Characterization of the α-mannosidase gene family in filamentous fungi: N-glycan remodelling for the development of eukaryotic expression systems

Although filamentous fungi are used extensively for protein expression, their use for the production of heterologous glycoproteins is constrained by the types of N-glycan structures produced by filamentous fungi as compared to those naturally found on the glycoproteins. Attempts are underway to engineer the N-glycan synthetic pathways in filamentous fungi in order to produce fungal expression strains which can produce heterologous glycoproteins carrying specific N-glycan structures. To fully realize this goal, a detailed understanding of the genetic components of this pathway in filamentous fungi is required. In this review, we discuss the characterization of the α-mannosidase gene family in filamentous fungi and its implications for the elucidation of the N-glycan synthetic pathway.     

丝状真菌次级代谢产物生物合成的表观遗传调控

丝状真菌产生的次级代谢产物是新药的重要来源之一,其生物合成过程受到众多因素的调控。最近的研究表明,表观遗传对多种丝状真菌次级代谢产物的生物合成具有调控作用。DNA和组蛋白的甲基化与乙酰化修饰是目前所知的丝状真菌主要的表观遗传调控形式。通过过表达或缺失相关表观修饰基因和利用小分子表观遗传试剂改变丝状真菌染色体的修饰形式,不仅可以提高多种已知次级代谢产物产量,而且可以通过激活沉默的生物合成基因簇诱导丝状真菌产生新的未知代谢产物。丝状真菌表观遗传学正逐渐成为真菌菌株改良的新策略以及挖掘真菌次级代谢产物合成潜力的强有力手段。     

丝状真菌二倍半萜化合物及其合成酶

相比其他萜类化合物,丝状真菌来源的二倍半萜化合物数量较少,但具有广泛的生理活性和药用价值。已经发现的丝状真菌二倍半萜合成酶均由萜类环化酶和异戊烯基转移酶两个结构域组成,表现出底物的非特异性和环化方式的多样性。本文重点叙述了丝状真菌来源的二倍半萜化合物以及其合成酶的结构与功能特征,并对丝状真菌二倍半萜化合物及其合成酶研究现状作简要概述。     

丝状真菌蛋白表达系统研究进展*

丝状真菌以其优秀的表达分泌能力和良好的环境适应能力,使得其在蛋白质表达领域应用越来越广泛。近几十年来,通过诱变、培养优化及遗传改造等手段,使得包含曲霉属、木霉属、青霉属等在内的丝状真菌被开发成高效表达宿主。为促进丝状真菌蛋白表达系统的开发,结合作者的研究工作,对工业上丝状真菌表达宿主、蛋白质表达元件及其改造策略进行综述,并探讨了当前丝状真菌表达系统开发过程中的不足之处,为新型丝状真菌表达系统的研究提供参考和启示。     

Escherichia coli tolQ mutants are resistant to filamentous bacteriophages that adsorb to the tips, not the shafts, of conjugative pili

The tolQ (previously fii) mutation in Escherichia coli K12 inhibits infection by filamentous bacteriophages f1 and IKe but not by RNA-containing phage f2. This work extends these observations to other plasmid-specific bacteriophages including various filamentous. RNA-containing, and lipid-containing isolates. Only tip-adsorbing filamentous phages were affected by tolQ and not shaft-adsorbing ones. Electron microscopy showed that RP4-specific filamentous phage Pf3 was one of the latter kind. Several tip-adsorbing filamentous phages inhibited conjugation between tolQ strains carrying their specific plasmids, implicating the phage receptors (conjugative pili) as mating organelles. tolQ mutant strains were as proficient as their parents in conjugation mediated by a wide range of plasmids.     

