Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum.
Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterized by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates of P. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 μM) of nitrate (NO(-) 3) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO(-) 3 and found a significant increase of growth when supplementing PAO1 and clinical isolates with ≥150 μM NO(-) 3 and 100 μM NO(-) 3, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO(-) 3. Activation of denitrification could be achieved by supplementation with as little as 62.5 μM of NO(-) 3 according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts, and enzyme activity of the four N-oxide reductases in PAO1 (Nar, Nir, Nor, Nos) further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO(-) 3 followed by a transient increase of NO(-) 2. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P. aeruginosa growth in infected endobronchial CF mucus.
Department of Clinical Microbiology Rigshospitalet, Copenhagen, Denmark ; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen Copenhagen, Denmark.
Laura
L
Line
Department of Clinical Microbiology Rigshospitalet, Copenhagen, Denmark ; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen Copenhagen, Denmark.
Morten
M
Alhede
Department of Clinical Microbiology Rigshospitalet, Copenhagen, Denmark ; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen Copenhagen, Denmark.
Mette
M
Kolpen
Marine Biological Section, Department of Biology, University of Copenhagen Copenhagen, Denmark ; Plant Functional Biology and Climate Change Cluster, University of Technology Sydney Sydney, NSW, Australia ; Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University Singapore, Singapore.
Michael
M
Kühl
Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen Copenhagen, Denmark.
Oana
O
Ciofu
Department of Clinical Microbiology Rigshospitalet, Copenhagen, Denmark ; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen Copenhagen, Denmark.
Thomas
T
Bjarnsholt
Department of Clinical Microbiology Rigshospitalet, Copenhagen, Denmark.
Claus
C
Moser
Graduate School of Life and Environmental Sciences, University of Tsukuba Tsukuba, Japan.
Masanori
M
Toyofuku
Graduate School of Life and Environmental Sciences, University of Tsukuba Tsukuba, Japan.
Nobuhiko
N
Nomura
Department of Clinical Microbiology Rigshospitalet, Copenhagen, Denmark ; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen Copenhagen, Denmark.
Niels
N
Høiby
Department of Clinical Microbiology Rigshospitalet, Copenhagen, Denmark.
Peter Ø
PØ
Jensen
Pseudomonas aeruginosa; cystic fibrosis; denitrification; growth