Date of Award

12-15-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Chung-Dar Lu

Second Advisor

Zehava Eichenbaum

Third Advisor

Irene Weber

Abstract

Pseudomonas aeruginosa can grow efficiently on spermine and other biogenic polyamines via the γ-glutamylpolyamine synthetase (GPS) pathway. Not only subjected to growth inhibition by spermine, the pauA2 mutant without a functional γ-glutamylpolyamine synthetase PauA2 became more sensitive to β-lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, the native form of PauA2 protein overexpressed in E. coli was purified to homogeneity for biochemical characterization. The specific activity of PauA2 was monitored by spectrophotometric measurements (660 nm) of the releasing phosphate from ATP in the presence of ammonium molybdate and malachite green. PauA2 displayed a sigmoid curve on Velocity-Concentration plot, indicating an allosteric modulation in the catalytic reaction. The apparent Km values were 0.2mM, 2.1mM, 6.1mM, and 1.1mM for ATP, L-glutamate, spermidine, and spermine, respectively. The obtained values of Hill coefficient were 3.4 and 5.4 for spermidine and spermine, respectively.

Although P. aeruginosa can degrade the spermine by PauA2, it seems likely that other mechanisms may alleviate the spermine toxicity in the absence of pauA2. All the pauA2 suppressors were isolated from spermine selection plates and shared common changes in various pathways including delayed growth rate, retarded swarming motility, and pyocyanin overproduction. Genome resequencing of a representative suppressor revealed a unique C599T mutation at the phoU gene that results in Ser200Leu substitution and a constitutive expression of the Pho regulon as evidenced by measurements of promotor activities and transcriptome analysis. All of the observed phenotypes could be complemented by a recombinant plasmid carrying the wild-type phoU gene. Also, accumulation of polyphosphate granules and spermine resistance in the suppressor mutant were reversed concomitantly when exopolyphosphatase PPX was overexpressed from a recombinant plasmid. Identical phenotypes were also observed in a ΔpauA2ΔphoU double knockout mutant.

In conclusion, we characterized the γ-glutamylspermine synthetase PauA2 as the essential enzyme and provide the foundations for PauA2 inhibitors screening as a potential antibacterial. Furthermore, we identified polyphosphate accumulation as a potential protection mechanism against spermine toxicity in P. aeruginosa.

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