Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates.

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solani AG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicillium strains with R. solani prepared in sterilized soil and when the Penicillium strains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicillium species against R. solani is discussed.

Enzymatic synthesis and hydrolysis of xylogluco-oligosaccharides using the first archaeal alpha-xylosidase from Sulfolobus solfataricus.

The first, recently identified, archaeal alpha-xylosidase from Sulfolobus solfataricus (XylS) shows high specificity for hydrolysis of isoprimeverose [alpha-D-xylopyranosyl-(1,6)-D-glucopyranose, (X)], the p-nitrophenyl-beta derivative of isoprimeverose, and xyloglucan oligosaccharides and has transxylosidic activity, forming, in a retaining mode, interesting alpha-xylosides. This article describes the synthesis of isoprimeverose, the disaccharidic repeating unit of xyloglucan, of the p-nitrophenyl-beta derivative of isoprimeverose, and of a trisaccharide based on isoprimeverose that is one of the trisaccharidic building blocks of xyloglucan. A substrate structure-activity relationship is recognized for both the hydrolysis and the synthesis reactions of XylS, it being a biocatalyst (i) active hydrolytically only on X-ending substrates liberating a xylose molecule and (ii) capable of transferring xylose only on the nonreducing end glucose of p-nitrophenyl-(PNP)-beta-D-cellobioside. The compounds synthesized by this enzyme are a starting point for enzymological studies of other new enzymes (i.e., xyloglucanases) for which suitable substrates are difficult to synthesize. This study also allows us to define the chemical characteristics of the xylose-transferring activity of this new archaeal enzyme, contributing to building up a library of different glycosidases with high specific selectivity for oligosaccharide synthesis.

Enzymatic synthesis of oligosaccharides by two glycosyl hydrolases of Sulfolobus solfataricus.

The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides. In this respect, the enzymological characteristics of the catalysts, in term of regioselectivity, substrate specificity, and operational stability, are of fundamental importance to improve the yields of the process and to widen the repertoire of the available products. Here, two methods of oligosaccharide synthesis performed by a glycosynthase and by an alpha-xylosidase from the hyperthermophilic archaeon Sulfolobus solfataricus are briefly reviewed. The approaches used and the biodiversity of the catalysts together are key features for their possible utilization in the synthesis of oligosaccharides.

Identification and molecular characterization of the first alpha -xylosidase from an archaeon.

We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.

A novel thermophilic glycosynthase that effects branching glycosylation.

A novel thermophilic glycosynthase that effects branching glycosylation has been obtained by mutation of the nucleophile in the active site of the glycosidase from Sulfolobus solfataricus. Two methods for the use of this mutant are reported.

Restoration of the activity of active-site mutants of the hyperthermophilic beta-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles.

The beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of the reaction, and Glu206, which acts as the general acid/base catalyst. The activities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly were restored by externally added nucleophiles. Sodium azide and sodium formate were used as external nucleophiles and the products of their reaction were characterized. Glu387Ala/Gly mutants were reactivated with 2, 4-DNP-beta-Glc substrate and the Glu387Gly mutant showed recovered activity, with the same nucleophiles, also on 2-NP-beta-Glc. The reaction catalyzed by the Glu387Gly mutant proceeded differently depending on the type of externally added nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, thereby yielding an inverting glycosidase. Sodium formate promoted the opposite behavior (retaining) in the mutant, producing 3-O-beta-linked disaccharide derivative of the substrates. A possible role of sodium formate as a biomimicking agent in replacing the natural nucleophile Glu387 is also discussed.

