Oxygen, Cyanide and Energy Generation in the Cystic Fibrosis Pathogen Pseudomonas aeruginosa

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Abstract

Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium that belongs to the γ-proteobacteria. This clinically challenging, opportunistic pathogen occupies a wide range of niches from an almost ubiquitous environmental presence to causing infections in a wide range of animals and plants. P. aeruginosa is the single most important pathogen of the cystic fibrosis (CF) lung. It causes serious chronic infections following its colonisation of the dehydrated mucus of the CF lung, leading to it being the most important cause of morbidity and mortality in CF sufferers. The recent finding that steep O2 gradients exist across the mucus of the CF-lung indicates that P. aeruginosa will have to show metabolic adaptability to modify its energy metabolism as it moves from a high O2 to low O2 and on to anaerobic environments within the CF lung. Therefore, the starting point of this review is that an understanding of the diverse modes of energy metabolism available to P. aeruginosa and their regulation is important to understanding both its fundamental physiology and the factors significant in its pathogenicity. The main aim of this review is to appraise the current state of knowledge of the energy generating pathways of P. aeruginosa. We first look at the organisation of the aerobic respiratory chains of P. aeruginosa, focusing on the multiple primary dehydrogenases and terminal oxidases that make up the highly branched pathways. Next, we will discuss the denitrification pathways used during anaerobic respiration as well as considering the ability of P. aeruginosa to carry out aerobic denitrification. Attention is then directed to the limited fermentative capacity of P. aeruginosa with discussion of the arginine deiminase pathway and the role of pyruvate fermentation. In the final part of the review, we consider other aspects of the biology of P. aeruginosa that are linked to energy metabolism or affected by oxygen availability. These include cyanide synthesis, which is oxygen-regulated and can affect the operation of aerobic respiratory pathways, and alginate production leading to a mucoid phenotype, which is regulated by oxygen and energy availability, as well as having a role in the protection of P. aeruginosa against reactive oxygen species. Finally, we consider a possible link between cyanide synthesis and the mucoid switch that operates in P. aeruginosa during chronic CF lung infection.

Section snippets

Introduction to Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium that belongs to the γ-proteobacteria (Holt and Kreig, 1984). This clinically challenging, opportunistic pathogen occupies a wide range of niches from an almost ubiquitous environmental presence to causing infections in a wide range of animals and plants (Van Delden and Iglewski, 1998; Tummler and Kiewitz, 1999; Stover et al., 2000). P. aeruginosa has a large genome of around 6.3 million base pairs encoding some 5270 predicted open

Oxygen and P. aeruginosa Infection of the Cystic Fibrosis Lung – the Scope of this Review

CF is caused by mutation of the gene encoding the CF transmembrane regulator (CFTR), which functions as a chloride channel in epithelial membranes (Collins, 1992). Several hypotheses link mutations in CFTR to development of chronic P. aeruginosa infections (Ratjen and Doring, 2003). A hypothesis for which there is increasing support is the isotonic fluid depletion/hypoxic mucus hypothesis (Fig. 1). This proposes that isotonic salt concentrations resulting from abnormal sodium absorption from

Means of Energy Generation in P. aeruginosa

It is in their methods of energy generation that bacteria demonstrate their exceptional metabolic versatility and diversity. Pseudomonads use a remarkably eclectic range of carbon and energy sources (Stanier et al., 1966). Irrespective of the energy source used, energy-generating reactions must accomplish the same metabolic functions, that is, to produce precursor metabolites, reducing power and the energy required by the cell in the form of ATP and Δp (proton and/or sodium electrochemical

Aerobic respiration in P. aeruginosa

Bacterial respiratory chains are complex organisations of electron-transfer components, which together can oxidise a broad array of substrates via substrate-specific dehydrogenases. These initial oxidations of redox couples with low negative redox potentials are linked to the four-electron reduction of oxygen to water by a sequence of electron transfer components that are common to all organisms. These include quinones, cytochromes and terminal oxidases and contain as redox centres haems, Fe-S

Anaerobic Respiration

P. aeruginosa is a denitrifying bacterium. It is able to carry out anaerobic respiration with N-oxides as terminal electron acceptors for anaerobic respiration. Denitrification is the sequential reduction of nitrate to N2 via nitrite, nitric oxide and nitrous oxide, a process that is catalysed by four enzymes: nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR) and nitrous oxide reductase (N2OR) (Figure 3, Figure 6; Zumft, 1997). The denitrifying enzymes provide

