A functional gltB gene is essential for utilization of acidic amino acids and expression of periplasmic glutaminase/asparaginase (PGA) by Pseudomonas putida KT2440.
Pseudomonas putida KT2440, a root-colonizing fluorescent pseudomonad, is capable of utilizing acidic amino acids (Asp and Glu) and their amides (Asn and Gln) as its sole source of carbon and nitrogen. The uptake of Gln and Asn is facilitated by a periplasmic glutaminase/asparaginase (PGA), which hydrolyses Asn and Gln to the respective dicarboxylates. Here, we describe transposon mutagenesis of P. putida KT2440 with a self-cloning promoter probe vector, Tn 5-OT182. Transconjugants defective in Glu-mediated PGA induction were selected for further studies. In most clones the transposon was found to have integrated into the gltB gene, which encodes the major subunit of the glutamate synthase (GOGAT). The transconjugants were nonmotile, no longer showed a chemotactic response towards amino acids, and could not survive prolonged periods of starvation. The acidic amino acids and their amides supported growth of the transconjugants only when supplied together with glucose, suggesting that the gltB-mutants had lost the ability to utilize amino acids as a carbon source. To confirm that gltB inactivation was the cause of this phenotype, we constructed a mutant with a targeted disruption of gltB. This strain behaved like the clones obtained by random mutagenesis, and failed to express not only PGA but also a number of other Glu-induced proteins. In contrast to wild-type cells, the gltB(-) strain accumulated considerable amounts of both Glu and Gln during long-term incubation.
Sonawane
A M
AM
Institute of Physiological Chemistry, Philipps University, 35033 Marburg, Germany. avinash@mailer.uni-marburg.de
Röhm
K H
KH
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D024342:Amino Acids, Acidic / Q000378:metabolism*; D001483:Base Sequence; D002455:Cell Division; D002633:Chemotaxis; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D009068:Movement; D016254:Mutagenesis, Insertional; D010641:Phenotype; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*
D024342:Amino Acids, Acidic; D004269:DNA, Bacterial; D000581:Amidohydrolases; C020654:glutamin-(asparagin-)ase