Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.     

Retention of soluble organic nutrients by a forested ecosystem

We document an example of a forested watershed at the Coweeta HydrologicLaboratory with an extraordinary tendency to retain dissolved organic matter(DOM) generated in large quantities within the ecosystem. Our objectives weretodetermine fluxes of dissolved organic C, N, and P (DOC, DON, DOP,respectively),in water draining through each stratum of the ecosystem and synthesizeinformation on the physicochemical, biological and hydrologic factors leadingtoretention of dissolved organic nutrients in this ecosystem. The ecosystemretained 99.3, 97.3, and 99.0% of water soluble organic C, N and P,respectively, produced in litterfall, throughfall, and root exudates. Exportsinstreamwater were 4.1 kg ha^–1yr^–1of DOC, 0.191 kg ha^–1 yr^–1 ofDON, and 0.011 kg ha^–1 yr^–1 ofDOP. Fluxes of DON were greater than those of inorganic N in all strata. MostDOC, DON, and DOP was removed from solution in the A and B horizons, with DOCbeing rapidly adsorbed to Fe and Al oxyhydroxides, most likely by ligandexchange. DON and DOC were released gradually from the forest floor over theyear. Water soluble organic C produced in litterfall and throughfall had adisjoint distribution of half-decay times with very labile and veryrefractory fractions so that most labile DOC was decomposed before beingleachedinto the mineral soil and refractory fractions dominated the DOC transportedthrough the ecosystem. We hypothesize that this watershed retained solubleorganic nutrients to an extraordinary degree because the soils have very highcontents of Fe and Al oxyhydroxides with high adsorption capacities and becausethe predominant hydrologic pathway is downwards as unsaturated flow through astrongly adsorbing A and B horizon. The well recognized retention mechanismsforinorganic nutrients combine with adsorption of DOM and hydrologic pathway toefficiently prevent leaching of both soluble inorganic andorganic nutrients in this watershed.     

Laccase production and wood degradation by a white-rot fungus Daedalea flavida

A comparative study has been conducted on seven white rot fungi to investigate their abilities to produce laccase and selectively degrade lignin. Laccase was produced constitutively on the different media tested. Of the different lignins, phenolic compounds and sugars involved, the highest laccase yield was obtained on indulin AT. Salicylic acid inhibited enzyme activity. A temperature of 20°C and 0.2% of indulin AT were found to be optimum for enzyme activity. No correlation was found between the amount of enzyme and fungal mass produced. During semisolid degradation of angiospermic wood sawdust, Daedalea flavida caused a total weight loss of 11%, with a lignin loss of 15.77% during two months of decay. Lignin removal was comparatively selective during the first month, during which time laccase production was also higher, indicating its probable role in lignin degradation.     

The uptake of nutrients by mature forest growth

The extremely low nutrient-status of various recently afforested moor soils, particularly Yorkshire (England)Calluna soils, raised the question of their ability to provide the possible nutrient-demands of continuous forest growth. This paper describes how, for calcium, potassium and phosphorus, uptake estimates have been calculated from existing nutrient-composition data, and discusses the silvicultural significance of such estimates, particularly in relation to nutrient-poor soils.All existing nutrient-composition data for whole individual mature temperate forest trees are summarized: using yield-data and a method of calculation critically discussed, they are made to provide nutrient-uptake estimates for one acre of such forest after periods of 50 and 100 years' growth. Mean estimates representing hardwood, other conifer and pine forest are then calculated showing: — the nutrients removed from the site via the various components of the timber-thinnings and clear-fellings, the nutrients taken up and immobilized within the various component organs of the growing forest, and the total nutrient-uptake from the site.The estimates show that timber exploited forest, by reason of its unavoidable continuous nutrient-removal from the site, differs fundamentally from unexploited virgin forest; in absolute terms this removal may be small, but its pedological and silvicultural importance upon any particular site, depends entirely upon the ability of the soil to replenish this loss. It is believed that up to now the failure to recognize this nutrient-removal upon nutrient-poor soils has diverted attention from an important factor initiating both soil-degradation and diminishing site-productivity.Soil nutrient-data presented show that the nutrient-uptakes of timber-producing forest of either confierous or hardwood species are so large compared with the nutrient-contents of moor soils that further overall soil-degradation and, sooner or later, diminished site-productivity are inevitable.Upon such soils, therefore, continuous timber production cannot be sustained by ploughing or by the growth of hardwoods alone; new silvicultural techniques are needed which can restore to the site, by means of suitable soil-ameliorants and methods yet to be evolved, the nutrients continually being removed via the timber.The paucity of knowledge concerning both the nutrient-demands of common tree species and the nutrient-status of the nutrient-poor soils upon which they are being extensively planted calls for an expansion of research upon these aspects if timber-production in this country is to be placed on a scientific, and not speculative, basis.     