Fermentation

P. aeruginosa has often been described as a non-fermentative bacterium. This is not the case, but it has only limited fermentative capacity. It has been long recognised that in the absence of oxygen and nitrate P. aeruginosa can use arginine as source of energy for growth using the arginine deiminase (ADI) pathway (Shoesmith and Sherris, 1960; Van der Wauven et al., 1984). This pathway catalyses the breakdown of l-arginine to l-ornithine, with the formation of an ATP (Fig. 7). However,

Anaerobic Metabolism in the Cystic Fibrosis Lung

Recent research has implicated the formation of anaerobic biofilms and consequently the operation of anaerobic respiratory pathways in the colonisation of and survival in the CF lung by P. aeruginosa.

In an elegant study, referred to earlier, Worlitzsch et al. (2002) showed that steep oxygen gradients exist in the mucus lining of the CF lung, whereas no equivalent gradients exist in the mucus of the healthy non-CF lung. There is evidence of raised oxygen consumption by CF epithelial cells (

Synthesis of the Respiratory Inhibitor Hydrogen Cyanide in P. aeruginosa

An intriguing aspect of the biology of P. aeruginosa is its ability to synthesise the respiratory inhibitor hydrogen cyanide, which can reach concentrations of 300 μM in laboratory cultures (Blumer and Haas, 2000; Zlosnik and Williams, 2004). The fact that cyanide is synthesised aerobically raises the interesting issue of how can the bacteria respire aerobically while producing cyanide. The fact that cyanide is made only under low oxygen conditions raises the issue of whether it is made in the

Mucoid Conversion of P. aeruginosa in the Cystic Fibrosis Lung: the Role of Oxygen and Energy Metabolism

The production of the exopolysaccharide alginate confers the well-described mucoid phenotype on P. aeruginosa. Alginate is a linear copolymer composed of β-d-mannuronic acid and α-l-guluronic acids and its production in bacteria was first described in P. aeruginosa in 1964 (Linker and Jones, 1964). Alginate is also known to be produced as an extracellular polysaccharide by A. vinelandi and other Pseudomonads (Gacesa, 1998).

In CF a typical pattern of P. aeruginosa infection has long been

Conclusion

P. aeruginosa has broad-ranging energy-generating pathways which will be important in the adaptation of P. aeruginosa to the niche of the CF lung. The recent demonstration of oxygen gradients across and anaerobic conditions in the depths of the mucus layer of the CF lung suggest that a full range of energy generating pathways will function in P. aeruginosa during colonisation and chronic infection of the CF lung. This information, coupled with the HCN production in the mucus layer and the

References (339)

  • B.C. Berks et al.

    Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions

    Biochim. Biophys. Acta

    (1995)
  • C. Braun et al.

    Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide

    J. Biol. Chem.

    (1991)
  • T. Brittain et al.

    Complex formation between the copper protein, azurin and the cytochrome c peroxidase of Pseudomonas aeruginosa

    J. Inorg. Biochem.

    (1992)
  • E.D. Brown et al.

    Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli

    J. Biol. Chem.

    (1992)
  • K. Brown et al.

    Revisiting the catalytic CuZ cluster of nitrous oxide (N2O) reductase. Evidence of a bridging inorganic sulfur

    J. Biol. Chem.

    (2000)
  • M.M. Brysk et al.

    Biosynthesis of cyanide from [2-14C-15N]glycine in Chromobacterium violaceum

    Biochim. Biophys. Acta

    (1969)
  • M.W. Calhoun et al.

    The cytochrome oxidase superfamily of redox-driven proton pumps

    Trends Biochem. Sci.

    (1994)
  • J. Cao et al.

    Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast

    J. Biol. Chem.

    (1992)
  • D.C. Carter et al.

    Crystal structure of Azotobacter cytochrome c5 at 2.5 A resolution

    J. Mol. Biol.

    (1985)
  • G. Cecchini et al.

    Succinate dehydrogenase and fumarate reductase from Escherichia coli

    Biochim. Biophys. Acta

    (2002)
  • D. Chen et al.

    Cloning, sequence analysis, and expression of the genes encoding the two subunits of the methylotrophic bacterium W3A1 electron transfer flavoprotein

    J. Biol. Chem.

    (1994)
  • H.E. Christensen

    Cloning and characterisation of the gene encoding cytochrome c4 from Pseudomonas stutzeri

    Gene

    (1994)
  • R. Cipollone et al.

