Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N^δ-cis-anhydromevalonyl-N^δ-hydroxy-l-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.     

TLR4 recognizes Pseudallescheria boydii conidia and purified rhamnomannans

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.     

Diversity and biotechnological potential of the sponge-associated microbial consortia

Sponges are well known to harbor diverse microbes and represent a significant source of bioactive natural compounds derived from the marine environment. Recent studies of the microbial communities of marine sponges have uncovered previously undescribed species and an array of new chemical compounds. In contrast to natural compounds, studies on enzymes with biotechnological potential from microbes associated with sponges are rare although enzymes with novel activities that have potential medical and biotechnological applications have been identified from sponges and microbes associated with sponges. Both bacteria and fungi have been isolated from a wide range of marine sponge, but the diversity and symbiotic relationship of bacteria has been studied to a greater extent than that of fungi isolated from sponges. Molecular methods (e.g., rDNA, DGGE, and FISH) have revealed a great diversity of the unculturable bacteria and archaea. Metagenomic approaches have identified interesting metabolic pathways responsible for the production of natural compounds and may provide a new avenue to explore the microbial diversity and biotechnological potential of marine sponges. In addition, other eukaryotic organisms such as diatoms and unicellular algae from marine sponges are also being described using these molecular techniques. Many natural compounds derived from sponges are suspected to be of bacterial origin, but only a few studies have provided convincing evidence for symbiotic producers in sponges. Microbes in sponges exist in different associations with sponges including the true symbiosis. Fungi derived from marine sponges represent the single most prolific source of diverse bioactive marine fungal compounds found to date. There is a developing interest in determining the true diversity of fungi present in marine sponges and the nature of the association. Molecular methods will allow scientists to more accurately identify fungal species and determine actual diversity of sponge-associated fungi. This is especially important as greater cooperation between bacteriologists, mycologists, natural product chemists, and bioengineers is needed to provide a well-coordinated effort in studying the diversity, ecology, physiology, and association between bacteria, fungi, and other organisms present in marine sponges.     

What is the Fungal Diversity of Marine Ecosystems in Europe?

In 2001 the European Register of Marine Species 1.0 was published (Costello et al. 2001 and http://erms.biol.soton.ac.uk/, and latterly: http://www.marbef.org/data/stats.php) [Costello MJ, Emblow C, White R, 2001. European register of marine species: a check list of the marine species in Europe and a bibliography of guides to their identification. Collection Patrimoines Naturels50, 463p.]. The lists of species (from fungi to mammals) were published as part of a European Union Concerted action project (funded by the European Union Marine Science and Technology (MAST) research programme) and the updated version (ERMS 2) is EU-funded through the Marine Biodiversity and Ecosystem Functioning (MARBEF) Framework project 6 Network of Excellence. Among these lists, a list of the fungi isolated and identified from coastal and marine ecosystems in Europe was included (Clipson et al. 2001) [Clipson NJW, Landy ET, Otte ML, 2001. Fungi. In@ Costelloe MJ, Emblow C, White R (eds), European register of marine species: a check-list of the marine species in Europe and a bibliography of guides to their identification. Collection Patrimoines Naturels50: 15-19.]. This article deals with the results of compiling a new taxonomically correct and complete list of all fungi that have been reported occurring in European marine waters.     

Antagonisms, mutualisms and commensalisms affect outbreak dynamics of the southern pine beetle

Feedback from community interactions involving mutualisms are a rarely explored mechanism for generating complex population dynamics. We examined the effects of two linked mutualisms on the population dynamics of a beetle that exhibits outbreak dynamics. One mutualism involves an obligate association between the bark beetle, Dendroctonus frontalis and two mycangial fungi. The second mutualism involves Tarsonemus mites that are phoretic on D. frontalis (“commensal”), and a blue-staining fungus, Ophiostoma minus. The presence of O. minus reduces beetle larval survival (“antagonistic”) by outcompeting beetle-mutualistic fungi within trees yet supports mite populations by acting as a nutritional mutualist. These linked interactions potentially create an interaction system with the form of an endogenous negative feedback loop. We address four hypotheses: (1) Direct negative feedback: Beetles directly increase the abundance of O. minus, which reduces per capita reproduction of beetles. (2) Indirect negative feedback: Beetles indirectly increase mite abundance, which increases O. minus, which decreases beetle reproduction. (3) The effect of O. minus on beetles depends on mites, but mite abundance is independent of beetle abundance. (4) The effect of O. minus on beetles is independent of beetle and mite abundance. High Tarsonemus and O. minus abundances were strongly correlated with the decline and eventual local extinction of beetle populations. Manipulation experiments revealed strong negative effects of O. minus on beetles, but falsified the hypothesis that horizontal transmission of O. minus generates negative feedback. Surveys of beetle populations revealed that reproductive rates of Tarsonemus, O. minus, and beetles covaried in a manner consistent with strong indirect interactions between organisms. Co-occurrence of mutualisms embedded within a community may have stabilizing effects if both mutualisms limit each other. However, delays and/or non-linearities in the interaction systems may result in large population fluctuations. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Biosynthesis of photodynamically active rubellins and structure elucidation of new anthraquinone derivatives produced by Ramularia collo-cygni

Here we present the first isolation of the anthrachinone derivative rubellin A out of mycelium and culture filtrate of Ramularia collo-cygni. Furthermore, two compounds, rubellin E and 14-dehydro rubellin D were isolated and their structures elucidated. In comparison to the other rubellins, rubellin A shows increased photodynamic oxygen activation. By incorporating both [1-(13)C]-acetate and [2-(13)C]-acetate into the rubellins, we showed that such anthraquinone derivatives were biosynthesised via the polyketide pathway. The labelling pattern after being fed [U-(13)C(6)]-glucose proved the existence of fungal folding mode of the poly-beta-keto chain. The ability to produce rubellins is not limited to R. collo-cygni in the anamorph genus Ramularia.     

The current and future applications of microorganism in the bioremediation of cyanide contamination

Inorganic cyanide and nitrile compounds are distributed widely in the environment, chiefly as a result of anthropogenic activity but also through cyanide synthesis by a range of organisms including higher plants, fungi and bacteria. The major source of cyanide in soil and water is through the discharge of effluents containing a variety of inorganic cyanide and nitriles. Here the fate of cyanide compounds in soil and water is reviewed, identifying those factors that affect their persistence and which determine whether they are amenable to biological degradation. The exploitation of cyanides by a variety of taxa, as a mechanism to avoid predation or to inhibit competitors has led to the evolution in many organisms of enzymes that catalyse degradation of a range of cyanide compounds. Microorganisms expressing pathways involved in cyanide degradation are briefly reviewed and the current applications of bacteria and fungi in the biodegradation of cyanide contamination in the field are discussed. Finally, recent advances that offer an insight into the potential of microbial systems for the bioremediation of cyanide compounds under a range of environmental conditions are identified, and the future potential of these technologies for the treatment of cyanide pollution is discussed.     

Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans.