different dikaryons, resulting in genotypic variation in these mitotic cell lineages.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.