Induction of the pho regulon and polyphosphate synthesis against spermine stress in Pseudomonas aeruginosa
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Yu-Chih Peng1, ChienYi Lu1, Guoqing Li2
1Department of Biology, Georgia State University, Atlanta, GA, 30303, USA.
Abstract:
Growth of Pseudomonas aeruginosa on spermine requires a functional γ-glutamylpolyamine synthetase PauA2. Not only subjected to growth inhibition by spermine, the pauA2 mutant became more sensitive to β-lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant, which alleviated toxicity, were isolated from selection plates containing spermine. These suppressors share common phenotypic changes including delayed growth rate, retarded swarming motility, and pyocyanin overproduction. Genome resequencing of a representative suppressor revealed a unique C599 T mutation at the phoU gene that results in Ser200 Leu substitution and a constitutive expression of the Pho regulon. Identical phenotypes were also observed in a ΔpauA2ΔphoU double knockout mutant and complemented by the wild-type phoU gene. Accumulation of polyphosphate granules and spermine resistance in the suppressor were reversed concomitantly when expressing exopolyphosphatase PPX from a recombinant plasmid, or by the introduction of deletion alleles in pstS pstC for phosphate uptake, phoB for Pho regulation, and ppk for polyphosphate synthesis. In conclusion, this study identifies polyphosphate accumulation due to an activated Pho regulon and phosphate uptake by the phoU mutation as a potential protection mechanism against spermine toxicity.
Insights
Pseudomonas aeruginosa pauA2 mutants exhibit spermine toxicity and increased antibiotic sensitivity. A phoU mutation confers resistance by activating the Pho regulon, leading to polyphosphate accumulation.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas aeruginosa requires functional gamma-glutamylpolyamine synthetase (PauA2) for growth on spermine.
- A pauA2 mutant shows increased sensitivity to beta-lactam antibiotics in human serum, highlighting PauA2's role beyond spermine metabolism.
- Identifying mechanisms of spermine resistance is crucial for understanding P. aeruginosa pathogenesis and developing therapeutic strategies.
Purpose of the Study:
- To investigate the genetic basis of spermine resistance in P. aeruginosa pauA2 mutants.
- To explore the Pho regulon's role in conferring resistance to spermine toxicity.
- To evaluate PauA2 as a potential drug target.
Main Methods:
- Isolation and characterization of suppressors of the pauA2 mutant.
- Genome resequencing to identify mutations in suppressor strains.
- Construction and analysis of double knockout mutants (ΔpauA2ΔphoU) and complemented strains.
- Assessment of polyphosphate accumulation and reversal of resistance phenotypes.
Main Results:
- A C599T mutation in the phoU gene, leading to Ser200Leu substitution, was identified in suppressors, causing constitutive Pho regulon expression.
- The phoU mutation resulted in polyphosphate granule accumulation and spermine resistance, mimicking phenotypes of ΔpauA2ΔphoU mutants.
- Reversal of spermine resistance and polyphosphate accumulation was achieved by expressing exopolyphosphatase or altering phosphate uptake/regulation pathways.
Conclusions:
- Spermine resistance in P. aeruginosa pauA2 mutants is mediated by polyphosphate accumulation.
- Constitutive activation of the Pho regulon and enhanced phosphate uptake via phoU mutations drive this protective mechanism.
- The interplay between polyamine metabolism and phosphate homeostasis offers potential therapeutic targets.