Aspergillus species are among the most important filamentous fungi in terms of industrial use and because of their pathogenic or toxin-producing features. The genomes of several Aspergillus species ...have become publicly available in this decade, and genomic analyses have contributed to an integrated understanding of fungal biology. Stress responses and adaptation mechanisms have been intensively investigated using the accessible genome infrastructure. Mitogen-activated protein kinase (MAPK) cascades have been highlighted as being fundamentally important in fungal adaptation to a wide range of stress conditions. Reverse genetics analyses have uncovered the roles of MAPK pathways in osmotic stress, cell wall stress, development, secondary metabolite production, and conidia stress resistance. This review summarizes the current knowledge on the stress biology of Aspergillus species, illuminating what we have learned from the genomic data in this "post-genomic era."
Abstract
Aspartate/alanine exchange transporter (AspT) is a secondary transporter isolated from the lactic acid bacterium Tetragenococcus halophilus D10 strain. This transporter cooperates with ...aspartate decarboxylase to produce proton-motive force through decarboxylative phosphorylation. A method that successfully analyzes the AspT mechanism could serve as a prototype for elucidating the substrate transport mechanism of other exchange transporters; therefore, the purpose of this study was to search for conditions that improve the thermal stability of AspT for 3D structure analysis. We used the fluorescence size-exclusion chromatography-based thermostability assay to evaluate conditions that contribute to AspT stability. We found that the AspT thermostability was enhanced at pH 5.0 to 6.0 and in the presence of Na+ and Li+. Pyridoxal phosphate, a coenzyme of aspartate decarboxylase, also had a thermostabilizing effect on AspT. Under the conditions obtained from these results, it was possible to increase the temperature at which 50% of dimer AspT remained by 14°C. We expect these conditions to provide useful information for future structural analysis of AspT.
Graphical Abstract
Graphical Abstract
Although α-1,3-glucan is a major cell wall polysaccharide in filamentous fungi, its biological functions remain unclear, except that it acts as a virulence factor in animal and plant pathogenic ...fungi: it conceals cell wall β-glucan on the fungal cell surface to circumvent recognition by hosts. However, cell wall α-1,3-glucan is also present in many of non-pathogenic fungi. Recently, the universal function of α-1,3-glucan as an aggregation factor has been demonstrated. Applications of fungi with modified cell wall α-1,3-glucan in the fermentation industry and of in vitro enzymatically-synthesized α-1,3-glucan in bio-plastics have been developed. This review focuses on the recent progress in our understanding of the biological functions and biosynthetic mechanism of cell wall α-1,3-glucan in fungi. We briefly consider the history of studies on α-1,3-glucan, overview its biological functions and biosynthesis, and finally consider the industrial applications of fungi deficient in α-1,3-glucan.
Bacteria with an actinomycetes-like morphology have recently been discovered, and the class Ktedonobacteria was created for these bacteria in the phylum Chloroflexi. They may prove to be a valuable ...resource with the potential to produce unprecedented secondary metabolites. However, our understanding of their diversity, richness, habitat, and ecological significance is very limited. We herein developed a 16S rRNA gene-targeted, Ktedonobacteria-specific primer and analyzed ktedonobacterial amplicons. We investigated abundance, diversity, and community structure in forest and garden soils, sand, bark, geothermal sediment, and compost. Forest soils had the highest diversity among the samples tested (1181–2934 operational taxonomic units OTUs; Chao 1 estimate, 2503–5613; Shannon index, 4.21–6.42). A phylogenetic analysis of representative OTUs revealed at least eight groups within unclassified Ktedonobacterales, expanding the known diversity of this order. Ktedonobacterial communities markedly varied among our samples. The common mesic environments (soil, sand, and bark) were dominated by diverse phylotypes within the eight groups. In contrast, compost and geothermal sediment samples were dominated by known ktedonobacterial families (Thermosporotrichaceae and Thermogemmatisporaceae, respectively). The relative abundance of Ktedonobacteria in the communities, based on universal primers, was ≤0.8%, but was 12.9% in the geothermal sediment. These results suggest that unknown diverse Ktedonobacteria inhabit common environments including forests, gardens, and sand at low abundances, as well as extreme environments such as geothermal areas.
Abstract
The aspartate:alanine exchanger family of membrane transporters includes industrially important transporters such as succinate exporter and glutamate exporter. No high-resolution structure ...is available from this family so far, and the transport mechanism of these transporters also remains unclear. In the present study, we focus on the oligomeric status of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus, which is the prototype of this family. To investigate the oligomeric structure of AspT, we established a system that produces high yields of highly purified AspT and determined the oligomeric structure of AspT by analysis with size exclusion chromatography coupled with multi-angle light scattering and blue native PAGE and by comparison of the wild-type AspT with a single-cysteine mutant that forms spontaneous inter-molecular thiol crosslinking. All the results consistently support the notion that AspT is a homodimer in solutions and in membranes.
Graphical Abstract
Graphical Abstract
An aspartate:alanine antiporter (AspT) from the lactic acid bacterium Tetragenococcus halophilus catalyzes the electrogenic aspartate
:alanine
exchange reaction. Our previous kinetic analyses of ...transport reactions mediated by AspT in reconstituted liposomes suggested that, although the substrate transport reactions are physiologically coupled, the putative binding sites of L-aspartate (-Asp) and L-alanine (-Ala) are independently located on AspT. By using the fluorescent probe Oregon Green maleimide (OGM), which reacts specifically with cysteine, we also found that the presence of L-Asp changes the conformation of AspT. In this study, we conducted an OGM labeling assay in the presence of L-Ala. The labeling efficiency of single cysteine mutants (G62C and P79C) in transmembrane helix 3 of the AspT showed novel patterns depending on the presence of L-Ala or analogs. A concentration-dependent shift of AspT from the conformation in the presence of one substrate to that specific to the substrate added subsequently (L-Ala or L-Asp) was observed. Moreover, size-exclusion-chromatography-based thermostability assays indicated that the thermal stability of AspT in the presence of L-Ala differed from that in the presence of L-Asp. From these results, we concluded that L-Ala binding yields a conformation different from the apo or L-Asp binding conformations.