The uptake of inorganic nutrients by heterotrophic bacteria

It is now well known that heterotrophic bacteria account for a large portion of total uptake of both phosphate (60% median) and ammonium (30% median) in freshwaters and marine environments. Less clear are the factors controlling relative uptake by bacteria, and the consequences of this uptake on the plankton community and biogeochemical processes, e.g., new production. Some of the variation in reported inorganic nutrient uptake by bacteria is undoubtedly due to methodological problems, but even so, uptake would be expected to vary because of variation in several parameters, perhaps the most interesting being dissolved organic matter. Uptake of ammonium by bacteria is very low whereas uptake of dissolved free amino acids (DFAA) is high in eutrophic estuaries (the Delaware Bay and Chesapeake Bay). The concentrations and turnover of DFAA are insufficient, however, in oligotrophic oceans where bacteria turn to ammonium and nitrate, although the latter only as a last resort. I argue here that high uptake of dissolved organic carbon, which has been questioned, is necessary to balance the measured uptake of dissolved inorganic nitrogen (DIN) in seawater culture experiments. What is problematic is that this DIN uptake exceeds bacterial biomass production. One possibility is that bacteria excrete dissolved organic nitrogen (DON). A recent study offers some support for this hypothesis. Lysis by viruses would also release DON.While ammonium uptake by heterotrophic bacteria has been hypothesized to affect phytoplankton community structure, other impacts on the phytoplankton and biomass production (both total and new) are less clear and need further work. Also, even though bacteria account for a very large fraction of phosphate uptake, how this helps to structure the plankton community has not been examined. What is clear is that the interactions between bacterial and phytoplankton uptake of inorganic nutrients are more complicated than simple competition.     

Cycling of organic and inorganic sulphur in a chestnut oak forest

Summary Sulfur (S) cycling in a chestnut oak forest on Walker Branch Watershed, Tennessee, was dominated by geochemical processes involving sulfate. Even though available SO ₄ ^2- was present far in excess of forest nutritional requirements, the ecosystem as a whole accumulated 60% of incoming SO₄–S. Most (90%) of this accumulation occurred by SO ₄ ^2- adsorption in sesquioxide-rich subsurface soils, with a relatively minor amount accumulating and cycling as SO ₄ ^2- within vegetative components. Organic sulfates are thought to constitute a large proportion of total S in surface soils, also, and to provide a pool of readily mineralized available S within the ecosystem.Research sponsored by Division of Biomedical and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with Union Carbide Corporation. Soil ester sulfate work sponsored by contract RP-1813-1 with the Electric Power Research Institute. Publication No. 1990, Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830     

Direct uptake and degradation of DNA by lysosomes

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered “RNautophagy,” an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term “DNautophagy,” is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.     

