Cyanide-insensitive Respiratory Pathway Research Articles

Molecular characterization and gene expression modulation of the alternative oxidase in a scuticociliate parasite by hypoxia and mitochondrial respiration inhibitors

Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus). In addition to a cytochrome pathway (CP), the ciliate can use a cyanide-insensitive respiratory pathway, which indicates the existence of an alternative oxidase (AOX) in the mitochondrion. Although AOX activity has been described in P. dicentrarchi, based on functional assay results, genetic evidence of the presence of AOX in the ciliate has not previously been reported. In this study, we conducted genomic and transcriptomic analysis of the ciliate and identified the AOX gene and its corresponding mRNA. The AOX gene (size 1,106 bp) contains four exons and three introns that generate an open reading frame of 915 bp and a protein with a predicted molecular weight of 35.6 kDa. The amino acid (aa) sequence of the AOX includes an import signal peptide targeting the mitochondria and the protein is associated with the inner membrane of the mitochondria. Bioinformatic analysis predicted that the peptide is a homodimeric glycoprotein, although monomeric forms may also appear under native conditions, with EXXH motifs associated with the diiron active centers. The aa sequences of the AOX of different P. dicentrarchi isolates are highly conserved and phylogenetically closely related to AOXs of other ciliate species, especially scuticociliates. AOX expression increased significantly during infection in the host and after the addition of CP inhibitors. This confirms the important physiological roles of AOX in respiration under conditions of low levels of O2 and in protecting against oxidative stress generated during infection in the host.

Cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 involves a malate : quinone oxidoreductase and an associated cyanide-insensitive electron transfer chain

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to grow with cyanide as the sole nitrogen source. Membrane fractions from cells grown under cyanotrophic conditions catalysed the production of oxaloacetate from L-malate. Several enzymic activities of the tricarboxylic acid and glyoxylate cycles in association with the cyanide-insensitive respiratory pathway seem to be responsible for the oxaloacetate formation in vivo. Thus, in cyanide-grown cells, citrate synthase and isocitrate lyase activities were significantly higher than those observed with other nitrogen sources. Malate dehydrogenase activity was undetectable, but a malate:quinone oxidoreductase activity coupled to the cyanide-insensitive alternative oxidase was found in membrane fractions from cyanide-grown cells. Therefore, oxaloacetate production was linked to the cyanide-insensitive respiration in P. pseudoalcaligenes CECT5344. Cyanide and oxaloacetate reacted chemically inside the cells to produce a cyanohydrin (2-hydroxynitrile), which was further converted to ammonium. In addition to cyanide, strain CECT5344 was able to grow with several cyano derivatives, such as 2- and 3-hydroxynitriles. The specific system required for uptake and metabolization of cyanohydrins was induced by cyanide and by 2-hydroxynitriles, such as the cyanohydrins of oxaloacetate and 2-oxoglutarate.

Regulation of Alternative Oxidase at the Transcription Stage in Aspergillus niger Under the Conditions of Citric Acid Production

The citric acid-producing fungus Aspergillus niger WU-2223L possesses a cyanide-insensitive respiratory pathway catalyzed by alternative oxidase. The regulation of the alternative oxidase under the conditions of citric acid production was determined from the transcription level of the alternative oxidase gene (aox1). PCR and Southern blot analyses revealed that there is only one copy of aox1 on the chromosome of WU-2223L and no homologous gene of aox1. To confirm the regulation stage of alternative oxidase, alternative oxidase activities and aox1 transcription levels were measured under several cultivation conditions, including those for citric acid production. On each cultivation day, the changes in the specific activity of the alternative oxidase were found to be comparable to those in the transcription level of aox1. These results indicate that the activity of the alternative oxidase encoded by aox1 is regulated at the transcription stage under the conditions tested for A. niger WU-2223L.

The effects of mutation of the anr gene on the aerobic respiratory chain of Pseudomonas aeruginosa.

