Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration

Abstract

Pseudomonas aeruginosa in the lungs of cystic fibrosis patients grows to high densities in mucopurulent material that is depleted in oxygen. Some have concluded that growth in these circumstances is dependent on anaerobic nitrate respiration. Here we present data in favour of the alternative hypothesis that microaerobic respiration is the predominant mode of P. aeruginosa growth in the cystic fibrosis lung. We found that P. aeruginosa strain PAO1 and a mucoid derivative of strain PAO1 each grew at dissolved oxygen concentrations of less than 3 microM. This is lower than the concentration of oxygen that has been measured in hypoxic cystic fibrosis mucous. A transcriptome analysis comparing cells grown under aerobic conditions (185 microM dissolved oxygen) with cells grown with 20 microM or 3 microM dissolved oxygen, or anaerobically with nitrate, revealed that overlapping sets of genes are expressed depending on oxygen availability. This suggests that P. aeruginosa responds to changes in oxygen concentration along a continuum rather than having a discrete low oxygen regulon. Any one of three high affinity terminal oxidases that P. aeruginosa encodes supported microaerobic growth. A triple mutant lacking all three of these oxidases failed to grow at low oxygen and formed abnormal biofilms.

Fig. 1

Diagram of Pseudomonas aeruginosa branched aerobic respiratory chain (adapted from Comolli and Donohue, 2002). The terminal oxidases predicted to have a high affinity for oxygen are the bd-type oxidase (cyanide insensitive), cbb₃-1 and cbb₃-2.

Fig. 2

Growth of Pseudomonas aeruginosa PAO1 at different concentrations of oxygen. Oxygen was supplied in the gas phase at concentrations of 0.4% (A), 2% (B) or 20% (C). Culture optical density (circles) and dissolved oxygen concentrations (triangles) are plotted over time after inoculation into CF sputum medium. Dissolved oxygen is reported in mg per l; for uninoculated medium this is 5.88 ± 0.13 (20% oxygen) 0.81 ± 0.035 mg (2% oxygen) and 0.3 ± 0.01 (0.4% oxygen).

Fig. 3

Numbers of genes that are differentially expressed in response to growth at different concentrations of oxygen. Gene expression profiles of cells grown at the indicated concentration of oxygen were compared with that of cells grown with 20% oxygen. A 2.5-fold cut-off and a P-value threshold of 0.001 were applied to transcriptome data. A. Numbers of genes expressed at higher levels relative to 20% oxygen. B. Numbers of genes expressed at lower levels relative to 20% oxygen.

Fig. 4

Double and triple high affinity terminal oxidase mutants form defective biofilms. Images were obtained after 48 h and 96 h growth of the wild-type strain PAO1 (A and D), the cbb₃-1 cbb₃-2 double mutant PAO1373 (B and E) and the cioA cbb₃-1 cbb₃-2 triple mutant PAO1387 (C and F) in continuous flow chambers. Each square on the grid is 46 µM per side.

Fig. 5

The triple high affinity oxidase mutant loses viability in biofilms. Flow cell biofilms for wild type and the cioA cbb₃-1 cbb₃-2 triple mutant PAO1987. SD, side dimension (x,z-plane); TD, top down view (x,y-plane). GFP-labelled biofilms were grown for 96 h at which time dead cells were counterstained with propidium iodide. The combined green (GFP) and red (propidium iodide) channels are presented.