Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate...

An electron capture gas-chromatographic technique was developed to continuously measure nitrate (NO3-) reduction during in vitro complementation tests with extracts from Pseudomonas aeruginosa mutants deficient in both assimilatory and dissimilatory nitrate reduction as a result of a single genetic mutation. The procedure involves coupling nitrate reduction to nitrous oxide (N2O) evolution via a series of reactions specific to the denitrification pathway. The assay was dependent on nitrate concentration, and optimal activity was obtained with a final concentration of 0.2% potassium nitrate. The reduction exhibited a stoichiometry of 2:1 (NO3-/N2O), and succinate was the best electron source for the reaction, followed by glucose, pyruvate, and malate. The technique can also be used for continuously monitoring nitrate reduction. The optimal nitrite concentration in the nitrite reductase assay was 0.025%. The initial complementation studies of mutant extracts demonstrated that at least two genes are shared between the two nitrate reduction pathways in P. aeruginosa.