Journal article
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 KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442.
Genetic evidence as well as sequence analyses of the DNA regions flanking nasB suggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction of nasB 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 glutamate synthase.
A detailed physiological characterization of the gltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.
Language: | English |
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Publisher: | American Society for Microbiology |
Year: | 2000 |
Pages: | 3368-3376 |
ISSN: | 10985530 , 00219193 and 10678832 |
Types: | Journal article |
DOI: | 10.1128/JB.182.12.3368-3376.2000 |
ORCIDs: | Sternberg, Claus , Kilstrup, Mogens and Molin, Søren |
Amino Acid Sequence DNA Transposable Elements Electrophoresis, Gel, Two-Dimensional Enzyme Activation Gene Expression Regulation, Bacterial Glutamate Synthase Luciferases Molecular Sequence Data Mutagenesis, Insertional Nitrate Reductase Nitrate Reductases Nitrates Nitrogen Operon Pseudomonas putida