Post-fire uptake of nutrients by diverse ephemeral herbs in chamise chaparral

Summary Diverse populations of ephemeral herbs form the dominant element of community biomass in the first year of growth following a fall burn in chamise chaparral. Ephemeral herbs constituted 337 kg ha^-1 of above-ground biomass after the first season of post-fire growth. This was 64% of the total, with the majority of the remaining biomass being in resprouts of Adenostoma fasciculatum. Ephemeral herb biomass following fire in other stands was as high as 1117 kg ha^-1. Nutrient contents of ephemeral herbs were 6.68 kg N ha^-1, 0.71 kg P ha^-1, 10.05 kg K ha^-1, 4.75 kg Ca ha^-1 and 0.91 kg Mg ha^-1. These were 55, 54, 81, 71 and 70% respectively of the above-ground totals. In the second year following fire, the total herb biomass was 40% higher, but the nutrient pool in above-ground biomass of these herbs was only 30–60% of what it had been the first year. Resprouts of A. fasciculatum and short-lived wood shrubs constituted more than 90% of above-ground biomass at this stage of postfire succession.     

Endocytotic uptake of nutrients in carnivorous plants

Carnivorous plants trap, digest and absorb animals in order to supplement their mineral nutrition. Nutrients absorbed by the plant include different nitrogen species, phosphate, potassium, trace elements and small organic compounds. Uptake is usually thought to be performed via specific channels, but this study provides evidence that endocytosis is involved as well. Traps of the carnivorous plants Nepenthes coccinea, Nepenthes ventrata, Cephalotus follicularis, Drosophyllum lusitanicum, Drosera capensis, Dionaea muscipula, Aldrovanda vesiculosa, Genlisea violacea × lobata, Sarracenia psittacina and Sarracenia purpurea were stained with methylene blue in order to identify possible sites of uptake. The permeable parts of the traps were incubated with fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and other fluorescent endocytosis markers, combined with the soluble protein BSA or respiratory inhibitors. Uptake was studied by confocal microscopy. In Nepenthes, small fluorescent vesicles became visible 1 h after incubation with FITC-BSA. These vesicles fused to larger compartments within 30 h. A similar behaviour was found in the related genera Drosera, Dionaea, Aldrovanda and Drosophyllum but also in Cephalotus with glands of different evolutionary origin. In Genlisea and Sarracenia, no evidence for endocytosis was found. We propose that in many carnivorous plants, nutrient uptake by carriers is supplemented by endocytosis, which enables absorption and intracellular digestion of whole proteins. The advantage for the plant of reducing secretion of enzymes for extracellular digestion is evident.     

Growth responses to nutrients of an auxotroph and a prototroph of a predatory fungus

The growth responses of a prototroph and an auxotroph of Arthrobotrys dactyloides were compared, on several nutrient media. The prototroph can utilize a variety of nitrogenous compounds almost equally, but the auxotroph has an exacting requirement for several amino acids together. Both strains utilize sucrose best among the three sugars tested as a C-source but the prototroph responds strongly to phospholipid, in addition. They differ in their needs for purines, pyrimidines and B vitamins.     

2,4-Dichlorophenol degradation by the soil fungus Mortierella sp

We investigated the degradation pathways and kinetics of 2,4-dichlorophenol (DCP) by an endemic soil fungus, Mortierella sp. (Zygomycetes). Mortierella sp. degraded 32% of added DCP (final concentration, 250 microM) within 1 h. We identified four aromatic metabolites and found two DCP degradation pathways (a hydroxylation pathway and a dechlorination pathway). This is the first report of a dechlorination pathway in Zygomycetes.     

Degradation of the antibiotics norfloxacin and ciprofloxacin by a white-rot fungus and identification of degradation products

More than 90% of the antibiotics ciprofloxacin (CIPRO) and norfloxacin (NOR) at 2 mg L⁻¹ were degraded by Trametes versicolor after 7 days of incubation in malt extract liquid medium. In in vitro assays with purified laccase (16.7 nkat mL⁻¹), an extracellular enzyme excreted constitutively by this fungus, 16% of CIPRO was removed after 20 h. The addition of the laccase mediator 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt led to 97.7% and 33.7% degradation of CIPRO and NOR, respectively. Inhibition of CIPRO and NOR degradation by the cytochrome P450 inhibitor 1-aminobenzotriazole suggests that the P450 system also plays a role in the degradation of the two antibiotics. Transformation products of CIPRO and NOR were monitored at different incubation times by triple-quadrupole and quadrupole time-of-flight mass spectrometry, and can be assigned to three different reaction pathways: (i) oxidation of the piperazinyl substituent, (ii) monohydroxylation, and (iii) formation of dimeric products.     