    Cyanide detoxification by recombinant bacterial rhodanese

    Chemosphere

    (2006)
  • R. Cipollone et al.

    Characterization of a rhodanese from the cyanogenic bacterium Pseudomonas aeruginosa

    Biochem. Biophys. Res. Commun.

    (2004)
  • B.J. Clawson et al.

    Preliminary report on the production of hydrocyanic acid by bacteria

    J. Biol. Chem.

    (1913)
  • A.R. Crofts et al.

    Structure and function of the cytochrome bc1 complex of mitochondria and photosynthetic bacteria

    Curr. Opin. Struct. Biol.

    (1998)
  • H. Cuypers et al.

    Multiple nosZ promoters and anaerobic expression of nos genes necessary for Pseudomonas stutzeri nitrous oxide reductase and assembly of its copper centers

    Biochim. Biophys. Acta

    (1995)
  • M. Dermastia et al.

    Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics

    J. Biol. Chem.

    (1991)
  • J.A. Duine

    The PQQ story

    J. Biosci. Bioeng.

    (1999)
  • M. Elias et al.

    C-terminal periplasmic domain of Escherichia coli quinoprotein glucose dehydrogenase transfers electrons to ubiquinone

    J. Biol. Chem.

    (2001)
  • M.D. Elias et al.

    Occurrence of a bound ubiquinone and its function in Escherichia coli membrane-bound quinoprotein glucose dehydrogenase

    J. Biol. Chem.

    (2004)
  • N. Ellfolk et al.

    Structural and functional features of Pseudomonas cytochrome c peroxidase

    Biochim. Biophys. Acta

    (1991)
  • H.G. Enoch et al.

    The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli

    J. Biol. Chem.

    (1975)
  • B. Entsch et al.

    Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa

    Biochim. Biophys. Acta

    (1989)
  • G. Finocchiaro et al.

    Molecular cloning and nucleotide sequence of cDNAs encoding the alpha-subunit of human electron transfer flavoprotein

    J. Biol. Chem.

    (1988)
  • V. Fulop et al.

    Crystallization and preliminary X-ray analysis of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa

    J. Mol. Biol.

    (1993)
  • C. Abergel et al.

    Crystallization and preliminary crystallographic study of an extremophile cytochrome c4 from Thiobacillus ferrooxidans

    Acta Crystallogr. D Biol. Crystallogr.

    (2000)
  • J.L. Abrahamson et al.

    Proline dehydrogenase from Escherichia coli K12. Properties of the membrane-associated enzyme

    Eur. J. Biochem.

    (1983)
  • D.F. Ackerley et al.

    Substrate specificity of the nonribosomal peptide synthetase PvdD from Pseudomonas aeruginosa

    J. Bacteriol.

    (2003)
  • R.P. Ambler et al.

    The amino acid sequence of the dihaem cytochrome c4 from the bacterium Azotobacter vinelandii

    Biochem. J.

    (1984)
  • H. Arai et al.

    Cascade regulation of the two CRP/FNR-related transcriptional regulators (ANR and DNR) and the denitrification enzymes in Pseudomonas aeruginosa

    Mol. Microbiol.

    (1997)
  • H. Arai et al.

    Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa

    Microbiology

    (2003)
  • R.A. Askeland et al.

    Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

    Appl. Environ. Microbiol.

    (1983)
  • R.M. Atlas et al.

    Microbial Ecology: Fundamentals and Applications

    (1997)
  • T. Atlung et al.

    Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation

    J. Bacteriol.

    (1997)
  • A.J. Bater et al.

    Allohydroxy-d-proline dehydrogenase. An inducible membrane-bound enzyme in Pseudomonas aeruginosa PA01

    Arch. Microbiol.

    (1977)
  • H. Baur et al.

    Sequence analysis and expression of the arginine-deiminase and carbamate-kinase genes of Pseudomonas aeruginosa

    Eur. J. Biochem.

    (1989)
  • A.S. Bayer et al.

    Oxygen-dependent up-regulation of mucoid exopolysaccharide (alginate) production in Pseudomonas aeruginosa

    Infect. Immun.

    (1990)
  • A.S. Bayer et al.

    Oxygen-dependent differences in exopolysaccharide production and aminoglycoside inhibitory-bactericidal interactions with Pseudomonas aeruginosa – implications for endocarditis

    J. Antimicrob. Chemother.

    (1989)
  • L. Bedzyk et al.

    The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification

    J. Bacteriol.

    (1999)
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