The anr gene of Pseudomonas aeruginosa encodes a transcriptional regulator of anaerobic gene expression, homologous to the Fnr protein of Escherichia coli. We report here that Anr has a role in regulating the activity of the aerobic respiratory chain of P. aeruginosa. Strains with internal deletions in their anr gene had lowered levels of membrane bound cytochromes whilst the activity of the cytochrome c oxidase, cytochrome co (likely to be a cytochrome cbb3-type oxidase), and the cyanide-insensitive respiratory pathway was markedly higher than in the wild-type strains. These data, and the finding that provision of multiple copies of the anr gene led to severe repression of these respiratory activities, suggest that Anr is a repressor of aerobic respiratory pathways and possibly the terminal oxidases themselves. In contrast, Anr activated cytochrome c peroxidase, a respiratory chain linked enzyme induced under low oxygen conditions.

Characterization of the mitochondrial respiratory pathways in Candida albicans

Candida albicans is an opportunistic oral pathogen. The flexibility of this microorganism in response to environmental changes includes the expression of a cyanide-resistant alternative respiratory pathway. In the present study, we characterized both conventional and alternative respiratory pathways and determined their ADP/O ratios, inhibitor sensitivity profiles and the impact of the utilization of either pathway on susceptibility to commonly used antimycotics. Oxygen consumption by isolated mitochondria using NADH or malate/pyruvate as respiratory substrates indicated that C. albicans cells express both cytoplasmic and matrix NADH–ubiquinone oxidoreductase activities. The ADP/O ratio was higher for malate/pyruvate (2.2±0.1), which generate NADH in the matrix, than for externally added NADH (1.4±0.2). In addition, malate/pyruvate respiration was rotenone-sensitive, and an enzyme activity assay further confirmed that C. albicans cells express Complex I activity. Cells grown in the presence of antimycin A expressed the cyanide-insensitive respiratory pathway. Determination of the respiratory control ratio (RCR) and ADP/O ratios of mitochondria from these cells indicated that electron transport from ubiquinone to oxygen via the alternative respiratory pathway was not coupled to ATP production; however, an ADP/O ratio of 0.8 was found for substrates that donate electrons at Complex I. Comparison of antifungal susceptibility of C. albicans cells respiring via the conventional or alternative respiratory pathways showed that respiration via the alternative pathway does not reduce the susceptibility of cells to a series of clinically employed antimycotics (using Fungitest®), or to the naturally occurring human salivary antifungal peptide, histatin 5.

Contribution of cyanide-insensitive respiratory pathway, catalyzed by the alternative oxidase, to citric acid production in Aspergillus niger.

In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX). In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 microM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A. niger.

Partial male sterility in transgenic tobacco carrying an antisense gene for alternative oxidase under the control of a tapetum-specific promoter

The alternative oxidase of plant mitochondria is the terminal oxidase of the cyanide-insensitive respiratory pathway and is encoded by a nuclear gene. A 1 kb genomic fragment including exon 3 of the alternative oxidase was amplified by PCR from the genome of Arabidopsis thaliana. This fragment was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. The pollen viability in three transgenic plants ranged from 2% to 60%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. Immunolocalization of alternative oxidase protein in the immature flower bud section indicated that expression of alternative oxidase protein in tapetum of the transgenic plant was much lower than that of the non-transformant. The histological observation and protein gel-blot analysis showed that the development of pollen grains in the transgenic plant did not progress after the degradation of the tapetum, and the amount of alternative oxidase in pollen grains of the transgenic plant became lower than that of the non-transformant. These results suggested that the alternative oxidase activity in the tapetum has a significant effect on the pollen development.

The effects of mutation of the anr gene on the aerobic respiratory chain of Pseudomonas aeruginosa

The anr gene of Pseudomonas aeruginosa encodes a transcriptional regulator of anaerobic gene expression, homologous to the Fnr protein of Escherichia coli. We report here that Anr has a role in regulating the activity of the aerobic respiratory chain of P. aeruginosa. Strains with internal deletions in their anr gene had lowered levels of membrane bound cytochromes whilst the activity of the cytochrome c oxidase, cytochrome co (likely to be a cytochrome cbb 3-type oxidase), and the cyanide-insensitive respiratory pathway was markedly higher than in the wild-type strains. These data, and the finding that provision of multiple copies of the anr gene led to severe repression of these respiratory activities, suggest that Anr is a repressor of aerobic respiratory pathways and possibly the terminal oxidases themselves. In contrast, Anr activated cytochrome c peroxidase, a respiratory chain linked enzyme induced under low oxygen conditions.

A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase

Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N, N, N′, N′-tetrarnethyl- p-phenylenediarnine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c-type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway's terminal oxidase is a quinol oxidase.