Cdc42/Rho GTPases in fungi: variations on a common theme

Cdc42/Rho GTPases are universally important regulators of cellular morphogenesis. Whereas their functions are well characterized in budding yeast, they are only beginning to be understood in filamentous fungi. The recent systematic analysis of Cdc42, Rac1 and Rho function in Aspergillus niger provides the first global perspective on their respective roles in hyphal morphogenesis. Surprisingly, the partitioning of these roles between Cdc42 and Rac1 seems to vary even among related fungi. These observations highlight the variable use of a common signalling module in filamentous fungi.     

Existence of cytoplasmic inclusions in acinic cells of the exocrine pancreas of the hyperglycemic obese mouse]

Peculiar cytoplasmic inclusions with crystalline pattern are described in the obese hyperglycaemic mouse pancreas acinar cells. They are generally associated with filamentous structures. However, crystalline and filamentous inclusions have also been observed in isolated forms. These inclusions are not digested by pronase. Since they have also been encountered in normal mice, it seems that the hyperglycaemic syndrome does not play a role in inducing their formation.     

Comparison of morphogenetic networks of filamentous fungi and yeast.

Fungi generally display either of two growth modes, yeast-like or filamentous, whereas dimorphic fungi, upon environmental stimuli, are able to switch between the yeast-like and the filamentous growth mode. Signal transduction pathways have been elucidated in the budding yeast Saccharomyces cerevisiae, establishing a morphogenetic network that links cell-cycle events with cellular morphogenesis. Recent molecular genetic studies in several filamentous fungal model systems revealed key components required for distinct steps from fungal spore germination to the maintenance of polar hyphal growth, mycelium formation, and nuclear division. This allows a mechanistic comparison of yeast-like and hyphal growth and the establishment of a core model morphogenetic network for filamentous growth including signaling via the cAMP pathway, Rho modules, and cell cycle kinases. Appreciating similarities between morphogenetic networks of the unicellular yeasts and the multicellular filamentous fungi will open new research directions, help in isolating the central network components, and ultimately pave the way to elucidate the central differences (of many) that distinguish, e.g., the growth mode of filamentous fungi from that of their yeast-like relatives, the role of cAMP signaling, and nuclear division.     

活性污泥丝状膨胀的模拟试验与控制对策研究

以新鲜的生活污水作为水样,采用同步培菌法,在培养活性污泥的基础上,模拟一定的条件促进丝状菌生长直至形成丝状膨胀,并探索消除丝状膨胀的控制措施,同时通过对丝状菌进行分析鉴定及生活特性显微观察,确定产生丝状膨胀的丝状菌的种类。因为实验能够较好地接近污水厂的实际情况,其结论对污水厂曝气池的正常运行具有实际指导作用。     

The challenge for identifying the fungi living inside mushrooms: the case of truffle inhabiting mycelia

The Tuber ascomata (truffles) are a microhabitat for bacteria, viruses, and fungi (yeasts and filamentous fungi). In this survey, we tried to develop a method that would make it possible to define the mycobiome of the truffle-inhabiting filamentous fungi using culture independent molecular methods. The nested quantitative Real-Time PCR allowed us to demonstrate that each truffle is home to multiple species of filamentous fungi and that their DNA is present within the healthy ascoma at the ratio of 10^?6 compared to that of the truffle. Probably due to their insignificant presence, Denaturing Gradient Gel Electrophoresis of the amplification of ITS amplicons showed only those of the host. Based on the results, the possibilities of being able to detect the fungicolous fungi present in very small amounts within a fungal host are discussed.     

The fascinating biology behind phage display: filamentous phage assembly

With the recently awarded Nobel Prize to the inventor of Phage Display, George Smith, the technique has once more gained attention. However, one should not forget about the biology behind the method. Almost always ignored is how the structure of this bacterial virus is assembled. In contrast to lytic phages, filamentous phages are constantly being extruded through the bacterial membranes without lysis. Such filamentous phages are found in all aquatic environments, such as rivers and lakes, in the deep sea, in arctic ice, in hot springs and, associated with their hosts, in plants and animals including humans. While most filamentous phages infect Gram‐negative hosts, inoviruses of Gram‐positive hosts have also been described. Despite being among the minority within the phage family with an estimate of less than 5%, filamentous phages are real parasites as they exist at the expense of the host, but do not kill it. In contrast to lytic bacteriophages, filamentous phages are assembled in the host’s membrane and extruded across the cellular envelope while the bacterium continues to grow. In this review, we focus on this complex and yet poorly understood process of assembly and secretion of filamentous phages.     