丝状真菌Amorphotheca resinae ZN1的糠醛降解代谢分析

木质纤维素在预处理过程产生的降解产物对后续的酶水解和微生物发酵过程产生了强烈的抑制。因此,这些抑制物的脱除即所谓的"脱毒"步骤是正常进行后续酶解和发酵的前提条件。我们对本实验室筛选的丝状真菌Amorphotheca resinae ZN1的糠醛的代谢路径进行了研究。丝状真菌A.resinae ZN1转化糠醛的降解代谢途径可以简述为:糠醛首先快速地转化为毒性较低的糠醇;在有氧条件下,糠醇又再度生成不致对微生物产生危害的低浓度糠醛,糠醛继续氧化为糠酸。推测糠酸可能继续进入TCA循环,进而完成糠醛的完全降解。研究结果为将来加快丝状真菌A.resinae ZN1生物脱毒速率、改善木质纤维素生物转化的限速步骤提供了重要的实验依据。     

Metabolic synergies in the biotransformation of organic and metallic toxic compounds by a saprotrophic soil fungus

Applied Microbiology and Biotechnology - The saprotrophic fungus Penicillium griseofulvum was chosen as model organism to study responses to a mixture of hexachlorocyclohexane (HCH) isomers...     

Receptor- and non-receptor-mediated uptake and degradation of insulin by hepatocytes

Native insulin inhibits the binding and degradation of ¹²⁵I-labelled insulin in parallel. Half-maximal inhibition of degradation occurs with 10nm-insulin, a hormone concentration sufficient to saturate the insulin receptor. The proportion of bound hormone that is degraded increases as the insulin concentration is increased, suggesting that low-affinity uptake is functionally related to degradation. Since only a small fraction (approx. 10%) of the overall degradation occurs at the plasma membrane, or in the extracellular medium, translocation of bound hormone into the cell is the predominant mechanism mediating the degradation of insulin. In the presence of 0.6nm-insulin, a concentration at which most cell-associated hormone is receptor-bound, chloroquine increases the amount of ¹²⁵I-labelled insulin retained by hepatocytes. However, chloroquine increases the retention of degradation products of insulin in incubations containing sufficient hormone (6nm) to saturate the receptor and permit occupancy of low-affinity sites. Glucagon does not compete for the interaction of ¹²⁵I-labelled insulin (1nm) with the insulin receptor. In contrast, 20μm-glucagon inhibits 75% of the uptake of insulin (0.1μm) by low-affinity sites. A fraction of the cell-bound radioactivity is not intact insulin throughout a 90min association reaction at 37°C. During dissociation, fragments of ¹²⁵I-labelled insulin are released to the medium more rapidly than is intact hormone. The production and transient retention of degradation products of the hormone complicates the characterization of the insulin receptor by equilibrium or kinetic methods of assay. It is proposed that insulin degradation occurs by receptor- and non-receptor-mediated pathways. The latter may be related to the action of glutathione–insulin transhydrogenase, with which both insulin and glucagon interact.     

Limitation of lowland riverine bacterioplankton by dissolved organic carbon and inorganic nutrients

Flow regulation in lowland rivers has reduced the amount of allochthonous dissolved organic carbon (DOC) entering main channels through less frequent wetting of benches, flood runners and floodplains. The hypothesis tested was that lowland riverine bacterioplankton are DOC limited when flow events are absent and simulating an increase in assimilable DOC similar to that expected during an environmental flow will lead to heterotrophic dominance. Experiments took place in the Namoi River, a highly regulated lowland river in Australia. Specifically, in situ microcosms were used to examine the responses of bacterioplankton and phytoplankton to various additions of DOC as glucose or leaf leachate, with and without additions of inorganic nutrients. The results indicated that ambient DOC availability limited the bacterioplankton for the three seasons over which we conducted the experiments. When DOC was added alone, dissolved oxygen concentrations decreased primarily because of increased bacterial respiration and bacterioplankton growth generally increased relative to controls. Additions of DOC alone led to a pattern of decreased chlorophyll a concentration relative to controls, except for willow leachate. Additions of inorganic nutrients alone increased chlorophyll a concentrations above controls, indicating limitation of phytoplankton. These findings support our hypothesis. Based on the present results, environmental flows should increase the duration of allochthonously driven heterotrophic dominance, thus shifting regulated lowland rivers to more natural (pre-regulation) conditions for greater periods.     

