Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

粗糙脉孢菌丝氨酸/苏氨酸激酶家族基因敲除库纤维素酶表达分泌研究

【目的】蛋白磷酸化在丝状真菌细胞对外界纤维素酶诱导信号感应以及信号胞内的传导过程中有着重要的作用,而蛋白磷酸化是由蛋白激酶来完成的。为了挖掘在丝状真菌纤维素酶表达过程中发挥重要作用的激酶基因,对粗糙脉孢菌丝氨酸/苏氨酸家族的61株蛋白激酶单基因突变体的纤维素酶表达分泌情况进行了分析测定。【方法】在以微晶纤维素为唯一碳源的条件下,7株单基因突变体胞外分泌蛋白产量有显著变化,随后,对这7株突变体胞外蛋白进行了详细的SDS-PAGE分析和内切-β-1,4-葡聚糖酶酶活、β-葡萄糖苷酶酶活、外切纤维素酶酶活以及木聚糖酶酶活的测定。【结果】突变株W14、W38、W87和W40胞外分泌蛋白含量提高了30%以上,除了突变株W14外,其它突变体的内切-β-1,4-葡聚糖酶酶活分别显著提高了62%、42%和42%。而突变株W85、W26和W46胞外分泌蛋白含量降低了50%以上,相对应的内切-β-1,4-葡聚糖酶酶活也分别下降了86%、75%和84%。【结论】这些关于粗糙脉孢菌丝氨酸/苏氨酸家族蛋白激酶基因的挖掘,为进一步深入研究蛋白激酶在纤维素酶诱导表达调控中的分子机理奠定了基础。     

C24(28)-甾醇还原酶参与粗糙脉孢菌极性生长及早期发育

麦角甾醇是真菌细胞膜的主要固醇类物质,其生物合成是一个复杂的酶促反应过程, 其中C24(28)-甾醇还原酶是麦角甾醇合成途径中的关键酶,对C24(28)-甾醇还原酶功能的研究有助于阐明麦角甾醇对真菌极性生长的影响.本文对粗糙脉胞菌C24(28)-甾醇还原酶蛋白（Erg-2基因编码）序列的同源性分析表明,在子囊菌门的3个物种中,C24(28)-甾醇还原酶具有很高的保守性.根据同源重组基因敲除原理,通过电转化、分生孢子过膜以及PCR鉴定的方法获得了Erg-2基因缺失突变株（Erg-2KO）,进一步利用斜面生长法并结合细胞壁染色进行突变株表型分析发现,与野生型相比,Erg-2KO(Ku70RIP背景)在生长初期菌丝生长缓慢,而后期与野生型无显著差异.这些结果表明,C24(28)-甾醇还原酶对N. crassa早期的生长和发育至关重要.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

形成素结合蛋白调节粗糙脉孢菌菌丝极性生长发育

微丝骨架在真菌菌丝极性生长中具有重要的功能,而其动态解聚 聚合特性是其实现功能的前提.形成素作为肌动蛋白结合蛋白,是微丝骨架动态调控因子之一,而形成素结合蛋白对于形成素发挥功能非常关键,但是对其在真菌极性生长发育中的功能还未见报道.本文以丝状真菌粗糙脉孢菌为材料,利用同源重组基因敲除技术,通过电击转化、分生孢子过膜以及PCR鉴定的方法,获得了形成素结合蛋白基因缺失突变菌株（FBPKO）.进一步利用平板生长方法并结合细胞壁染色对突变菌株的表型进行分析. 结果显示, 与野生型相比,在分生孢子接种后24 h内突变菌株FBPKO的菌丝生长明显减慢且分支异常.这些结果表明, 形成素结合蛋白调节着粗糙脉孢菌菌丝早期的极性生长发育.     

The respiratory response to heat shock in Neurospora crassa

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.     

脉孢菌lca-1基因调控无性产孢及类胡萝卜素的合成

类胡萝卜素是很多生物细胞内重要的抗氧化剂,具有保护细胞免受紫外线伤害的功能。粗糙脉孢菌是少数几个类胡萝卜素合成基因比较清楚的真菌之一,为了深入了解该菌类胡萝卜素合成调控机制,通过对粗糙脉孢菌基因突变体库中6,087株突变体进行筛选,新发现6个基因敲除突变体营养生长正常,但类胡萝卜素的合成降低,其中表型较好的1个突变体,其无性产孢量与类胡萝卜素合成量均明显降低。鉴定发现该突变体所缺失的基因编码一种依赖ATP的染色体重建复合体的ATP酶链ISW1,将该基因命名为lca-1。进一步测定发现lca-1基因的突变导     

全基因组水平扫描鉴定粗糙脉孢菌糖转运蛋白及其在酿酒酵母己糖发酵中的评价

木质纤维素降解真菌粗糙脉孢菌天然具有吸收利用多种单糖和寡糖的能力,但是目前基因组中注释的预测糖转运蛋白仍然有过半功能未知。本研究从全基因组水平系统分析了粗糙脉孢菌预测糖转运蛋白的转运底物。研究发现两个转运蛋白(NCU01868和NCU08152)具有转运多种己糖底物的功能,因此分别命名为NcHXT-1和NcHXT-2。利用荧光共振能量转移技术(FRET)确认了NcHXT-1/-2具有葡萄糖转运功能。在己糖转运蛋白全缺酿酒酵母EBY.VW4000中分别过表达NcHXT-1/-2,能恢复其在葡萄糖、半乳糖或甘露糖的液体培养基中生长并生成乙醇的能力。NcHXT-1/-2在很多纤维素降解真菌中均具有保守的同源蛋白。本研究通过全基因组扫描鉴定,发现了两个保守的丝状真菌己糖转运蛋白,为真菌降解利用木质纤维素及酵母利用单糖发酵提供了新的改造靶点。     

