Bioenergetics of simultaneous oxygen and nitrate respiration and nitric oxide production in a Pseudomonas aeruginosa agar colony biofilm

Pseudomonas aeruginosa is a biofilm forming pathogen commonly associated with infection of the cystic fibrosis (CF) lung, chronic wounds and indwelling medical devices. P. aeruginosa is a facultative aerobe that can use nitrate (NO₃⁻) found in healthy and infected tissues and body fluids to generate energy through denitrification. Further, P. aeruginosa the expression of denitrification genes has been found in specimens from people with CF. The main aim of this study was to determine the relative energy contribution of oxygen (O₂) respiration and denitrification in single Pseudomonas aeruginosa PAO1 biofilm colonies under different O₂ concentrations to estimate the possible relative importance of these metabolic processes in the context of biofilm infections. We showed that the used strain PAO1 in biofilms denitrified with nitrous oxide (N₂O), and not nitrogen (N₂), as the end product in our incubations. From simultaneous O₂ and N₂O microprofiles measured with high spatial resolution by microsensors in agar colony biofilms under air, N₂ and pure O₂, the rates of aerobic respiration and denitrification were calculated and converted to ATP production rates. Denitrification occurred both in the oxic and anoxic zones, and became increasingly dominant with decreasing O₂ concentrations. At O₂ concentrations characteristic for tissues and wounds (20–60 μM), denitrification was responsible for 50% of the total energy conservation in the biofilm. In addition the formation of nitric oxide (NO), a precursor of N₂O and an important regulator of many cellular processes, was strongly influenced by the local O₂ concentrations. NO production was inhibited under pure O₂, present under anoxia (∼1 μM) and remarkably high (up to 6 μM) under intermediate O₂ levels, which can be found in infected tissues. Possible impacts of such NO levels on both the host and the biofilm bacteria are discussed.