Polarized growth in fungi--interplay between the cytoskeleton, positional markers and membrane domains

One kind of the most extremely polarized cells in nature are the indefinitely growing hyphae of filamentous fungi. A continuous flow of secretion vesicles from the hyphal cell body to the growing hyphal tip is essential for cell wall and membrane extension. Because microtubules (MT) and actin, together with their corresponding motor proteins, are involved in the process, the arrangement of the cytoskeleton is a crucial step to establish and maintain polarity. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, actin-mediated vesicle transportation is sufficient for polar cell extension, but in S. pombe, MTs are in addition required for the establishment of polarity. The MT cytoskeleton delivers the so-called cell-end marker proteins to the cell pole, which in turn polarize the actin cytoskeleton. Latest results suggest that this scenario may principally be conserved from S. pombe to filamentous fungi. In addition, in filamentous fungi, MTs could provide the tracks for long-distance vesicle movement. In this review, we will compare the interaction of the MT and the actin cytoskeleton and their relation to the cortex between yeasts and filamentous fungi. In addition, we will discuss the role of sterol-rich membrane domains in combination with cell-end marker proteins for polarity establishment.     

Impacts of differential consumption by the grazing fish, Plecoglossus altivelis, on the benthic algal composition in the Chikuma River, Japan

Grazing effects of ayu, Plecoglossus altivelis Temminck et Schegel, on the benthic algal assemblages were investigated in the Chikuma River, Japan. Comparison of the algal composition on boulders with and without intensively grazed patches indicated that fish grazing decreased the abundance of diatoms and prostrate filamentous cyanobacteria and caused upright filamentous cyanobacteria to predominate. Differential consumption by ayu was estimated by comparing the relative abundance of algae in the stomach contents of ayu and that in the algal assemblages within the grazed patches. The results showed that ayu consumed the prostrate filamentous cyanobacteria proportionally to their abundance, whereas they ingested diatoms and the upright filamentous cyanobacteria in a larger and lower quantity, respectively, than that expected from their abundance. Differential consumption would involve the change in the algal composition toward the predominance of upright filamentous cyanobacteria under fish grazing conditions.     

In situ filamentous communities from the Ediacaran (approx. 563 Ma) of Brazil

Precambrian filamentous microfossils are common and diverse. Nevertheless, their taxonomic assignment can be difficult owing to their overall simple shapes typically lacking in diagnostic features. Here, we report in situ communities of well-preserved, large filamentous impressions from the Ediacaran Itajaí Basin (ca 563 Ma) of Brazil. The filaments are uniserial (unbranched) and can reach up to 200 µm in width and up to 44 mm in length. They occur as both densely packed or sparsely populated surfaces, and typically show a consistent orientation. Although simple in shape, their preferred orientation suggests they were tethered to the seafloor, and their overall flexibility (e.g. bent, folded and twisted) supports a biological (rather than sedimentary) affinity. Biometric comparisons with modern filamentous groups further support their biological affinity, suggesting links with either large sulfide-oxidizing bacteria (SOB) or eukaryotes. Other morphological and palaeoecological characteristics further corroborates their similarities with modern large filamentous SOB. Their widespread occurrence and association with complex Ediacaran macrobiota (e.g. frondose organisms, Palaeopascichnus) suggest that they probably played an important role in the ecological dynamics of these early benthic communities by providing firm substrates for metazoans to inhabit. It is further hypothesized that the dynamic redox condition in the latest Ediacaran, with the non-continuous rise in oxygen concentration and periods of hypoxia, may have created ideal conditions for SOB to thrive.     

