Purification and characterization of an extracellular polygalacturonase from the thermophilic fungus,Thermomyces lanuginosus

A polygalacturonase was purified from the thermophilic fungus, Thermomyces lanuginosus to apparent homogeneity by ultrafiltration, acetone precipitation and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60 °C. The apparent K_M with potassium pectate was 0.67 mg/ml and the V_max was 7.2 × 10⁵ mol/min/mg protein. The apparent molecular weight of the enzyme was 59 kDa and it contained approximately 10% carbohydrate. The enzyme was completely stable at room temperature (32 ± 3 °C) and retained about 50% activity at 50 °C for 6 h. The zymogram of the purified enzyme revealed two activity bands, one of which was a major one. Polyclonal antibodies raised against the enzyme did not show any immunological relatedness with other mesophilic polygalacturonases.     

Development of strategies for the incorporation of biological pesticides into the integrated management of locusts and grasshoppers

• 1 Effective biological pesticides based on oil formulation of deuteromycete fungal spores have been developed for use against locusts and grasshoppers. The isolate IMI 330189 of Metarhizium anisopliae (flavoviride) var. acridum has been registered, extensively field tested and its operating characteristics explored. It should form an powerful component technology in the integrated management of locust and grasshopper pests.
• 2 The particular advantages of Metarhizium anisopliae were found to be efficacy and persistence, low vertebrate toxicity, little environmental impact, conservation of natural enemies and potential for recycling. Additional socio-economic advantages include the possibility of local production, ease of disposal and versatility in use. The principal disadvantages relate to operating characteristics such as slower speed of kill and slightly greater lability in storage than chemical pesticides.
• 3 Strategies are being developed to integrate biological control agents into locust and grasshopper management schemes; for Metarhizium the accent is placed on: (i) treating the pest before it invades crops and (ii) situations with a high premium on environmental issues.
• 4 For some pest situations, fast-acting chemical pesticides will still be necessary for crop protection.
• 5 A cheaper biological agent, such as Nosema locustae, with the capacity to persist in the pest insect population would be useful. Research is recommended on the long-term impact of Nosema in Africa.
• 6 An evaluation of the utility of the manual destruction of egg pods leads to the conclusion that we should consider the possibility of importing egg parasitoids, such as Scelio parvicornis from Australia, into Africa.
• 7 Further development work is needed to clarify the economics and politics of locust and grasshopper control; to improve the regulatory framework for biopesticides; to inform key decision makers of the availability and potential of Metarhizium; and to implement the bio-intensive IPM strategies described.

     

Testing the association between residential fungus and health using ergosterol measures and cough recordings

Questionnaire surveys in several countries have consistently detected an association between symptoms and residential mould growth. Confirmation by objective measures would strengthen the argument for causality. To address this issue, quantitative and qualitative fungal measures (airborne ergosterol and viable fungi in dust) were compared to respiratory symptoms (n = 403) and nocturnal cough recordings (n = 145) in Canadian elementary schoolchildren during the winter of 1993–1994. There was a 25 percent to 50 percent relative increase in symptom prevalence when mould was reported to be present (p < 0.05). However, neither symptoms nor recorded cough was related to objective measures of mould. In conclusion, the inability to find an association between objective measures of fungus and health suggest that either these objective measures, or the traditionally used questionnaire data are inaccurate. This discrepancy limits the acceptance of a causal relation between indoor fungal growth and illness.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effect of the nitrogen source on caffeine degradation by Aspergillus tamarii

AIMS: To evaluate caffeine degradation and nitrogen requirements during Aspergillus tamarii growth in submerged culture. METHODS AND RESULTS: Aspergillus tamarii spores produced on a coffee infusion agar medium added with sucrose were used. Several caffeine and ammonium sulphate concentrations (0-1 and 0-1.36 g l-1, respectively) were tested simultaneously on fungal biomass production and caffeine degradation. An additional caffeine pulse (4 g l-1) was added for all experiments after 48 h of fermentation. Results revealed that when using 0.90 g l-1 of caffeine and 0.14 g l-1 of ammonium sulphate, biomass production and caffeine degradation were enhanced. Highest biomass production (Xmax = 9.87 g l-1) with a specific growth rate (micro) of 0.073 h-1 and caffeine degradation rate of 0.033 g l-1 h-1, was observed under these conditions. CONCLUSIONS: Caffeine degradation as well as biomass production were characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: These studies set the stage for future characterization studies of intracellular enzymes involved in caffeine degradation. Moreover, results observed may help in the biotreatment of residues from the coffee agroindustry.     

Iron gathering by zoopathogenic fungi

Iron is a metal required by most microorganisms and is prominently used in the transfer of electrons during metabolism. The gathering of iron is, then, an essential process and its fulfillment becomes a crucial pathogenetic event for zoopathogenic fungi. Iron is rather unavailable because it occurs on the earth's surface in its insoluble ferric form in oxides and hydroxides. In the infected host iron is bound to proteins such as transferrin and ferritin. Solubilization of ferric iron is the major problem confronting microorganisms. This process is achieved by two major mechanisms: ferric reduction and siderophore utilization. Ferric reductase is frequently accompanied by a copper oxidase transport system. There is one example of direct ferric iron transport apparently without prior reduction. Ferric reduction may also be accomplished by low molecular mass compounds. Some fungi have evolved a process of iron acquisition involving the synthesis of iron-gathering compounds called siderophores. Even those fungi that do not synthesize siderophores have developed permeases for transport of such compounds formed by other organisms. Fungi can also reductively release iron from siderophores and transport the ferrous iron often by the copper oxidase transport system. There is a great diversity of iron-gathering mechanisms expressed by pathogenic fungi and such diversity may be found even in a single species.     

Element distribution in mycorrhizal and nonmycorrhizal roots of the halophyte Aster tripolium determined by proton induced X-ray emission

Summary. The salt aster (Aster tripolium L.) colonized by the arbuscular mycorrhizal fungus Glomus intraradices Sy167 and noncolonized control plants were grown in a greenhouse for nine months with regular fertilization by Hoagland nutrient solution supplemented with 2% NaCl. Mycorrhizal roots showed a high degree of mycorrhizal colonization of 60–70% and formed approximately 25% more dry weight and much less aerenchyma than the nonmycorrhizal controls. Cryosectioning essentially preserved the root cell structures and apparently did not cause significant ion movements within the roots during cuttings. The experimental conditions, however, did not allow to discriminate between fungal and plant structures within the roots. Quantification of proton-induced X-ray emission (PIXE) data revealed that in control roots, Na⁺ was mainly concentrated in the outer epidermal and exodermal cells, whereas the Cl^– concentration was about the same in all cells of the roots. Cross sections of roots colonized by the mycorrhizal fungus did not show this Na1 gradient in the concentration from outside to inside but contained a much higher percentage of NaCl among the elements determined than the controls. PIXE images are also presented for the four other elements K, P, S, and Ca. Both in colonized and control roots, the concentration of potassium was high, probably for maintaining homoeostasis under salt stress. This is seemingly the first attempt to localize both Na⁺ and Cl^– in a plant tissue by a biophysical method and also demonstrates the usefulness of PIXE analysis for such kind of investigation.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.     

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.     

Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used.