Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP-transport system

Abstract

In this study, we investigated an SBP (DctPAm) of a tripartite ATP-independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7T. Deletion of dctPAm as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7T, if cultivated on mineral salt medium supplemented with d-glucose, d-galactose, l-arabinose, d-fucose, d-xylose or d-gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctPAm using the broad host vector pBBR1MCS-5::dctPAm. Furthermore, an uptake assay with radiolabeled [14C(U)]-d-glucose clearly showed that the deletion of dctPAm, dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of KD performing thermal shift assays showed a shift in the melting temperature of DctPAm in the presence of d-gluconic acid (KD 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above-mentioned carbohydrates d-galactonate (KD 10.72 ± 1.4 µM), d-fuconic acid (KD 13.50 ± 1.6 µM) and d-xylonic acid (KD 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.