A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase.

Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c-type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway's terminal oxidase is a quinol oxidase.

Isolation, selection and characterization of a cyanide-degrading fungus from an industrial effluent

Amongst 14 fungi isolates from a cyanide-containing industrial effluent, a strain of Fusarium oxysporum developed a particularly high tolerance for cyanide when incubated in a medium containing cyanide. Tolerance apparently involves the ability of the fungus to convert cyanide to non-toxic formamide by formamide hydro-lyase enzyme. The contribution of the alternate cyanide insensitive respiratory system to ensure the formamide hydro-lyase induction was assessed. When the cytochromes were blocked and the cyanide-insensitive respiratory pathway accounted for 100% of the oxygen uptake associated with respiration, formamide hydro-lyase activity was positively correlated with activity of alternate pathway.

Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.

Inhibitors of cyanide-insensitive respiratory pathway induce male sterility in broccoli (Brassica oleracea).

(...) All three major specific inhibitors; salicylhydroxamic acid (SHAM), propyl gallate and disulfiram (tetraethylthiuram disulfide), were effective in inducing male sterility whenever they were applied to the flower buds with anthers less than about 2 mm in length, which those pollen developing stages corresponded to be earlier than early microspore stage. These chemically induced male sterile flowers which were characterized by shorter filaments and a reduction in the number of total and fertile pollen grains per flower, were similar to the cytoplasmic male sterile flowers. (...)

The Role of Cu in Respiration of Pea Plants and Heterotrophically Growing Scenedesmus Cells

In Scenedesmus about half of NADH oxidation proceeds via a cyanide-sensitive and the other half via a cyanide-insensitive respiratory pathway. In contrast, respiration is completely cyanide sensitive in pea indicating that the alternative respiratory pathway is absent. Cu deficiency in pea plants and in heterotrophically grown Scenedesmus cells interferes with respiratory activity of mitochondria. In both organisms, the cyanide-sensitive NADH oxidation was strongly decreased during cultivation in low Cu media. Cu sensitivity was also observed for the alternative respiratory pathway in Scenedesmus. These results suggest that a Cu-containing component is involved in the alternative respiratory pathway. This is the main reason why alternative respiration cannot be regarded as a compensation for low cytochrome-oxidase activities during Cu starvation. The Cu dependency of the cyanide-sensitive respiration was located at the site of cytochrome oxidase. A strong coordination of the biosynthesis of the Cu-containing cytochrome-oxidase complex was evident. When the endogenous Cu pool was low, formation of cytochrome aa3, another component of cytochrome oxidase, was also decreased.

Temperature-controlled alternative respiration and outgrowth rate from conidia ofNeurospora crassa

Incubation of conidia of wild typeNeurospora crassa at temperatures ranging from 25 to 46°C modulates their respiratory type. Between 37 and 41°C, the transient activity of the cyanide-insensitive respiratory pathway parallels, with a maximal extrusion of protons into the medium, the optimal rate of germ tube outgrowth.

Oxidation of durohydroquinone via the cyanide-insensitive respiratory pathway in higher plant mitochondria

FEBS LettersVolume 92, Issue 2 p. 317-320 Full-length articleFree Access Oxidation of durohydroquinone via the cyanide-insensitive respiratory pathway in higher plant mitochondria S. Huq, S. Huq Department of Botany, Imperial College, University of London, Prince Consort Road, London SW7 2BB EnglandSearch for more papers by this authorJ.M. Palmer, J.M. Palmer Department of Botany, Imperial College, University of London, Prince Consort Road, London SW7 2BB EnglandSearch for more papers by this author S. Huq, S. Huq Department of Botany, Imperial College, University of London, Prince Consort Road, London SW7 2BB EnglandSearch for more papers by this authorJ.M. Palmer, J.M. Palmer Department of Botany, Imperial College, University of London, Prince Consort Road, London SW7 2BB EnglandSearch for more papers by this author First published: August 15, 1978 https://doi.org/10.1016/0014-5793(78)80778-9Citations: 16AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume92, Issue2August 15, 1978Pages 317-320 ReferencesRelatedInformation

Contribution of a Cyanide-insensitive Alternate Respiratory System to Increases in Formamide Hydro-lyase Activity and to Growth in Stemphylium loti in Vitro

