Expression of multiplecbb₃cytochromecoxidase isoforms by combinations of multiple isosubunits inPseudomonas aeruginosa

Abstract

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa has five terminal oxidases for aerobic respiration and uses them under different growth conditions. Two of them are cbb 3 -type cytochrome c oxidases encoded by the gene clusters ccoN1O1Q1P1 and ccoN2O2Q2P2, which are the main terminal oxidases under high-and low-oxygen conditions, respectively. P. aeruginosa also has two orphan gene clusters, ccoN3Q3 and ccoN4Q4, encoding the core catalytic CcoN isosubunits, but the roles of these genes have not been clarified. We found that 16 active cbb 3 isoforms could be produced by combinations of four CcoN, two CcoO, and two CcoP isosubunits. The CcoN3-or CcoN4-containing isoforms were produced in the WT cell membrane in response to nitrite and cyanide, respectively. The strains carrying these isoforms were more resistant to nitrite or cyanide under low-oxygen conditions. These results indicate that P. aeruginosa gains resistance to respiratory inhibitors using multiple cbb 3 isoforms with different features, which are produced through exchanges of multiple core catalytic isosubunits.

Interdependency of respiratory metabolism and phenazine-associated physiology in Pseudomonas aeruginosa PA14

“…To this end, we generated a mutant, referred to as "∆HMS∆phz", that is unable to produce or modify any of PA14's known phenazines ( Figure 6A). We 315 also created the ∆HMS∆phz∆cco mutant, which we expected to show defects in 15 phenazine reduction under certain conditions. We then grew these mutants on agar plates each containing one pure phenazine compound and measured phenazine reduction on our carbon sources of interest.…”

“…Each of these terminal oxidases possesses unique characteristics, including different expression patterns and affinities for O2 (11)(12)(13)(14). Our picture of P. aeruginosa's ETC is further complicated by the fact that its "Cco" complexes, which belong to the cbb3 family 100 of terminal oxidases, can contain subunits encoded by multiple, redundant operons present at distinct sites in the genome (15). These heterocomplexes (i.e., isoforms) have specific roles under different conditions (15) and contribute differentially to biofilm physiology and redox state (7).…”

“…Our picture of P. aeruginosa's ETC is further complicated by the fact that its "Cco" complexes, which belong to the cbb3 family 100 of terminal oxidases, can contain subunits encoded by multiple, redundant operons present at distinct sites in the genome (15). These heterocomplexes (i.e., isoforms) have specific roles under different conditions (15) and contribute differentially to biofilm physiology and redox state (7). In addition, work from our group has implicated specific Cco terminal oxidase isoforms in phenazine utilization in biofilms grown on a complex 105 medium (7) Though metabolism is often conceptualized as a modular process in which the oxidation of individual carbon sources can be paired with the respiration of electron acceptors via 6 common, central pathways, this is an oversimplification.…”

An orphancbb3-type cytochrome oxidase subunit supportsPseudomonas aeruginosabiofilm growth and virulence

“…While the cco1 and cco2 operons, which are chromosomally adjacent, each contain four genes encoding a functional Cco complex (consisting of subunits N, O, P, and Q), the two additional partial operons ccoN3Q3 and ccoN4Q4 each contain homologs coding for only the Q and catalytic N subunits ( Figure 1B ). Expression of the ccoN3Q3 operon is induced under anaerobic denitrification conditions (Alvarez-Ortega & Harwood 2007), and by nitrite exposure during growth under 2% O 2 (Hirai et al 2016). During aerobic growth in liquid cultures, ccoN4Q4 is induced by cyanide, which is produced in stationary phase (Hirai et al 2016).…”

“…Expression of the ccoN3Q3 operon is induced under anaerobic denitrification conditions (Alvarez-Ortega & Harwood 2007), and by nitrite exposure during growth under 2% O 2 (Hirai et al 2016). During aerobic growth in liquid cultures, ccoN4Q4 is induced by cyanide, which is produced in stationary phase (Hirai et al 2016). However, additional expression studies indicate that ccoN4Q4 transcription is influenced by redox conditions, as this operon is induced by O 2 limitation (Alvarez-Ortega & Harwood 2007) and slightly downregulated in response to pyocyanin, a redox-active antibiotic produced by P. aeruginosa (Dietrich, Price-Whelan, et al 2006).…”