RESUMEN
An oxalate-fermenting brown rot fungus, Fomitopsis palustris, secretes large amounts of oxalic acid during wood decay. Secretion of oxalic acid is indispensable for the degradation of wood cell walls, but almost nothing is known about the transport mechanism by which oxalic acid is secreted from F. palustris hyphal cells. We characterized the mechanism for oxalate transport using membrane vesicles of F. palustris. Oxalate transport in F. palustris was ATP dependent and was strongly inhibited by several inhibitors, such as valinomycin and NH(4)(+), suggesting the presence of a secondary oxalate transporter in this fungus. We then isolated a cDNA, FpOAR (Fomitopsis palustris oxalic acid resistance), from F. palustris by functional screening of yeast transformants with cDNAs grown on oxalic acid-containing plates. FpOAR is predicted to be a membrane protein that possesses six transmembrane domains but shows no similarity with known oxalate transporters. The yeast transformant possessing FpOAR (FpOAR-transformant) acquired resistance to oxalic acid and contained less oxalate than the control transformant. Biochemical analyses using membrane vesicles of the FpOAR-transformant showed that the oxalate transport property of FpOAR was consistent with that observed in membrane vesicles of F. palustris. The quantity of FpOAR transcripts was correlated with increasing oxalic acid accumulation in the culture medium and was induced when exogenous oxalate was added to the medium. These results strongly suggest that FpOAR plays an important role in wood decay by acting as a secondary transporter responsible for secretion of oxalate by F. palustris.
Asunto(s)
Coriolaceae/enzimología , Coriolaceae/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Oxalatos/metabolismo , Adenosina Trifosfato/metabolismo , Análisis por Conglomerados , Coriolaceae/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/genética , Inhibidores Enzimáticos/metabolismo , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , Compuestos de Amonio Cuaternario/metabolismo , Vesículas Secretoras/enzimología , Análisis de Secuencia de ADN , Homología de Secuencia , Valinomicina/metabolismo , Madera/metabolismo , Madera/microbiologíaRESUMEN
Nano-black lipid membranes (nano-BLMs) were obtained by functionalization of highly ordered porous alumina substrates with an average pore diameter of 60nm based on a self-assembled alkanethiol submonolayer followed by spreading of 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane on the hydrophobic substrate. By means of impedance spectroscopy, we analyzed the influence of the self-assembled alkanethiol submonolayer on the electrical properties of the nano-BLMs as well as their long-term stability. We were able to stably integrate nano-BLMs into a flow through system, which allowed us to readily exchange buffer solutions several times and accounts for mass transport phenomena. The ionophore valinomycin was successfully inserted into nano-BLMs and its transport activity monitored as a function of different potassium and sodium ion concentrations reflecting the specificity of valinomycin for potassium ions.