Biotechnological potential of pectinolytic complexes of fungi

Plant cell wall-degrading enzymes, such as cellulases, hemicellulases and pectinases, have been extensively studied because of their well documented biotechnological potential, mainly in the food industry. In particular, lytic enzymes from filamentous fungi have been the subject of a vast number of studies due both to their advantages as models for enzyme production and their characteristics. The demand for such enzymes is rapidly increasing, as are the efforts to improve their production and to implement their use in several industrial processes, with the goal of making them more efficient and environment-friendly. The present review focuses mainly on pectinolytic enzymes of filamentous fungi, which are responsible for degradation of pectin, one of the major components of the plant cell wall. Also discussed are the past and current strategies for the production of cell wall-degrading enzymes and their present applications in a number of biotechnological areas.     

Metabolite profiling of fungi and yeast: from phenotype to metabolome by MS and informatics

Filamentous fungi and yeast from the genera Saccharomyces, Penicillium, Aspergillus, and Fusarium are well known for their impact on our life as pathogens, involved in food spoilage by degradation or toxin contamination, and also for their wide use in biotechnology for the production of beverages, chemicals, pharmaceuticals, and enzymes. The genomes of these eukaryotic micro-organisms range from about 6000 genes in yeasts (S. cerevisiae) to more than 10,000 genes in filamentous fungi (Aspergillus sp.). Yeast and filamentous fungi are expected to share much of their primary metabolism; therefore much understanding of the central metabolism and regulation in less-studied filamentous fungi can be learned from comparative metabolite profiling and metabolomics of yeast and filamentous fungi. Filamentous fungi also have a very active and diverse secondary metabolism in which many of the additional genes present in fungi, compared with yeast, are likely to be involved. Although the 'blueprint' of a given organism is represented by the genome, its behaviour is expressed as its phenotype, i.e. growth characteristics, cell differentiation, response to the environment, the production of secondary metabolites and enzymes. Therefore the profile of (secondary) metabolites--fungal chemodiversity--is important for functional genomics and in the search for new compounds that may serve as biotechnology products. Fungal chemodiversity is, however, equally efficient for identification and classification of fungi, and hence a powerful tool in fungal taxonomy. In this paper, the use of metabolite profiling is discussed for the identification and classification of yeasts and filamentous fungi, functional analysis or discovery by integration of high performance analytical methodology, efficient data handling techniques and core concepts of species, and intelligent screening. One very efficient approach is direct infusion Mass Spectrometry (diMS) integrated with automated data handling, but a full metabolic picture requires the combination of several different analytical techniques.     

The regulation of filamentous growth in yeast

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host-cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways-rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)-also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior.     

Varying Virulence in Rabbits Infected with Different Filamentous Types of Histoplasma capsulatum

Histoplasma capsulatum filamentous primary isolates and their subcultures are separable into two distinct colonial types (A and B) having different microscopic characteristics. Yeast forms of the A and B types and the parent (P) strains from which they are derived are microscopically indistinguishable. Critically standardized inocula of living P, A, and B yeasts from one strain of H. capsulatum (G-184) were injected intravenously into 12 rabbits. Each type produced progressively debilitating disease, but in varying degrees. Of the 12 animals, 6 died within 2 to 14 weeks. A persisting copious nasal exudate, beginning at or before 1 week, was cultured weekly at 26 C on Mycosel (BBL) agar. Pure cultures of A and B filamentous type colonies were recovered from exudates of animals receiving A and B yeasts, respectively, whereas both filamentous types were isolated from rabbits injected with P yeasts, with B predominating. Only A and B yeasts thus maintained their filamentous integrity during animal passage. It was noted that dissemination of H. capsulatum through the nares of infected rabbits represents a possible hazard to laboratory personnel heretofore unrecognized. It is also a possible means of cross-infecting or sensitizing or cross-infecting and sensitizing animals housed in the same room, if A and B yeasts prove not to be antigenically identical.