Interactions Between Hyphosphere-Associated Bacteria and the Fungus <Emphasis Type="Italic">Cladosporium herbarum</Emphasis> on Aquatic Leaf Litter

We investigated microbial interactions of aquatic bacteria associated with hyphae (the hyphosphere) of freshwater fungi on leaf litter. Bacteria were isolated directly from the hyphae of fungi from sedimented leaves of a small stream in the National Park “Lower Oder,” Germany. To investigate interactions, bacteria and fungi were pairwise co-cultivated on leaf-extract medium and in microcosms loaded with leaves. The performance of fungi and bacteria was monitored by measuring growth, enzyme production, and respiration of mono- and co-cultures. Growth inhibition of the fungus Cladosporium herbarum by Ralstonia pickettii was detected on leaf extract agar plates. In microcosms, the presence of Chryseobacterium sp. lowered the exocellulase, endocellulase, and cellobiase activity of the fungus. Additionally, the conversion of leaf material into microbial biomass was retarded in co-cultures. The respiration of the fungus was uninfluenced by the presence of the bacterium.     

Distribution of O-glycosylhydrolases in marine fungi of the Sea of Japan and the Sea of Okhotsk: Characterization of exocellular N-acetyl-β-D-glucosaminidase of the marine fungus <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The capacity to produce exocellular enzymes was studied for 92 samples of fungi from various marine habitats in the Sea of Okhotsk (78 strains) and the Sea of Japan (14 strains). Strains producing highly active glycanases and glycosidases were found. Synthesis of O-glycosylhydrolases was stimulated by addition of laminaran to the nutrient medium. Highly purified N-acetyl-β-D-glucosaminidase was isolated from the marine fungus Penicillium canescens. The molecular weight of the enzyme determined by SDS-Na-electrophoresis was 68 kDa. The enzyme displayed maximum activity at pH 4.5 and temperature 45°C. Inactivation half-time of the enzyme at 50°C was 25 min. N-acetyl-β-D-glucosaminidase hydrolyzed both β-glucosaminide and β-galactosaminide bonds and possessed a high transglycosylating activity.     

Two glucuronoyl esterases of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The white-rot fungus Phanerochaete chrysosporium produces glucuronoyl esterase, a recently discovered carbohydrate esterase, during growth on sugar beet pulp. Two putative genes encoding this enzyme, ge1 and ge2, were isolated and cloned. Heterologous expression in Aspergillus vadensis, Pycnoporus cinnabarinus and Schizophyllum commune resulted in extracellular glucuronoyl esterase activity, demonstrating that these genes encode this enzymatic function. The amino acid sequence of GE1 was used to identify homologous genes in the genomes of twenty-four fungi. Approximately half of the genomes, both from ascomycetes and basidiomycetes, contained putative orthologues, but their presence could not be assigned to any of fungal class or subclass. Comparison of the amino acid sequences of identified and putative glucuronoyl esterases to other types of carbohydrate esterases (CE) confirmed that they form a separate family of CEs. These enzymes are interesting candidates for biotechnological applications such as the separation of lignin and hemicellulose.     

Supramolecular complexes of the <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> virulence protein VirE2

Virulence protein VirE2 from Agrobacterium tumefaciens is involved in plant infection by transferring a fragment of agrobacterial Ti plasmid ssT-DNA in complex with VirE2-VirD2 proteins into the plant cell nucleus. The VirE2 protein interactions with ssDNA and formation of VirE2 protein complexes in vitro and in silico have been studied. Using dynamic light scattering we found that purified recombinant protein VirE2 exists in buffer solution in the form of complexes of 2–4 protein molecules of 12–18 nm size. We used computer methods to design models of complexes consisting of two and four individual VirE2 proteins, and their dimensions were estimated. Dimensions of VirE2 complexes with ssDNA (550 and 700 nucleotide residues) were determined using transmission electron microscopy and dynamic light scattering. We found that in vitro, upon interaction with ssDNA recombinant protein, VirE2 is able to alter conformation of the latter by shortening the initial length of the ssDNA.     

