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The effect of a non-metabolizable analog on mandelate catabolism in Pseudomonas putida
Abstract:
DL-2,3,4,5,6-pentafluoromandelic acid (PFM) specifically inhibits the growth of Pseudomonas putida (ATCC 12633) on medium containing mandelate as sole carbon and energy source by competitive inhibition of mandelate dehydrogenase. PFM is not metabolized and is neither an inducer of the mandelate catabolic enzymes nor an antagonist of induction. Mutants resistant to the inhibitory effects of PFM (PFMr) were isolated; most prove to be superinducible, i.e. synthesize corrdinately the mandelate-specific catabolic enzymes at elevated levels following induction. In at least one case the PFMr mutation maps very near the structural genes that encode the enzymes functional in the first two steps of mandelate catabolism. It is reasoned that the PFMr mutation is of the promotor type. Resistance to substrate analogs such as PFM offers a general method for isolation of regulatory mutants in catabolic metabolism.
Insights
DL-2,3,4,5,6-pentafluoromandelic acid (PFM) inhibits Pseudomonas putida growth by blocking mandelate dehydrogenase. This study identifies a method to isolate regulatory mutants in catabolic metabolism using PFM resistance.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Pseudomonas putida utilizes mandelate as a carbon source.
- Mandelate metabolism involves specific catabolic enzymes.
- Understanding enzyme regulation is crucial for microbial physiology.
Purpose of the Study:
- To investigate the inhibitory mechanism of DL-2,3,4,5,6-pentafluoromandelic acid (PFM) on Pseudomonas putida.
- To isolate and characterize mutants resistant to PFM (PFMr).
- To explore the utility of PFM resistance as a method for isolating regulatory mutants.
Main Methods:
- Competitive inhibition assays using PFM and mandelate.
- Isolation and genetic mapping of PFM-resistant mutants (PFMr).
- Analysis of enzyme induction and regulation in wild-type and mutant strains.
Main Results:
- PFM specifically inhibits Pseudomonas putida growth by competitively inhibiting mandelate dehydrogenase.
- PFM is not metabolized and does not affect enzyme induction.
- Most isolated PFMr mutants were superinducible, showing elevated enzyme synthesis.
- PFMr mutations mapped near the structural genes, suggesting a promoter-type mutation.
Conclusions:
- PFM is a potent inhibitor of mandelate dehydrogenase in Pseudomonas putida.
- PFM resistance is a valuable tool for isolating regulatory mutants in microbial catabolism.
- The PFMr mutation likely affects the promoter controlling mandelate catabolic genes.