Vitamin K-deficient Mutants of Bacteria

Săsárman, A; Surdeanu, M; Portelance, V.; Dobardzic, R.; Sonea, S

doi:10.1038/224272a0

Cited by 15 publications

(16 citation statements)

References 2 publications

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“…Induction of protein expression by SASX41B E. coli, auxotrophic for ␦-aminolevulinic acid and therefore heme deficient (20), was as described by Berthold (21). Alternative oxidase protein production was induced by growth of cells without the heme precursor and with vigorous aeration (21).…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Cyanide-resistant Alternative Oxidase of Plant Mitochondria

Rhoads

Umbach

Sweet

et al. 1998

Journal of Biological Chemistry

149

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The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by ␣-keto acids. After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate. Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with ␣-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by ␣-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of ␣-keto acid activation and of the regulatory sulfhydryl/disulfide system.In all plants and many fungi, a cyanide-resistant, alternative respiratory pathway branches from the cytochrome pathway at the ubiquinone pool in mitochondria (1). Electron flow through this alternative pathway is not coupled to ATP synthesis at two of the three proton translocation sites. In specific thermogenic floral tissues of some plants, a high rate of electron flow through the alternative pathway generates heat and volatilizes compounds that attract insect pollinators (2). In non-thermogenic plant tissues, the alternative pathway may allow continued turnover of carbon skeletons through glycolysis and the tricarboxylic acid cycle when the ATP concentration is high and inhibits ATP synthase activity (3) so that the pathway can function as an energy overflow mechanism (4). However, because the alternative pathway can be active even when the cytochrome pathway is not saturated (5-7), it is likely that the two pathways are regulated in concert to balance ubiquinone pool oxidation/reduction and carbon skeleton turnover in response to cytosolic ATP levels (8, 9).In recent years, a great deal of progress has been made in understanding the post-translational regulation of the alternative oxidase, the terminal oxidase that comprises the alternative respiratory pathway. The alternative oxidase is a homodimeric protein in the inner mitochondrial membrane and the oxidation/reduction state of an intersubunit disulfide bond has been demonstrated to regulate its activity (10, 11). A second mechanism of activation of the alternative oxidase involves ␣-keto acids, notably pyruvate (12), which significan...

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Section: Methodsmentioning

confidence: 99%

Regulation of the Cyanide-resistant Alternative Oxidase of Plant Mitochondria

Rhoads

Umbach

Sweet

et al. 1998

Journal of Biological Chemistry

149

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“…The E. coli strains used were DH5a and the 5-aminolevulinic acid auxotroph SASX41B (HfrPO2A hemA41 metBI relAI) (15). The strains were grown on M9 minimal medium (14) (FP), ubiquinones, and cytochromes and finally are oxidized to form water (A), and in cyanide-resistant respiration, electrons deviate from the cytochrome pathway at ubiquinone and are oxidized by alternative oxidase (B).…”

Section: Methodsmentioning

confidence: 99%

Arabidopsis alternative oxidase sustains Escherichia coli respiration.

Kumar

Söll

1992

Proc. Natl. Acad. Sci. U.S.A.

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Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor.

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“…Mutations producing resistance include ones affecting the synthesis of heme (27,29), menaquinone (26,28), cytochromes (6,28), and the proton-translocating adenosine triphosphatase (ATPase) (19). Because these mutations do not alter the ribosomal site of aminoglycoside action nor appear to result in inactivation of the antibiotics, it has been assumed that resistance in such mutants is due to reduced uptake of the drugs into the cells.…”

mentioning

confidence: 99%

Role of the membrane potential in bacterial resistance to aminoglycoside antibiotics

Damper¹,

Epstein²

1981

Antimicrob Agents Chemother

154

133

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The electrical potential difference (A0) across the membrane of Escherichia coli was measured by the distribution of lipid-soluble cations and correlated with resistance to dihydrostreptomycin, where resistance is presumed due to reduced uptake of the drug. A good correlation between the two measured parameters was found under all conditions tested, which included effects of several mutations, inhibitors, changes in pH, and osmolarity. The most dramatic changes were seen when pH was varied; in wild-type strains resistance increased more than 100-fold, and Aip fell by 70 mV when pH was reduced from 8.5 to 5.5. These results were interpreted as support for a model in which the uptake of the polycationic aminoglycosides is electrogenic and therefore driven by AO. The factor common to mutations and conditions which increase resistance was a reduction in A4+. A simple model was developed which relates the miniimal inhibitory concentration to the rate of aminoglycoside uptake and the rate of growth.

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Vitamin K-deficient Mutants of Bacteria

Cited by 15 publications

References 2 publications

Regulation of the Cyanide-resistant Alternative Oxidase of Plant Mitochondria

Regulation of the Cyanide-resistant Alternative Oxidase of Plant Mitochondria

Arabidopsis alternative oxidase sustains Escherichia coli respiration.

Role of the membrane potential in bacterial resistance to aminoglycoside antibiotics

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