Environmental and industrial applications of Yarrowia lipolytica

Yarrowia lipolytica is a fungus that degrades hydrophobic substrates very efficiently. The fungus displays several important characteristics that have encouraged researchers to study various basic biological and biotechnological applications in detail. Although the organism has been used as model system for studying dimorphism, salt tolerance, heterologous protein expression, and lipid accumulation, there are no recent reviews on the environmental and industrial applications of this organism. Included here are applications in bioremediation of environments contaminated with aliphatic and aromatic compounds, organic pollutants, 2,4,6-trinitrotoluene, and metals. A variety of industrially important recent processes for the synthesis of β-hydroxy butyrate, l-dopa, and emulsifiers have also been reviewed. Production of unique inherent enzymes (inulinases, α-mannosidases), novel applications of esterases and lipases, and the use of the fungus for heterologous expression of biotechnologically relevant products have also been highlighted. The review while entailing a general overview focuses critically on some of the recent advances on the applications of this yeast. The examples cited here demonstrate the use of wild-type, mutant as well as genetically manipulated strains of Y. lipolytica for the development of different products, processes, and technologies. This also throws light on how a single organism can be versatile with respect to its metabolic abilities and how it can be exploited for a variety of purposes. This review will thus form a base for future developments in this field.     

Stimulation of alkalothermophilic Aspergillus terreusxylanase by low-intensity laser radiation

In this study, Aspergillus terreus was irradiated by a 7.3 mW He–Ne laser in the presence of crystal violet, toluidine blue O and hematoporphyrin as photosensitizers. Xylanases recovered from non-irradiated and irradiated fungi were purified and characterized. The maximum production of xylanase (42.2 U/ml) was obtained after 5 min of laser irradiation in the absence of the photosensitizer. The irradiation of the sensitized fungus diminished the production of xylanase. On purification using G-100, the specific activity of xylanase recovered from the irradiated fungus was 292 U/mg protein representing a 37-fold purification over the crude extract compared with 95.6 U/mg protein representing the 12.8-fold for the enzyme recovered from the non-irradiated fungus. The enzyme recovered from the irradiated fungus had lower molecular weight as compared with that recovered from the non-irradiated one. Characterization of the purified enzymes revealed that the enzyme recovered from the irradiated fungus was more thermostable and had a wider range of optimum reaction temperature (60–70°C) and pH (4.0–12.0), compared to the non-irradiated one.     

Irpex lacteus, a white rot fungus applicable to water and soil bioremediation

Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydrocarbons by the fungus Irpex lacteus were characterized and compared with those of other white rot fungi capable of rapid decolorization of poly R-478 and Remazol Brilliant Blue R dyes. I. lacteus was able to grow on mineral and complex media and efficiently colonized sterile and non-sterile soil by exploratory mycelium growing from a wheat straw inoculum. In shallow stationary cultures growing on high nitrogen mineral medium containing 45 mM ammonium as nitrogen source, the fungus produced lignin peroxidase (LIP), Mn-dependent peroxidase (MnP) and laccase simultaneously, the respective maximal activities of 70, 970 and 36 U/l being attained around day 18. Growing in nitrogen-limited medium (2.4 mM ammonium), no LIP was formed and levels of MnP and laccase decreased significantly. During growth in sterile soil, the fungus synthesized LIP and laccase but not MnP. I. lacteus efficiently removed three- and four-ringed PAHs from liquid media and artificially spiked soil. The variety of ligninolytic enzymes, robust growth, capability of soil colonization and resistance to inhibitory action of soil bacteria make I. lacteus a suitable fungal organism for use in bioremediation. Received: 30 March 2000 / Accepted: 19 May 2000     

Mannanases: microbial sources,production, properties and potential biotechnological applications

Mannans are the major constituents of the hemicellulose fraction in softwoods and show widespread distribution in plant tissues. The major mannan-degrading enzymes are β-mannanases, β-mannosidases and β-glucosidases. In addition to these, other enzymes such as α-galactosidases and acetyl mannan esterases, are required to remove the side chain substituents. The mannanases are known to be produced by a variety of bacteria, fungi, actinomycetes, plants and animals. Microbial mannanases are mainly extracellular and can act in wide range of pH and temperature because of which they have found applications in pulp and paper, pharmaceutical, food, feed, oil and textile industries. This review summarizes the studies on mannanases reported in recent years in terms of important microbial sources, production conditions, enzyme properties, heterologous expression and potential industrial applications.     

