Comparative analysis of surface coating properties of five hydrophobins from Aspergillus nidulans and Trichoderma reseei.
Ontology highlight
ABSTRACT:
SUBMITTER: Winandy L
PROVIDER: S-EPMC6089913 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In Aspergillus nidulans two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter. Here we analyzed four more hydrophobins, DewB-E, in A. nidulans and found that all six hydrophobins contribute to the hydrophobic surface of the conidiospores but only deletion of rodA caused loss of the rodlet structure. Analysis of the rodlets in the dewB-E deletion strains with atomic force microscopy revealed that the rodlets appeared less robust. Expression of DewA and DewB driven from the rodA promoter and secreted with the RodA secretion signal in a strain lacking RodA, restored partly the hydrophobicity. DewA and B were able to form rodlets to some extent but never reached the rodlet structure of RodA. The rodlet-lacking rodA-deletion strain opens the possibility to systematically study rodlet formation of other natural or synthetic hydrophobins.
| S-EPMC3983194 | biostudies-literature
Project description:Fungal hydrophobins are small amphiphilic proteins that can be used for coatings on hydrophilic and hydrophobic surfaces. Through the formation of monolayers, they change the hydrophobicity of a given surface. Especially, the class I hydrophobins are interesting for biotechnology, because their layers are stable at high temperatures and can only be removed with strong solvents. These proteins self-assemble into monolayers under physiological conditions and undergo conformational changes that stabilize the layer structure. Several studies have demonstrated how the fusion of hydrophobins with short peptides allows the specific modification of the properties of a given surface or have increased the protein production levels through controlled localization of hydrophobin molecules inside the cell. Here, we fused the Aspergillus nidulans laccase LccC to the class I hydrophobins DewA and DewB and used the fusion proteins to functionalize surfaces with immobilized enzymes. In contrast to previous studies with enzymes fused to class II hydrophobins, the DewA-LccC fusion protein is secreted into the culture medium. The crude culture supernatant was directly used for coatings of glass and polystyrene without additional purification steps. The highest laccase surface activity was achieved after protein immobilization on modified hydrophilic polystyrene at pH 7. This study presents an easy-to-use alternative to classical enzyme immobilization techniques and can be applied not only for laccases but also for other biotechnologically relevant enzymes.ImportanceAlthough fusion with small peptides to modify hydrophobin properties has already been performed in several studies, fusion with an enzyme presents a more challenging task. Both protein partners need to remain in active form so that the hydrophobins can interact with one another and form layers, and so the enzyme (e.g., laccase) will remain active at the same time. Also, because of the amphiphilic nature of hydrophobins, their production and purification remain challenging so far and often include steps that would irreversibly disrupt most enzymes. In our study, we present the first functional fusion proteins of class I hydrophobins from A. nidulans with a laccase. The resulting fusion enzyme is directly secreted into the culture medium by the fungus and can be used for the functionalization of hard surfaces.
| S-EPMC5066356 | biostudies-literature
Project description:BACKGROUND:Sugarcane is one of the world's most profitable crops. Waste steam-exploded sugarcane bagasse (SEB) is a cheap, abundant, and renewable lignocellulosic feedstock for the next-generation biofuels. In nature, fungi seldom exist as planktonic cells, similar to those found in the nutrient-rich environment created within an industrial fermenter. Instead, fungi predominantly form biofilms that allow them to thrive in hostile environments. RESULTS:In turn, we adopted an RNA-sequencing approach to interrogate how the model fungus, Aspergillus nidulans, adapts to SEB, revealing the induction of carbon starvation responses and the lignocellulolytic machinery, in addition to morphological adaptations. Genetic analyses showed the importance of hydrophobins for growth on SEB. The major hydrophobin, RodA, was retained within the fungal biofilm on SEB fibres. The StuA transcription factor that regulates fungal morphology was up-regulated during growth on SEB and controlled hydrophobin gene induction. The absence of the RodA or DewC hydrophobins reduced biofilm formation. The loss of a RodA or a functional StuA reduced the retention of the hydrolytic enzymes within the vicinity of the fungus. Hence, hydrophobins promote biofilm formation on SEB, and may enhance lignocellulose utilisation via promoting a compact substrate-enzyme-fungus structure. CONCLUSION:This novel study highlights the importance of hydrophobins to the formation of biofilms and the efficient deconstruction of lignocellulose.
