Inactivation of gltB abolishes expression of the assimilatory nitrate reductase gene (nasB) in Pseudomonas putida KT2442

By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase, luxAB, to genes of Pseudomonas putida K T2442 were generated. Insertion mutants that responded to ammonium deficien cy by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the tra nsposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions fl anking nasB suggest that the genes required for nitrate assimilation are no t clustered. We isolated three second-site mutants in which induction of na sB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glut amate synthase. A detailed physiological characterization of the gltB mutan ts revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation protei ns, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.