Heterologous expression of Neurospora crassa cbh1 gene in Pichia pastoris resulted in production of a neutral cellobiohydrolase I

The high production cost of cellulase is one of the limitations that hinder the commercialization of lignocellulose-based biorefineries. As one of the important cellulases, Neurospora crassa cellulase is not so intensively investigated as T. reesei cellulase. In this study, the cbh1gene (NCU07340) cloned from N. crassa was expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promotor. Six transformants with the highest resistance to G418 were selected by two rounds of transformant screening, among which the most robust producer of the recombinant cellobiohydrolase I (CBHI) has an Avicelase activity of 0.22 U/mL. After fermentation optimization, it was improved to 0.30 U/mL. Interestingly, the optimal temperature and pH of the recombinant CBHI were 60°C and 7.2, respectively, and it was quite stable within the wide ranges of temperature and pH. This work is a good example for the future improvement and optimization of N. crassa cellulase.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Effects of light on protein secretion in Neurospora crassa

The relative concentrations of secreted proteins in liquid cultures of Neurospora crassa differ in constant darkness compared to constant light (2500lx). Light reduces the concentrations of some polypeptides markedly and increases the concentrations of protein species of 67, 40, 18 and 13 kDa. The blind wc-2 mutant of Neurospora does not show light dependent differences in amounts of secreted proteins. One of the light-sensitive extracellular proteins is shown to be a protease of 17,5 kDa.     

粗糙脉孢菌C2H2转录因子家族基因敲除突变体产纤维素酶筛选分析

丝状真菌的木质纤维素水解酶基因的表达很大程度上受到转录水平的调控,对纤维素酶系调控转录因子的研究对提高纤维素酶的产量具有重要意义。为了挖掘纤维素酶表达新调控基因,本研究对粗糙脉孢菌锌指转录因子C₂H₂家族的57株基因敲除突变株在以2%结晶纤维素为唯一碳源的培养条件下进行产纤维素酶水平分析筛选。通过测定发酵液的蛋白、内切β-1,4-葡聚糖酶酶活、外切纤维素酶酶活、β-葡萄糖苷酶酶活、木聚糖酶酶活及生物量,发现突变株L-38、L-85、L-40、L-64、L-99、L-07、L-86在蛋白水平及内切β-1,4-葡聚糖酶酶活水平均比野生型有25%-77%不等的显著提高,而突变株L-87、L-06在蛋白水平及内切β-1,4-葡聚糖酶酶活水平均比野生型有65%-80%不等的显著降低。这些纤维素酶新调控基因的获得为后续相关表达调控的研究提供了新素材。     

链孢霉四分体分析连锁基因距离矫正的一种简便方法和计算数据的修正

本文探讨一个在链孢霉四分体分析中连锁基因距离矫正的简便方法。并提出在计算交换值时,当遇到着丝粒在两基因中间的类型时,应该把基因与着丝粒之间发生的四线双交换类型分别计入两个单交换内。 Abstract:The present paper is dealing with a simple method to correct the distance of linked genes in tetrad analysis in Neurospora crassa.It is suggested that the data of 4-thread double crossing over should be added in two single crossing over respectively in centromere maping when calculating crossover value of the two genes locating across the centromere.     

The aging biological clock in Neurospora crassa

The biological clock affects aging through ras‐1 (bd) and lag‐1, and these two longevity genes together affect a clock phenotype and the clock oscillator in Neurospora crassa. Using an automated cell‐counting technique for measuring conidial longevity, we show that the clock‐associated genes lag‐1 and ras‐1 (bd) are true chronological longevity genes. For example, wild type (WT) has an estimated median life span of 24 days, while the double mutant lag‐1, ras‐1 (bd) has an estimated median life span of 120 days for macroconidia. We establish the biochemical function of lag‐1 by complementing LAG1 and LAC1 in Saccharomyces cerevisiae with lag‐1 in N. crassa. Longevity genes can affect the clock as well in that, the double mutant lag‐1, ras‐1 (bd) can stop the circadian rhythm in asexual reproduction (i.e., banding in race tubes) and lengthen the period of the frequency oscillator to 41 h. In contrast to the ras‐1 (bd), lag‐1 effects on chronological longevity, we find that this double mutant undergoes replicative senescence (i.e., the loss of replication function with time), unlike WT or the single mutants, lag‐1 and ras‐1 (bd). These results support the hypothesis that sphingolipid metabolism links aging and the biological clock through a common stress response     

The GOLD Domain-containing Protein TMED1 Is Involved in Interleukin-33 Signaling

The proinflammatory danger signal IL-33, which is released from damaged or dying cells, achieves its effects via the IL-1R family member ST2L. The detection of IL-33 by ST2L initiates downstream signaling pathways that result in the activation of MAPKs and NF-κB. Here, we show that TMED1 associates with ST2L. Using a series of mutation and deletion constructs, we demonstrate that this interaction is mediated by the GOLD domain of TMED1 and the TIR domain of ST2L. Our findings also demonstrate that TMED1 is required for optimal IL-33-induced IL-8 and IL-6 production. This discovery provides additional support to the concept that the TMED family members are important players in innate immune signaling.