Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245

The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are ve ry similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacte r sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate re ductase assays with the artificial electron donors benzyl viologen and meth yl viologen, and assays with periplasmic cell-free extracts, it was conclud ed that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was ex pressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mu tant still expressed assimilatory nitrate reductase activity, but was devoi d of its periplasmic dissimilatory nitrate reductase activity.