| S-EPMC4950808 | biostudies-literature
Project description:The surface of Aspergillus fumigatus conidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. The RODA gene was previously characterized, and DeltarodA conidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). The RODB gene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp of A. nidulans. DeltarodB conidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of DeltarodA conidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the DeltarodA DeltarodB double mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.
| S-EPMC150101 | biostudies-literature
Project description:Resistance of Aspergillus fumigatus conidia to desiccation and their capacity to reach the alveoli are partly due to the presence of a hydrophobic layer composed of a protein from the hydrophobin family, called RodA, which covers the conidial surface. In A. fumigatus there are seven hydrophobins (RodA-RodG) belonging to class I and III. Most of them have never been studied. We constructed single and multiple hydrophobin-deletion mutants until the generation of a hydrophobin-free mutant. The phenotype, immunogenicity, and virulence of the mutants were studied. RODA is the most expressed hydrophobin in sporulating cultures, whereas RODB is upregulated in biofilm conditions and in vivo Only RodA, however, is responsible for rodlet formation, sporulation, conidial hydrophobicity, resistance to physical insult or anionic dyes, and immunological inertia of the conidia. None of the hydrophobin plays a role in biofilm formation or its hydrophobicity. RodA is the only needed hydrophobin in A. fumigatus, conditioning the structure, permeability, hydrophobicity, and immune-inertia of the cell wall surface in conidia. Moreover, the defect of rodlets on the conidial cell wall surface impacts on the drug sensitivity of the fungus.
| S-EPMC5872305 | biostudies-literature
Project description:BackgroundOur dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world's second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.ResultsAnalyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and ?-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, ?-glucosidases, ?-xylosidases, endoxylanases, xyloglucanases, and ?-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.ConclusionOur work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.
| S-EPMC4460134 | biostudies-literature
Project description:The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs.
| S-EPMC3591445 | biostudies-literature
Project description:Sterigmatocystin (ST) and the aflatoxins (AFs), related fungal secondary metabolites, are among the most toxic, mutagenic, and carcinogenic natural products known. The ST biosynthetic pathway in Aspergillus nidulans is estimated to involve at least 15 enzymatic activities, while certain Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius strains contain additional activities that convert ST to AF. We have characterized a 60-kb region in the A. nidulans genome and find it contains many, if not all, of the genes needed for ST biosynthesis. This region includes verA, a structural gene previously shown to be required for ST biosynthesis, and 24 additional closely spaced transcripts ranging in size from 0.6 to 7.2 kb that are coordinately induced only under ST-producing conditions. Each end of this gene cluster is demarcated by transcripts that are expressed under both ST-inducing and non-ST-inducing conditions. Deduced polypeptide sequences of regions within this cluster had a high percentage of identity with enzymes that have activities predicted for ST/AF biosynthesis, including a polyketide synthase, a fatty acid synthase (alpha and beta subunits), five monooxygenases, four dehydrogenases, an esterase, an 0-methyltransferase, a reductase, an oxidase, and a zinc cluster DNA binding protein. A revised system for naming the genes of the ST pathway is presented.
| S-EPMC39953 | biostudies-other
Project description:We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.
| S-EPMC1214207 | biostudies-literature
Project description:In Aspergillus nidulans a combination of null mutations in halA, encoding a protein kinase, and sltA, encoding a zinc-finger transcription factor having no yeast homologues, results in an elevated calcium requirement ('calcium auxotrophy') without impairing net calcium uptake. sltA(-) (+/-halA(-)) mutations result in hypertrophy of the vacuolar system. In halA(-)sltA(-) (and sltA(-)) strains, transcript levels for pmcA and pmcB, encoding vacuolar Ca(2+)-ATPase homologues, are highly elevated, suggesting a regulatory relationship between vacuolar membrane area and certain vacuolar membrane ATPase levels. Deletion of both pmcA and pmcB strongly suppresses the 'calcium auxotrophy'. Therefore the 'calcium auxotrophy' possibly results from excessive vacuolar calcium sequestration, causing cytosolic calcium deprivation. Null mutations in nhaA, homologous to Saccharomyces cerevisiae NHA1, encoding a plasma membrane Na(+)/H(+) antiporter effluxing Na(+) and K(+), and a non-null mutation in trkB, homologous to S. cerevisiae TRK1, encoding a plasma membrane high affinity K(+) transporter, also suppress the calcium auxotrophy.
| S-EPMC2884188 | biostudies-literature