培菌白蚁菌圃微生物降解木质纤维素的研究进展

白蚁及其共生微生物协同降解植物细胞壁的机理一直被世界各国科学家所关注。培菌白蚁作为高等白蚁,相比低等食木白蚁具有更多样化的食性,其利用外共生系统“菌圃”,对多种植物材料进行处理。本文综述了菌圃微生物降解木质纤维素的研究进展,以期为深入研究菌圃中木质纤维素降解过程及其机制,并挖掘利用菌圃降解木质纤维素的能力及仿生模拟菌圃开发新的生物质利用系统提供参考。培 菌白蚁在其巢内利用由植物材料修建的多孔海绵状结构——“菌圃”来培养共生真菌鸡枞菌Termitomyces spp.,形成了独特的木质纤维素食物降解和消化策略,使木质纤维素在培菌白蚁及其共生微生物协同作用下被逐步降解。幼年工蚁取食菌圃上的共生真菌菌丝组成的小白球和老年工蚁觅得食物并排出粪便堆积到菌圃上成为上层菌圃。这一过程中,被幼年工蚁取食的共生真菌释放木质素降解酶对包裹在植物多糖外部的木质素屏障进行解聚。菌圃微生物(包括共生真菌)对解聚的木质素基团进一步降解,将多糖长链或主链剪切成短链,使菌圃基质自下而上被逐步降解。最后下层的老熟菌圃被老年工蚁取食,其中肠的内源酶系及后肠微生物将这些短链进一步剪切和利用。因此,蚁巢菌圃及其微生物是培菌白蚁高效转化利用木质纤维素的基础。化学层面的研究表明,菌圃能够实现对植物次生物质解毒和植 物纤维化学结构解构。对共生真菌相关酶系的研究显示可能其在菌圃的植物纤维化学结构和植物次生物质的降解中发挥了作用,但不同属共生真菌间其效率和具体功能不尽相同。而菌圃中的细菌是否发挥了作用和哪些细菌类群发挥了作用等仍有待进一步的研究。相比于低等食木白蚁利用其后肠共生微生物降解木质纤维素,培菌白蚁利用菌圃降解木质纤维素具有非厌氧和能处理多种类型食物两大优势,仿生模拟菌圃降解木质纤维素的机制对林地表面枯枝落叶的资源化利用具有重要意义。     

The uptake of persistent organic pollutants by plants

In a field experiment, the transfer of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) from contaminated soil to maize (Zea mays L.), sunflower (Helianthus annuus), poplar (Populus nigra × P. maximowiczii) and willow (Salix × smithiana) and the distribution of PCB congeners in maize and sunflower was investigated. The former waste incinerator in Hradec Králové (Czech Republic) was chosen for the experiment. Results of plot screening showed heterogenous contamination by PCBs and PAHs. PCB soil contamination was evidently caused by Delor 106 or Aroclor 1260 stocking and PAH contamination by chemicals containing fluoranthene, benzo/b/fluoranthene, phenanthrene and pyrene. Tested plants were planted on a contaminated field site, in soil contaminated with 1530 μg/kg of total PCBs and 0.138 and 3.42 mg/kg of total PAHs. The results show that maize and sunflower roots accumulated the most PCBs from soil. These plants accumulated hexa- and heptachlorobiphenyl congeners more than tri-, tetra-, and pentachlorobiphenyl congeners. Total concentrations of PAHs in tested plants ranged from 0.096 to 1.34 mg/kg. The highest phenanthrene concentration was found in aboveground biomass of sunflower and the highest concentration of pyrene, in maize roots.