Enzymatic synthesis of 2-beta-D-galactopyranosyloxy ethyl methacrylate (GalEMA) by the thermophilic archeon Sulfolobus solfataricus.

beta-Glycosidases catalyze the synthesis of glycosides when a nucleophilic acceptor other than water is present in the reaction medium. We describe the enzymatyc synthesis of 2-beta-D-galactopyranosyloxyethyl methacrylate (GalEMA) starting from 2-hydroxyethyl methacrylate (HEMA) and p-nitrophenyl-beta-D-galactopyranoside (pNPG) using a beta-glycosidase activity present in the thermophilic archaeon Sulfolobus solfataricus. This thermophilic enzyme catalyzes the transfer of the galactopyranosyl unit from pNPG to the HEMA hydroxyl group by the formation of a new beta-glycosidic bond. The conditions of the biocatalytic system have been optimized to obtain high yield of GalEMA. (c) 1996 John Wiley & Sons, Inc.

Calcium-induced interaction and fusion of archaeobacterial lipid vesicles: a fluorescence study.

The lipids extracted from the membrane of the thermophilic archaeobacterium Sulfolobus solfataricus have an unusual bipolar structure. Each molecule is formed by two isoprenoid chains (with up to four cyclopentane groups per chain) ether-linked at both ends to glycerol or nonitol groups. These groups can be variably substituted, mainly with complex sugars. Fluorescence resonance energy transfer, aqueous contents mixing and calcein release assays were employed to assess whether bipolar lipid vesicles were able to undergo a calcium-induced fusion process. The possibility of getting fusion depends strongly on the phase behaviour of the lipids. With vesicles formed by the natural polar lipid extract (PLE), a mixture showing a complex polymorphic behaviour, the fusion process was observed above the temperature T congruent to 60 degrees C at 15 mM Ca2+. By contrast, no fusion was observed in vesicles of P2, a fraction displaying only the lamellar phase. A dramatic change of the fusion process was observed when egg PC or P2 was added to PLE. In this case only lipid mixing, but not a real fusion process occurred at T > or = 60 degrees C. The dependence of such a process on ionic conditions has also been studied. Additional experiments involving surface tension measurements on monolayers have been performed to assess the importance of a surface tension increase to get fusion. In contrast to other monopolar lipid systems, no detectable change in surface tension has been observed in our bipolar lipids even in cases in which the fusion process is present.

The glycolipid of Halobacterium trapanicum.

The structural elucidation of the polar lipids in Halobacterium trapanicum is reported with particular emphasis on a new sulfated disaccharide derivative of 2,3-di-O-phytanyl-sn-glycerol. The full structural designation of this glycolipid is 2,3-di-O-phytanyl-1-O- (mannopyranosyl-(2-sulfate)-alpha-D-1-2-glucopyranosyl-alpha-D)-sn-glyce rol. The value of glycolipid structures in the taxonomy of halophilic Archaea is also discussed.

Pyrobaculum aerophilum sp. nov., a novel nitrate-reducing hyperthermophilic archaeum.

A novel rod-shaped hyperthermophilic archaeum has been isolated from a boiling marine water hole at Maronti Beach, Ischia, Italy. It grew optimally at 100 degrees C and pH 7.0 by aerobic respiration as well as by dissimilatory nitrate reduction, forming dinitrogen as a final product. Organic and inorganic compounds served as substrates during aerobic and anaerobic respiration. Growth was inhibited by elemental sulfur. The cell wall was composed of a surface layer of hexameric protein complexes arranged on a p6 lattice. The core lipids consisted mainly of glycerol diphytanyl glycerol tetraethers with various degrees of cyclization. The G+C content was 52 mol%. The new isolate resembled members of the genera Thermoproteus and Pyrobaculum by its ability to form characteristic terminal spherical bodies ("golf clubs"). On the basis of its 16S rRNA sequence, the new isolate exhibited a close relationship to the genus Pyrobaculum. It is described as a new species, which we name Pyrobaculum aerophilum (type strain: IM2; DSM 7523).

Glycolipids from Thermotoga maritima, a hyperthermophilic microorganism belonging to Bacteria domain.