Traditionally, filamentous fungi and actinomycetes are well-known cellulolytic microorganisms that have been utilized in the commercial production of cellulase enzyme cocktails for industrial-scale ...degradation of plant biomass. Noticeably, the Ktedonobacteria lineage (phylum Chloroflexi) with actinomycetes-like morphology was identified and exhibited diverse carbohydrate utilization or degradation abilities. In this study, we performed genome-wide profiling of carbohydrate-active enzymes (CAZymes) in the filamentous Ktedonobacteria lineage. Numerous CAZymes (153–290 CAZymes, representing 63–131 glycoside hydrolases (GHs) per genome), including complex mixtures of endo- and exo-cellulases, were predicted in 15 available Ktedonobacteria genomes. Of note, 4–28 CAZymes were predicted to be extracellular enzymes, whereas 3–29 CAZymes were appended with carbohydrate-binding modules (CBMs) that may promote their binding to insoluble carbohydrate substrates. This number far exceeded other Chloroflexi lineages and were comparable to the cellulolytic actinomycetes. Six multi-modular extracellular GHs were cloned from the thermophilic Thermosporothrix hazakensis SK20-1T strain and heterologously expressed. The putative endo-glucanases of ThazG5-1, ThazG9, and ThazG12 exhibited strong cellulolytic activity, whereas the putative exo-glucanases ThazG6 and ThazG48 formed weak but observable halos on carboxymethyl cellulose plates, indicating their potential biotechnological application. The purified recombinant ThazG12 had near-neutral pH (optimal 6.0), high thermostability (60°C), and broad specificity against soluble and insoluble polysaccharide substrates. It also represented described a novel thermostable bacterial β-1,4-glucanase in the GH12 family. Together, this research revealed the underestimated cellulolytic potential of the Ktedonobacteria lineage and highlighted its potential biotechnological utility as a promising microbial resource for the discovery of industrially useful cellulases.
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•The Ktedonobacteria lineage (phylum Chloroflexi) with actinomycetes-like morphology harbored numerous CAZymes.•The Ktedonobacteria lineage harbored complex mixtures of endo-/exo-cellulases with potential biotechnological application.•The thermostable (60°C) ThazG12 represented a novel bacteria-derived β-1,4-glucanase in the GH12 family.
Signal transduction pathways regulating cell wall integrity (CWI) in filamentous fungi have been studied taking into account findings in budding yeast, and much knowledge has been accumulated in ...recent years. Given that the cell wall is essential for viability in fungi, its architecture has been analyzed in relation to virulence, especially in filamentous fungal pathogens of plants and humans. Although research on CWI signaling in individual fungal species has progressed, an integrated understanding of CWI signaling in diverse fungi has not yet been achieved. For example, the variety of sensor proteins and their functional differences among different fungal species have been described, but the understanding of their general and species-specific biological functions is limited. Our long-term research interest is CWI signaling in filamentous fungi. Here, we outline CWI signaling in these fungi, from sensor proteins required for the recognition of environmental changes to the regulation of cell wall polysaccharide synthesis genes. We discuss the similarities and differences between the functions of CWI signaling factors in filamentous fungi and in budding yeast. We also describe the latest findings on industrial applications, including those derived from studies on CWI signaling: the development of antifungal agents and the development of highly productive strains of filamentous fungi with modified cell surface characteristics by controlling cell wall biogenesis.
The hyphal surface of cells of filamentous fungi is covered with cell wall, which is mainly composed of polysaccharides. Since the cell wall is the first structure to come in contact with the ...infection host, the environment, and the fungus itself, the elucidation of the cell wall structure and biogenesis is essential for understanding fungal ecology. Among filamentous fungi, the genus Aspergillus is an important group in the industrial, food, and medical fields. It is known that Aspergillus species form hyphal pellets in shake liquid culture. The authors previously found the role of α-1,3-glucan in hyphal aggregation in Aspergillus species. In addition, extracellular polysaccharide galactosaminogalactan contributed to hyphal aggregation as well, and dual disruption of biosynthesis genes of α-1,3-glucan and galactosaminogalactan resulted in complete hyphal dispersion in shake liquid culture. The characteristic of mycelia to form pellets under liquid culture conditions was the main reason why the growth measurement methods used for unicellular organisms could not be applied. We reported that hyphal growth of the dual disruption mutant could be measured by optical density. A real-time plate reader could be used to determine the growth curve of the mycelial growth of the dual disruption mutant. This measurement approach not only provides basic microbiological insights in filamentous fungi, but also has the potential to be applied to high-throughput screening of anti-Aspergillus drugs.
Laccase1 (Lcc1) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L. edodes results in abnormal hyphal structure and thinner cell wall ...in mycelia. In this study, we observed the effects of Lcc1 on the hyphal morphology and cell wall structure of L. edodes. A thick cell wall and fibrous layer were clearly observed in the lcc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble β-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble β-1,3-1,6-glucan content increased after Lcc1 secretion in the WT. Taken together, these data suggest that the increased level of β-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength.
•Laccase (Lcc1) influences hyphal morphology in Lentinula edodes.•β-1,3-1,6-glucan reduced in Lcc1 silenced strain.•Chitin is synthesized earlier than β-1,3-1,6-glucan.•β-1,3-1,6-glucan contributes strength of hyphae.
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