Stemphylium loti, a pathogen of a cyanogenic plant, possesses a cyanide-insensitive alternate respiratory pathway. In the absence of cytochrome inhibitors, the alternate system had only a minor role in respiration. When S. loti was grown in medium amended with antimycin to block the cytochrome chain, the alternate system accounted for the total oxygen consumption associated with respiration.The contribution of the alternate respiratory system to increases in formamide hydro-lyase (FHL) activity and to growth in S. loti in vitro was assessed. FHL, induced by cyanide, converts cyanide to nontoxic formamide and is partially responsible for the tolerance of S. loti to high concentrations of cyanide in vitro. When the cytochromes were blocked and the cyanide-insensitive respiratory pathway accounted for 100% of the oxygen uptake associated with respiration, FHL activity, but not changes in dry weight, was positively correlated with activity of the alternate pathway. As the alternate pathway activity decreased with increasing concentrations of salicylhydroxamic acid, the level of FHL activity correspondingly decreased. The alternate respiratory system may provide for increases in FHL activity but not for growth. S. loti appears to have two mechanisms for cyanide tolerance in vitro: cyanide-insensitive respiration and FHL activity. The initial activity of FHL for detoxification of cyanide may depend on the alternate respiratory pathway when the cytochromes of the electron transport chain are blocked.

Involvement of iron in the biogenesis of the cyanide-insensitive respiration in the yeast Saccharomycopsis lipolytica

The involvement of iron in the biogenesis of the cyanide-insensitive respiration in the yeast Saccharomycopsis lipolytica has been established on the following basis: (1) endogenous metal chelation by either benzyl- or salicylhydroxamic acid, EDTA or nitrilotriacetate prevented the biogenesis of the cyanide-insensitive respiratory pathway in S. lipolytica. (2) Addition of Fe(III) during the biogenesis increased both the rate of the appearance of the alternative respiratory pathway and its extent. Neither Fe(II), nor Co(II), Cu(II), Al(III), La(III), Mn(II) or Mg(II) could substitute for Fe(III). (3) The biogenesis of the alternative respiratory pathway could be dissociated into two steps: (a) a first one, slow, cycloheximide-sensitive, temperature-dependent, iron-independent, leading to cells still fully cyanide-sensitive, presumably involving the de novo biosynthesis of an inactive protein moiety and (b) a second step, fast, iron-dependent, temperature-independent, cycloheximide-insensitive, leading to cells with a cyanide-insensitive respiration, presumably the activation by iron of the inactive precursor.

Upsurge in Respiration and Peroxide Formation in Potato Tubers as Influenced by Ethylene, Propylene, and Cyanide

A continuous application of ethylene (10 mul/l) and propylene (500 mul/l) to potato tubers (Solanum tuberosum L.) resulted in an upsurge of respiration and a concomitant rise in peroxides. When applied in 100% O(2), the effect of ethylene and propylene on respiration and peroxide formation was augmented. Hydrogen cyanide (500 mul/l) mimicked the action of ethylene and propylene inducing a respiratory rise and a corresponding increase in peroxides. As with ethylene, the effect of HCN was augmented in high O(2) tensions. The results support the suggestion that ethylene activates the cyanide-insensitive respiratory pathway.

Potentiation of wound induced formation of ipomeamarone by cyanide insensitive respiration in sweet potato (Ipomoea batatas) root slices

Summary Ipomeamarone, a furanosesquiterpenoid stress metabolite of sweet potato root, accumulated in slices treated with 5 mM HgCl 2 . A fivefold increase in HgCl 2 -induction of ipomeamarone accumulation was observed in tissue treated with either ethylene (100 ppm), cyanide (10 mM) or carbon monoxide (1 mM) in the dark. Ethylene and its analogues did not induce ipomeamarone accumulation in the absence of mercuric chloride. Potassium isothiocyanate (10 mM) and 8-hydroxyquinoline sulfate (10mM), known inhibitors of cyanide-insensitive respiration, repressed HgCl 2 and HgCl 2 -cyanide induction of ipomeamarone. Also, induction of ipomeamarone formation by HgCl 2 was potentiated by aging tissue slices 24 hours prior to treatment. These data are consistent with the hypothesis that ethylene and ethylene analogues potentiate ipomeamarone accumulation by evoking the cyanide-insensitive respiratory pathway.