Production and characterization of laccase from Cyathus bulleri and its use in decolourization of recalcitrant textile dyes

Many fungi (particularly the white rot) are well suited for treatment of a broad range of textile dye effluents due to the versatility of the lignin-degrading enzymes produced by them. We have investigated decolourization of a number of recalcitrant reactive azo and acid dyes using the culture filtrate and purified laccase from the fungus Cyathus bulleri. For this, the enzyme was purified from the culture filtrate to a high specific activity of 4,022 IU mg⁻¹ protein, produced under optimized carbon, nitrogen and C/N ratio with induction by 2,6-dimethylaniline. The protein was characterized as a monomer of 58±5.0 kDa with carbohydrate content of 16% and was found to contain all three Cu(II) centres. The three internal peptide sequences showed sequence identity (80–92%) with laccases of a number of white rot fungi. Substrate specificity indicated highest catalytic efficiency (k _cat/K _M) on guaiacol followed by 2,2′-azino-bis(3-ethylthiazoline-6-sulfonic acid) (ABTS). Decolourization of a number of reactive azo and acid dyes was seen with the culture filtrate of the fungus containing predominantly laccase. In spite of no observable effect of purified laccase on other dyes, the ability to decolourize these was achieved in the presence of the redox mediator ABTS, with 50% decolourization in 0.5–5.4 days.     

Comparative Characterization of Laminarinases from the Filamentous Marine Fungi Chaetomium Indicum Corda and Trichoderma Aureviride Rifai

Marine filamentous fungi (103 strains) isolated from various marine habitats were studied for their ability to produce extracellular O-glycosylhydrolases. Cultural filtrates of these strains were shown to contain a series of glycanases (laminarinases, amylases, cellulases, pustulanases) and glycosidases (β-glucosidases, N-acetyl-β-glucosaminidases, β-galactosidases, α-mannosidases).Two species of marine fungi from different habitats were chosen for isolation of laminarinases and detailed study on enzyme properties. The fungus Chaetomium indicum associated with the alga Fucus evanescens C. Agardh was collected near the Kuril Islands, and T. aureviride was sampled from bottom deposits of South China Sea. Properties of extracellular laminarinases were similar: temperature optimums (40–45 ^∘C), molecular masses (54–56 kDa), K _m (0.1–0.3 mg ml⁻¹). Temperature stability of laminarinase of C. indicum was significantly higher than those from T. aureveride. It is shown that these enzymes are specific to β-1,3-bonds in glucans, release predominantly glucose from laminaran and do not catalyze reaction of transglycosylation. Accoding to these data enzymes are exo-1,3-β-D-glucan-glucanohydrolases (EC 3.2.1.58). Inhibitor analysis demonstrated the significant role of tryptophan and tyrosine residues in the catalytic activity of enzymes. Molecules of T. aureviride laminarinase contained the functionally important thiol group.     

Signal Transduction Pathways in Mycorrhizal Associations: Comparisons with theRhizobium–Legume Symbiosis

A number of genera of soil fungi interact with plant roots to establish symbiotic associations whereby phosphate acquired by the fungus is exchanged for fixed carbon from the plant. Recent progress in investigating these associations, designated as mycorrhizae (sing., mycorrhiza), has led to the identification of specific steps in the establishment of the symbiosis in which the fungus and the plant interact in response to various molecular signals. Some of these signals are conserved with those of theRhizobium–legume nitrogen-fixing symbiosis, suggesting that the two plant–microbe interactions share a common signal transduction pathway. Nevertheless, only legume hosts nodulate in response toRhizobium,whereas the vast majority of flowering plants establish mycorrhizal associations. The key questions for the future are: what are the signal molecules produced by mycorrhizal fungi and how are they perceived by the plant?     

The effect of acetylated xylan and sugar beet pulp on the expression and secretion of enzymes by <Emphasis Type="Italic">Penicillium purpurogenum</Emphasis>

Sugar beet pulp is a natural carbon source composed mainly of pectin and cellulose, which is utilized and degraded by the ascomycete Penicillium purpurogenum. The fungus also grows on and degrades acetylated xylan which lacks cellulose and pectin. Both carbon sources have been used in our laboratory to grow the fungus and to purify different enzymes secreted to the medium. The enzymes involved in the complex process of degradation of these carbon sources by the fungus have been explored previously under non-denaturing conditions; multienzyme complexes were separated and some subunits identified by Western blots and mass spectrometry. In this work, proteomic profiles show that the secretome is composed of numerous proteins varying in pI and molecular weight. Some enzymes are common to both growth conditions, while others are specific for each carbon source. The results show that the carbon sources utilized exert strong regulatory control over the proteins secreted. This is the first secretome study from a lignocellulolytic Penicillium.     

