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Mutations affecting the synthesis of NADP-dependent glutamate dehydrogenase in Pseudomonas aeruginosa
C L Joannou1, P R Brown, R Tata
1Department of Biochemistry, King's College London, UK.
Abstract:
NADP-dependent glutamate dehydrogenase (NADP-GDH) was purified to homogeneity from Pseudomonas aeruginosa strain 8602 (PAC 1). The Mr determined by Sephadex gel filtration was 280,000; the subunit Mr determined by SDS-PAGE was 45,000. Mutant strains lacking NADP-GDH and glutamate synthase (Gdh-Glt-) required glutamate for growth. Transductants that lacked only NADP-GDH were indistinguishable from the wild-type strain in growth properties. It was concluded that NADP-GDH is not essential for growth of the wild-type organism and that glutamate formation via NAD-dependent glutamate dehydrogenase does not occur to a significant extent. A mutant strain, 39, producing high NADP-GDH activity, synthesized normal NADP-GDH and had the same intracellular glutamate concentrations as its parent. The mutation responsible for the synthesis of high levels of NADP-GDH was shown, by transduction, to be closely linked to the NADP-GDH structural gene (gdhA).
Insights
NADP-dependent glutamate dehydrogenase (NADP-GDH) is not essential for Pseudomonas aeruginosa growth. Glutamate synthase is required for growth when NADP-GDH is absent, indicating its crucial role.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Pseudomonas aeruginosa utilizes NADP-dependent glutamate dehydrogenase (NADP-GDH) for amino acid biosynthesis.
- The essentiality of NADP-GDH for growth and its relationship with glutamate synthase in P. aeruginosa were not fully understood.
Purpose of the Study:
- To investigate the role of NADP-GDH in Pseudomonas aeruginosa growth.
- To determine if NADP-GDH is essential for the organism's survival.
- To understand the interplay between NADP-GDH and glutamate synthase.
Main Methods:
- Purification of NADP-GDH from Pseudomonas aeruginosa strain 8602.
- Characterization of purified NADP-GDH using Sephadex gel filtration and SDS-PAGE.
- Construction and analysis of mutant strains lacking NADP-GDH and/or glutamate synthase.
- Transduction experiments to map mutations affecting NADP-GDH activity.
Main Results:
- NADP-GDH was purified, with a molecular weight of 280,000 and subunit molecular weight of 45,000.
- Mutants lacking both NADP-GDH and glutamate synthase required glutamate for growth.
- Transductants lacking only NADP-GDH exhibited wild-type growth properties.
- A mutant with high NADP-GDH activity synthesized normal enzyme and maintained similar intracellular glutamate levels.
Conclusions:
- NADP-dependent glutamate dehydrogenase is not essential for the growth of wild-type Pseudomonas aeruginosa.
- Glutamate formation via NAD-dependent glutamate dehydrogenase is not significant in this organism.
- Glutamate synthase plays a critical role in glutamate biosynthesis when NADP-GDH is absent.
- The mutation leading to high NADP-GDH expression is linked to the gdhA structural gene.