Mutation or Overexpression of a Terminal Oxidase Leads to a Cell Division Defect and Multiple Antibiotic Sensitivity in Pseudomonas aeruginosa

Abstract

Mutation of the cyanide-insensitive terminal oxidase ofPseudomonas aeruginosa leads to pleiotropic effects. A cio mutant and strains, including the wild-type, carrying the cioAB genes on a multicopy plasmid were temperature-sensitive and had a cell division defect, leading to the formation of non-septate, multinucleated filaments. Such strains of this intrinsically antibiotic-resistant bacterium were more sensitive to a range of antibiotics including chloramphenicol, ␤-lactams, quinolones, aminoglycosides, and macrolides. The effect of cio mutation on ⌬p-dependent accumulation of chloramphenicol suggested that antibiotic sensitivity resulted from loss of or damage to a multidrug efflux pump. The ability of reducing agents and catalase to suppress the temperature-sensitive phenotype and of catalase to partially suppress antibiotic sensitivity suggested that increased levels of reactive oxygen species might be the cause of the observed phenotypes. Consistent with this was the increased sensitivity of strains to H 2 O 2 and their increased protein carbonyl content, an indicator of oxidative protein modification. The temperature-dependent synthesis of a specific catalase was absent in the cio mutant and in strains carrying multiple plasmid-borne copies of cioAB. We propose that reduced catalase levels result in oxidative modification and consequent loss of function of proteins involved in a range of cellular functions. How mutation or overexpression of the cyanide-insensitive terminal oxidase leads to a loss of catalase activity is unknown at present.Respiratory adaptability is likely to be an important component of the growth and survival of Pseudomonas aeruginosa in the diverse ecological niche in which it is found. This Gramnegative, opportunistic pathogen is able to respire and grow under a variety of aerobic and anaerobic conditions (1). Although it preferentially obtains energy via aerobic respiration, it is well adapted to conditions of limited oxygen supply (2). It is capable of anaerobic growth with nitrate as a terminal electron acceptor, and in its absence it is able to ferment arginine, generating ATP by substrate level phosphorylation (1-3).Examination of the genome sequence of P. aeruginosa suggests that its aerobic respiratory chain is extensively branched and is terminated by at least 5 terminal oxidases (4).1 Of four putative terminal oxidases belonging to the heme-copper oxidase superfamily, three are predicted to be cytochrome c oxidases, and one is a quinol oxidase. The fifth oxidase is the cyanide-insensitive oxidase (CIO), 2 which is encoded by the cioAB operon (5, 6). CioA and CioB are homologous to the two subunits of the cytochrome bd quinol oxidases, CydA and CydB, of Escherichia coli, Azotobacter vinelandii, and other bacteria (7) and as such show no homology to members of the ubiquitous heme-copper oxidase superfamily (8). Histidine and methionine residues identified in E. coli cytochrome bd as being ligands to the low spin heme b 558 and high spin b 595 are conserved, as is a periplasmic...

Regulation of expression of the cyanide-insensitive terminal oxidase in Pseudomonas aeruginosa

“…So O 2 could be an inducing signal that is working in combination either with another stationary-phase induction signal or with an exponential-phase repressing signal. We favour the latter, as we have recently found that an increase in exponentialphase CIO activity, as a result of the cioAB genes being present on a multicopy plasmid, leads to a number of detrimental effects on the growth and physiology of P. aeruginosa (Tavankar et al, 2003). This suggests a requirement for tight regulation of the CIO in exponentialphase cultures.…”

“…Mutation of cioAB leads to temperature sensitivity for growth, difficulty exiting stationary phase, cell division defects and multiple antibiotic sensitivity, probably due to damage to a multidrug efflux pump. This may be partly explained by increases in oxidative stress levels in CIO-defective strains as a result of the inability of these strains to synthesize a specific catalase, leading to oxidative protein damage (Tavankar et al, 2003).…”

“…Mutation of cioAB leads to temperature sensitivity for growth, difficulty exiting stationary phase, cell division defects and multiple antibiotic sensitivity, probably due to damage to a multidrug efflux pump. This may be partly explained by increases in oxidative stress levels in CIO-defective strains as a result of the inability of these strains to synthesize a specific catalase, leading to oxidative protein damage (Tavankar et al, 2003).Adaptation to low-O 2 or anaerobic environments may be significant in cystic fibrosis patients infected with mucoid strains of P. aeruginosa, where alginate has been shown to restrict diffusion of O 2 , thus creating a microaerobic or anaerobic environment, where nitrate levels are increased (Hassett, 1996). Furthermore, in cystic fibrosis patients with established lung disease, P. aeruginosa is located within hypoxic mucopurulent masses in airway lumen and steep O 2 gradients are present within the mucus on cystic fibrosis epithelial surfaces prior to infection (Worlitzsch et al, 2002).…”

cbb 3 -Type Cytochrome c Oxidases, Aerobic Respiratory Enzymes, Impact the Anaerobic Life of Pseudomonas aeruginosa PAO1

“…Thus, cell morphological changes affect anaerobic biofilm formation via unknown mechanisms. It has been reported that the mutation or overexpression of the terminal oxidase CIO results in a cell division defect in P. aeruginosa (34). cbb 3 oxidases may also affect cell morphology via a common unknown mechanism under anoxic conditions.…”

Investigation of the physiological relationship between the cyanide-insensitive oxidase and cyanide production in Pseudomonas aeruginosa

“…Using cells from an exponential-phase culture, in which CIO activity is at a minimum (Cooper et al, 2003), all strains had equivalent resistance to cyanide, except the cioAB-overexpressing strain, which has increased CIO activity levels (Fig. 2) (Cunningham et al, 1997;Tavankar et al, 2003). However, when cells from a stationary-phase culture were used, in which the CIO activity is maximum, strains possessing a functional CIO had MICs two-to fourfold higher than cio mutants (Fig.…”