Two novel glycolipids with a very rare alpha(1-->4) diglucosyl structure have been isolated from the thermophilic bacterium Thermotoga maritima. The structures of these compounds, on the basis of chemical procedures and spectroscopic studies (FAB-MS and NMR), were shown to be: 1(3),2-dipalmitoyl-3(1)-[glucopyranosyl-(6-decanoyl)-alpha-D-(1-->4)- glucopyranosyl-alpha-D]-glycerol (Glycolipid 1) and 1(3),2-dipalmitoyl-3(1)-[glucopyranosyl-alpha-D-(1-->4)-glucopyranosyl- alpha-D]-glycerol (Glycolipid 2).

Determination of hydride transfer stereospecificity of NADH-dependent alcohol-aldehyde/ketone oxidoreductase from Sulfolobus solfataricus.

This paper describes the determination of stereospecificity of hydride transfer reaction of an alcohol dehydrogenase isolated from the archaebacterium Sulfolobus solfataricus. The 1H-NMR and EI-MS data indicate that the enzyme transfers the pro-R hydrogen from coenzyme to substrate and is therefore an A-specific dehydrogenase.

Asymmetric reduction of ketones with resting cells of Sulfolobus solfataricus.

The method of resting cells has been of interest in the development of biocatalysts applied to organic reactions.This article deals with the use of resting cells of a thermophilic archaebacterium Sulfolobus solfataricus, in the asymmetric reduction of acyclic, cyclic, and aromatic ketones. The system allows the continuous regeneration of endogenous coenzyme with the coupled substrate approach. The results indicate that the direction of hydride attack was equatorial on the re face of the carbonyl group of substrates producing (S)-alcohols with a good optical yield. A convenient system for the reuse of resting cells has been set out to synthesize (S)-alcohols on a preparative scale.

Calditol tetraether lipids of the archaebacterium Sulfolobus solfataricus. Biosynthetic studies.

Lipids from the archaebacterium Sulfolobus solfataricus are based on 72-membered macrocyclic tetraethers made up from two C40 diol units differently cyclized and either two glycerol moieties or one glycerol moiety and a unique branched-chain nonitol named calditol (glycerodialkylnonitol tetraethers, GDNTs). To elucidate the biosynthesis of calditol and related tetraethers, labelled precursors, [U-14C,1(3)-3H]glycerol, [U-14C,2-3H]glycerol, D-[1-14C,6-3H]glucose, D-[6-14C,1-3H]glucose, D-[1-14C,2-3H]glucose, D-[1-14C,6-3H]fructose and D-[1-14C]galactose, were fed to S. solfataricus. Without regard to stereochemistry or phosphorylation, incorporation experiments provided evidence that the biosynthesis of calditol occurs via an aldolic condensation between dihydroxyacetone and fructose, through a 2-oxo derivative of calditol as an intermediate. The latter is in turn reduced and then alkylated to yield the GDNTs. The biogenetic origins of both glycerol and C40 isoprenoid moieties of GDNTs are also discussed.

A simple chromatographic procedure for the detection of cyclized archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids.

Archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40, C40 lipid by TLC. The core lipids of examples from the genera Methanobacterium, Methanobrevibacter, Methanogenium and Methanoplanus did not contain cyclized forms of glycerol-bisdiphytanyl-glycerol tetraethers, whereas the core lipids of Methanosarcina barkeri contained glycerol-bisdiphytanyl-glycerol tetraethers with from one to three cyclopentane rings in each C40 isopranoid chain.

1H and 13C NMR assignment of benzothiophenquinones from the sulfur-oxidizing archaebacterium Sulfolobus solfataricus.

From Sulfolobus solfataricus, a sulfur-oxidizing thermophilic member of archaebacteria, three unusual benzothiophenquinones were isolated. Detailed NMR studies on these quinones, including multipulse mono-dimensional and two-dimensional techniques, were performed to obtain carbon and proton assignments, one-bond, geminal and vicinal coupling constants and T1 relaxation times. This report extends the known quinone composition of thermophilic archaebacteria and further supports the concept that these biomolecules can serve as a useful chemotaxonomic tool.