Phenazines Regulate Nap-Dependent Denitrification in Pseudomonas aeruginosa Biofilms

Area of Science:

• Microbiology
• Bacterial Physiology
• Biofilm Research

Background:

• Microbes in biofilms, like Pseudomonas aeruginosa, face substrate limitations, particularly oxygen.
• Phenazines, P. aeruginosa antibiotics, help balance intracellular redox state in biofilms.
• Understanding biofilm physiology is crucial for treating infections.

Purpose of the Study:

• To investigate the induction of denitrification genes in phenazine-null (Δphz) mutant P. aeruginosa biofilms under aerobic conditions without nitrate.
• To elucidate the specific denitrification pathway utilized and its regulation by phenazines.
• To explore the spatial distribution of denitrification pathway steps within biofilms.

Main Methods:

• Analysis of gene expression in Δphz mutant biofilms grown aerobically.
• Identification of promoter sequences regulating nap and nir operons.
• Assessment of phenazine's differential effects on nap gene expression.
• Investigation of denitrification pathway enzyme activity across biofilm depths.

Main Results:

• Denitrification genes are induced in Δphz mutant biofilms even without nitrate, suggesting a bet-hedging strategy.
• The induced pathway involves periplasmic Nap and downstream Nir, Nor, Nos, differing from anaerobic pathways.
• Specific phenazines differentially affect nap gene expression.
• Individual denitrification steps are catalyzed at different biofilm depths, indicating potential metabolic cross-feeding.

Conclusions:

• P. aeruginosa employs bet-hedging by inducing denitrification pathways to anticipate nitrate availability and manage redox state under electron acceptor limitation.
• The aerobic denitrification pathway in P. aeruginosa biofilms is distinct from its anaerobic counterpart.
• Metabolic cooperation between subpopulations within biofilms may facilitate complete denitrification.