Premium
Genetic inactivation of the H + ‐translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans is facilitated by insertion of the ndh gene from Escherichia coli
Author(s) -
Finel Moshe
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00831-9
Subject(s) - paracoccus denitrificans , escherichia coli , oxidoreductase , chemistry , biochemistry , gene , biology , enzyme
The H + ‐translocating NADH:ubiquinone oxidoreductase (NDH1) is probably an obligatory enzyme in Paracoccus denitrificans and disruption of its genes may be lethal to this organism. In order to overcome this problem and delete the nqo8 and nqo9 genes of NDH1, it was necessary to render the enzyme non‐essential. This was achieved by constructing a deletion plasmid in which most of the coding regions of nqo8 and nqo9 were replaced by the ndh gene of Escherichia coli that encodes an alternative NADH:ubiquinone oxidoreductase (NDH2), and a kanamycin resistance gene. Subsequent homologous recombination gave rise to a mutant the membranes of which catalyzed rotenone‐insensitive NADH oxidation, but which did not oxidize deamino‐NADH. Hence, this mutant expressed active and membrane‐bound NDH2, and lacked NDH1 activity.