title: Characterization of membrane-bound dehydrogenases from Gluconobacter oxydans 621H via whole-cell activity assays using multideletion strains.
abstract: Gluconobacter oxydans, like all acetic acid bacteria, has several membrane-bound dehydrogenases, which oxidize a multitude of alcohols and polyols in a stereo- and regio-selective manner. Many membrane-bound dehydrogenases have been purified from various acetic acid bacteria, but in most cases without reporting associated sequence information. We constructed clean deletions of all membrane-bound dehydrogenases in G. oxydans 621H and investigated the resulting changes in carbon utilization and physiology of the organism during growth on fructose, mannitol, and glucose. Furthermore, we studied the substrate oxidation spectra of a set of strains where the membrane-bound dehydrogenases were consecutively deleted using a newly developed whole-cell 2,6-dichlorophenolindophenol (DCPIP) activity assay in microtiter plates. This allowed a detailed and comprehensive in vivo characterization of each membrane-bound dehydrogenase in terms of substrate specificity. The assays revealed that general rules can be established for some of the enzymes and extended the known substrate spectra of some enzymes. It was also possible to assign proteins whose purification and characterization had been reported previously, to their corresponding genes. Our data demonstrate that there are less membrane-bound dehydrogenases in G. oxydans 621H than expected and that the deletion of all of them is not lethal for the organism.
authors.affiliation: Lehrstuhl für Mikrobiologie, Technische Universität München, Emil-Ramann-Strasse 4, 85354 Freising, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Peters
authors.forename: Markus
authors.initials: M
authors.lastname: Mientus
authors.forename: David
authors.initials: D
authors.lastname: Kostner
authors.forename: Anja
authors.initials: A
authors.lastname: Junker
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Liebl
authors.forename: Armin
authors.initials: A
authors.lastname: Ehrenreich
mesh_terms: D015086:2,6-Dichloroindophenol / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002244:Carbon; D015086:2,6-Dichloroindophenol; D010088:Oxidoreductases