Enzymatic characterization and in vivo function of five terminal oxidases in Pseudomonas aeruginosa

Abstract

The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo(3)-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa3-type cytochrome c oxidase (aa3), and two cbb(3)-type cytochrome c oxidases (cbb(3)-1and cbb(3)-2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant strains of P. aeruginosa that express only one aerobic terminal oxidase to investigate the enzymatic characteristics and in vivo function of each enzyme. The Km values of Cyo, CIO, and aa3 for oxygen were similar and were 1 order of magnitude higher than those of cbb(3)-1 and cbb(3)-2, indicating that Cyo, CIO, and aa3 are low-affinity enzymes and that cbb(3)-1 and cbb(3)-2 are high-affinity enzymes. Although cbb(3)-1 and cbb(3)-2 exhibited different expression patterns in response to oxygen concentration, they had similar Km values for oxygen. Both cbb(3)-1 and cbb(3)-2 utilized cytochrome c4 as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb(3)-1 and cbb(3)-2 generate a proton gradient across the cell membrane with similar efficiencies. The electron transport chain of aa3 had the highest proton translocation efficiency, whereas that of CIO had the lowest efficiency. The enzymatic properties of the terminal oxidases reported here are partially in agreement with their regulatory patterns and may explain the environmental adaptability and versatility of P. aeruginosa.

FIG 1

Growth profiles of the quadruple terminal oxidase mutant strains of P. aeruginosa. (A) Growth under aerobic conditions. The strains were cultivated in 40 ml LB medium in 300-ml Erlenmeyer flasks with shaking at 180 rpm. (B) Growth under microaerobic conditions (2% O₂). The strains were cultivated in 150 ml LB medium in 300-ml square bottles. The medium was bubbled with a gas mixture consisting of 2% O₂ and 98% N₂ at a gas flux rate of 150 ml/min. The data shown are representatives from at least three independent experiments. The same plots in linear scale are shown in Fig. S4 in the supplemental material.

FIG 2

Oxygen consumption activities of the membrane fractions of the quadruple terminal oxidase mutant strains. Bo, Ci, Aa, Cb1, and Cb2 indicate membrane fractions of QXBo, QXCi, QXAaS2, QXCb1, and QXCb2, respectively. A 0.5 mM concentration of NADH (A, B, and C) and a 0.1 mM concentration of TMPD reduced by 5 mM ascorbate (D) were used as electron donors. A 1 mM concentration of KCN (B) or 10 μg/ml antimycin A (C) was added to the reaction mixture to inhibit the heme-copper oxidases or the cytochrome bc₁ complex, respectively. The data are means of results from three independent experiments. Error bars indicate standard deviations from the means.

FIG 3

Effect of knockout of the cytochrome c genes on growth rates. Growth profiles of the derivatives of QXCb1 (A) and QXCb2 (D). Complementation of QXCb1Δcc4 (B), QXCb1ΔPA5491 (C), QXCb2Δcc4 (E), and QXCb2ΔPA5491 (F). The strains were grown aerobically in 40 ml LB medium in 100-ml Erlenmeyer flasks with shaking at 200 rpm. The data shown are representatives from three independent experiments. The same plots in linear scale are shown in Fig. S5 in the supplemental material.

FIG 4

Functional heterologous expression of CIO in E. coli MB43, which lacks type 1 NADH dehydrogenase and three quinol oxidases. MB43 was transformed with pUC-cioAB or pUC-cydAB, and the transformants were cultivated in 100 ml LB medium in 300-ml square bottles. The medium was bubbled with air at a gas flux rate of 500 ml/min for aerobic conditions or with a gas mixture consisting of 2% O₂ and 98% N₂ at a gas flux rate of 150 ml/min for microaerobic conditions. The data shown are representatives from two independent experiments. The doubling times ± standard deviations are indicated in brackets. The same plots in linear scale are shown in Fig. S6 in the supplemental material.