Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn 168 in NS1 and Asn 459 in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg 368 and Arg 87 respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn 168 /Arg 368 and Asn 459 /Arg 87 residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn 168 /Arg 368 and Asn 459 /Arg 87 pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discusse

Entomopathogenic Fungus as a Biological Control for an Important Vector of Livestock Disease: The Culicoides Biting Midge

BACKGROUND: The recent outbreak of bluetongue virus in northern Europe has led to an urgent need to identify control measures for the Culicoides (Diptera: Ceratopogonidae) biting midges that transmit it. Following successful use of the entomopathogenic fungus Metarhizium anisopliae against larval stages of biting midge Culicoides nubeculosus Meigen, we investigated the efficacy of this strain and other fungi (Beauveria bassiana, Isaria fumosorosea and Lecanicillium longisporum) as biocontrol agents against adult C. nubeculosus in laboratory and greenhouse studies. METHODOLOGY/FINDINGS: Exposure of midges to 'dry' conidia of all fungal isolates caused significant reductions in survival compared to untreated controls. Metarhizium anisopliae strain V275 was the most virulent, causing a significantly decrease in midge survival compared to all other fungal strains tested. The LT(50) value for strain V275 was 1.42 days compared to 2.21-3.22 days for the other isolates. The virulence of this strain was then further evaluated by exposing C. nubeculosus to varying doses (10(8)-10(11) conidia m(-2)) using different substrates (horse manure, damp peat, leaf litter) as a resting site. All exposed adults were found to be infected with the strain V275 four days after exposure. A further study exposed C. nubeculosus adults to 'dry' conidia and 'wet' conidia (conidia suspended in 0.03% aq. Tween 80) of strain V275 applied to damp peat and leaf litter in cages within a greenhouse. 'Dry' conidia were more effective than 'wet' conidia, causing 100% mortality after 5 days. CONCLUSION/SIGNIFICANCE: This is the first study to demonstrate that entomopathogenic fungi are potential biocontrol agents against adult Culicoides, through the application of 'dry' conidia on surfaces (e.g., manure, leaf litter, livestock) where the midges tend to rest. Subsequent conidial transmission between males and females may cause an increased level of fungi-induced mortality in midges thus reducing the incidence of disease

Microbial degradation of furanic compounds: biochemistry, genetics, and impact

Microbial metabolism of furanic compounds, especially furfural and 5-hydroxymethylfurfural (HMF), is rapidly gaining interest in the scientific community. This interest can largely be attributed to the occurrence of toxic furanic aldehydes in lignocellulosic hydrolysates. However, these compounds are also widespread in nature and in human processed foods, and are produced in industry. Although several microorganisms are known to degrade furanic compounds, the variety of species is limited mostly to Gram-negative aerobic bacteria, with a few notable exceptions. Furanic aldehydes are highly toxic to microorganisms, which have evolved a wide variety of defense mechanisms, such as the oxidation and/or reduction to the furanic alcohol and acid forms. These oxidation/reduction reactions constitute the initial steps of the biological pathways for furfural and HMF degradation. Furfural degradation proceeds via 2-furoic acid, which is metabolized to the primary intermediate 2-oxoglutarate. HMF is converted, via 2,5-furandicarboxylic acid, into 2-furoic acid. The enzymes in these HMF/furfural degradation pathways are encoded by eight hmf genes, organized in two distinct clusters in Cupriavidus basilensis HMF14. The organization of the five genes of the furfural degradation cluster is highly conserved among microorganisms capable of degrading furfural, while the three genes constituting the initial HMF degradation route are organized in a highly diverse manner. The genetic and biochemical characterization of the microbial metabolism of furanic compounds holds great promises for industrial applications such as the biodetoxifcation of lignocellulosic hydrolysates and the production of value-added compounds such as 2,5-furandicarboxylic acid

High-affinity nitrate/nitrite transporters NrtA and NrtB of aspergillus nidulans exhibit high specificity and different inhibitor sensitivity

The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a noncompetitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters