Pseudomonas aeruginosa Dnr-regulated denitrification in microoxic conditions

Abstract

Pseudomonas aeruginosa causes acute and chronic infections, such as those that occur in the lungs of people with cystic fibrosis (CF). In infection environments, oxygen (O₂) concentrations are often low. The transcription factor Anr (anaerobic regulation of arginine deiminase and nitrate reduction) responds to low O₂ by upregulating genes necessary for P. aeruginosa fitness in microoxic and anoxic conditions. Anr regulates Dnr (dissimilative nitrate respiration regulator), a gene encoding a transcriptional regulator that promotes the expression of genes required for using nitrate as an alternative electron acceptor during denitrification. In CF sputum, transcripts involved in denitrification are highly expressed. While Dnr is necessary for the anoxic growth of P. aeruginosa in CF sputum and artificial sputum media (ASMi), the contribution of denitrification to P. aeruginosa fitness in oxic conditions has not been well described. Here, we show that P. aeruginosa requires dnr for fitness in ASMi, and the requirement for dnr is abolished when nitrate is excluded from the media. Additionally, we show that P. aeruginosa consumes nitrate in lysogeny broth (LB) under microoxic conditions. Furthermore, strains without a functioning quorum sensing regulator LasR, which leads to elevated Anr activity, consume nitrate in LB even in normoxia. There was no growth advantage for P. aeruginosa when nitrate was present at concentrations from 100 to 1,600 µM. However, P. aeruginosa consumption of nitrate in oxic conditions created a requirement for Dnr and Dnr-regulated NorCB, likely due to the need to detoxify nitric oxide. These studies suggest that Anr- and Dnr-regulated processes may impact P. aeruginosa physiology in many common culture conditions.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen commonly isolated from low-oxygen environments such as the lungs of people with cystic fibrosis. While the importance of P. aeruginosa energy generation by denitrification is clear in anoxic environments, the effects of denitrification in oxic cultures are not well understood. Here, we show that nitrate is consumed in microoxic environments and, in some strains, in normoxic environments. While nitrate does not appear to stimulate microoxic growth rate or yield, it does impact physiology. We show that the regulators Anr (anaerobic regulation of arginine deiminase and nitrate reduction) and Dnr (dissimilative nitrate respiration regulator), which are best known for their roles in anoxic conditions, contribute to P. aeruginosa fitness in common laboratory media in the presence of oxygen.

Keywords: Anr; Dnr; Pseudomonas aeruginosa; denitrification; microoxia; nitrate; nitric oxide.