The activity of nonspecific esterases and glutathione-S-transferase in <Emphasis Type="Italic">Locusta migratoria</Emphasis> larvae infected with the fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> (Ascomycota,Hypocreales)

The activity of nonspecific esterases and glutathione-S-transferase in whole body homogenates, hemolymph plasma, and fat body of the larvae of the locust Locusta migratoria was analyzed during development of infection with the fungus Metarhizium anisopliae. The lethal dose of the fungus (LC₈₀) was found to enhance the activity of detoxifying enzymes in the whole body homogenate of the larvae on the 3rd day after infection. The activity of nonspecific esterases and glutathione-S-transferase in the plasma and fat body of the infected larvae increased on the 3rd day but dropped to the control levels by the 6th day, during the acute period of infection. The detoxifying enzymes may participate in defense reactions at the early stage of the acute fungal infection.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

Digestive responses of two omnivorous rodents (<Emphasis Type="Italic">Peromyscus maniculatus</Emphasis> and <Emphasis Type="Italic">P. alstoni</Emphasis>) feeding on epigeous fungus (<Emphasis Type="Italic">Russula occidentalis</Emphasis>)

The sporocarps of hypogeous and epigeous fungi are important dietary items for forest dwelling rodents in temperate and tropical forests throughout the world. However, results of some pioneering works have demonstrated that fungi cannot be considered as nutritionally high-quality food items for some mycophagous small rodents. According to these studies, when mycophagous rodents feed on fungus, they showed a minimal digestibility, but whether this applies to most rodent species that include fungi in their diets is unknown. In this study, we experimentally evaluated body mass changes and feed preferences in captive deer (Peromyscus maniculatus) and volcano (P. alstoni) mice when fed on epigeous fungus (Russula occidentalis). In experiment 1, the animals were fed with fungus as the only feedstuff in comparison to regular rodent chow and oat. In experiment 2, the animals were fed with fungus in a free-choice arrangement together with equal amounts of rodent chow and oat. Both species lost ∼15% of their body mass within 4 days when fed on fungus alone, but gained 5–10% body mass during the same time period when ingesting oat and rodent chow, respectively, as the only feedstuff. However, in contrast, in the free-choice arrangement with all three feedstuffs, both species gained 20–30% body mass, and showed the highest feed preference for fungus followed by oat and rodent chow. In addition, apparent digestibility of energy and nitrogen were analyzed in both rodent species, which were 50–60% for fungus, whereas approximately 90–94% for rodent chow and oat. According to our results, animals need to supplement their diets with alternative high-quality food items in order to maintain and increase their body mass, suggesting that epigeous fungi are only of moderate nutritional value for small rodents. Futures studies should focus on exploring the importance of a mixture of fungal species in the diet of small mycophagous rodents.     

<Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> from rice husks forming sclerotia in agar culture

Sclerotia were formed in agar culture by a fungus with clamp connections isolated from rice husks at Tsukuba, Japan. The sclerotia were brown, globose to ellipsoidal, small, up to 200 μm in diameter, and composed of external rind tissue and internal medulla tissue. Such tiny sclerotia have not been commonly reported among basidiomycetous fungi in the literature. The fungus was identified as Coprinopsis cinerea on the basis of morphological characteristics together with molecular analyses. Three reference strains of C. cinerea formed sclerotia similarly under identical cultural conditions.     

Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics

The archetypal white-rot fungus Phanerochaete chrysosporium has been shown to degrade a variety of persistent environmental pollutants. Many of the enzymes responsible for pollutant degradation, which are normally involved in the degradation of wood, are extracellular. Thus, P. chrysosporium is able to degrade toxic or insoluble chemicals more efficiently than other microorganisms. P. chrysosporium has a range of oxidative and reductive mechanisms and uses highly reactive, nonspecific redox mediators which increase the number of chemicals that can be effectively degraded. This review gives an overview of the enzymes that are believed to be important for bioremediation and briefly discusses the degradation of some individual chemicals. Received: 25 April 2000 / Received revision: 05 June 2000 / Accepted: 04 July 2000     

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe³⁺-reductants. Phenolates were the major compounds with Fe³⁺-reducing activity in both fungi and displayed Fe³⁺-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe³⁺ and H₂O₂ (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum—a model brown rot fungus—other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.