Mutations in Escherichia coli that effect sensitivity to oxygen

Jamison, Chaille; Adler, Howard I.

doi:10.1128/jb.169.11.5087-5094.1987

Cited by 28 publications

(13 citation statements)

References 39 publications

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“…Several independent mutants in the nrdA and -B genes of E. coli showing temperature or oxygen-sensitive phenotypes have been described (6,(14)(15)(16). This strongly suggests that the chromosomal nrdE and -F genes are silent under normal circumstances.…”

Section: Results Proteins Encoded By Nrde and Nrdf Have Ribonucleotidementioning

confidence: 99%

A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.

Jordan

Pontis

Atta

et al. 1994

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

108

139

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The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the Rl and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S.typhimurium nrdE and nrdF genes (RlE and R2F Ribonucleotide reductases catalyze the synthesis of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis. At least three separate classes of enzymes are known, each with a distinct protein structure but all requiring a protein radical for catalysis (1). The long-studied aerobic Escherichia coli enzyme is the prototype for class I enzymes, also present in all higher organisms and some other microorganisms. E. coli genes nrdA and nrdB encode the a and 8 polypeptide chains, respectively, that form the Rl (a2) and R2 (132) proteins that constitute the enzyme (2). Each protomer of the Rl dimer (2 x 85.7 kDa) contains one substrate-binding site with redox-active thiols involved in the reduction of the substrate ribonucleoside diphosphate, and two separate types of allosteric sites: one, the activity site, controls the overall activity of the enzyme, with ATP as a positive effector and dATP as a negative effector; the other, the substrate-specificity site, controls the specificity of the enzyme, with ATP and dATP favoring pyrimidine reduction, dTTP favoring GDP reduction, and dGTP favoring ADP reduction (3).The R2 dimer (2 x 43.4 kDa) contains two dinuclear iron centers with associated stable tyrosyl free radicals, located at Tyr-122 of the polypeptide chain (4). The drug hydroxyurea scavenges this radical and thereby inactivates the enzyme. Class II and III enzymes lack the tyrosyl radical. Class II enzymes, with the Lactobacillus leichmanni enzyme as a prototype, employ adenosylcobalamin as a radical generator, whereas class III enzymes use S-adenosylmethionine together with iron for this purpose.Salmonella typhimurium contains an active class I enzyme with amino acid sequences 96.5% and 98.4% identical to the E. coli Rl and R2 proteins, respectively (A.J., unpublished results). Recent genetic evidence involving complementation of nrd mutants of E. coli suggested the presence in S. typhimurium of the genes, nrdE and nrdF, coding for a second class I enzyme (5). These genes are also present on the chromosome of E. coli but are, under standard growth conditions, expressed only when present on a plasmid. The amino acid sequences deduced for the corresponding proteins showed a limited identity with other class I enzymes but contained many of their catalytically important residues.We have purified and characterized the two proteins encoded by the cloned genes nrdE and nrdF from S. typhimurium. Each protein is a homodimer. Together they catalyze the reductio...

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Section: Results Proteins Encoded By Nrde and Nrdf Have Ribonucleotidementioning

confidence: 99%

A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.

Jordan

Pontis

Atta

et al. 1994

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

108

139

View full text Add to dashboard Cite

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“…This suggests that facultative aerobes like E. coli may possess multiple regulatory mechanisms which respond to changes in oxygen availability and that different mechanisms may have different maxima. For example, enzymes such as superoxide dismutase are required under high oxygen concentrations (22), whereas several proteins are expressed as part of the fnr-activated regulon under strictly anaerobic conditions (8,23). In addition, there also exist mechanisms that activate proteins such as cytochrome d in response to decreasing oxygen concentrations (13).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli

Khosla

Bailey

1989

J Bacteriol

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The gene coding for the VitreoscUla hemoglobin (VHb) molecule has been cloned and functionally expressed in Escherichia coli. By using a plasmid-encoded gene as well as single-copy integrants, the oxygen-dependent VHb gene (VHb) promoter was shown to be functional in E. coli. The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation). Direct analysis of mRNA levels as well as the use of gene fusions with lacZ showed that oxygen-dependent regulation occurred at the level of transcription. Transcriptional activity decreased substantially under anaerobic conditions, suggesting the presence of a regulatory mechanism that is maximally induced under hypoxic but not completely anaerobic conditions in E. coli. Primer extension analysis was used to identify the existence of two overlapping promoters within a 150-base-pair region upstream of the structural VHb gene. The oxygen-dependent activity of both promoters was qualitatively similar, suggesting the existence of a common mechanism by which available oxygen concentrations influence expression from the two promoters. Analysis of promoter activity in crp and cya mutants showed that both cyclic AMP and catabolite activator protein were required for full activity of the promoter. The VHb promoter contained a region of signfficant homology to the catabolite activator protein-binding site near the E. coli lac promoter.

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“…Or, deoxyribonucleotides might be formed by other enzymes. The second alternative was favored strongly from genetic experiments (11)(12)(13) demonstrating that mutations in the nrdA or nrdB gene, coding for protein B1 or B2, did not affect the anaerobic growth of the bacteria. These experiments did, however, not distinguish between a different ribonucleotide reductase or a completely different mode of synthesis-e.g., via the deoxyribose 5-phosphate aldolase pathway (15).…”

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confidence: 99%

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Fontecave

Eliasson

Reichard

1989

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

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Escherichia coli obtains the deoxyribonucleotides required for DNA synthesis from ribonucleotides through the activity of the enzyme ribonucleoside diphosphate reductase (1-3). A closely related enzyme has the same function in mammalian cells. Both enzymes consist of two nonidentical subunits, proteins B1 and B2 (4) of E. coli and the corresponding mammalian proteins Ml and M2 (5). B2 and M2 contain a tyrosyl radical (6-8) that has arisen by oxidation of a specific tyrosine residue of the polypeptide chain. An oxygenrequiring enzyme system catalyzes the formation of this radical in E. coli (9, 10). However, E. coli grows well also under anaerobic conditions. This poses the question how deoxyribonucleotides are synthesized in the absence of oxygen. One could imagine that during anaerobiosis an electron acceptor other than oxygen might function in radical generation. Or, deoxyribonucleotides might be formed by other enzymes. The second alternative was favored strongly from genetic experiments (11-13) demonstrating that mutations in the nrdA or nrdB gene, coding for protein B1 or B2, did not affect the anaerobic growth of the bacteria. These experiments did, however, not distinguish between a different ribonucleotide reductase or a completely different mode of synthesis-e.g., via the deoxyribose 5-phosphate aldolase pathway (15).Here we first establish from isotope experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. We then describe the existence of a different reductase in extracts from anaerobically grown bacteria. The enzyme is not inhibited by antibodies raised against protein B1 or B2 and is only moderately sensitive to hydroxyurea, a drug that strongly inhibits the aerobic reductase, but is rapidly inactivated by air. The substrate of the reaction appears to be CTP and not CDP, as for the aerobic reaction. MATERIALS AND METHODSBacterial Strains. The E. coli K-12 strain AB1157 and its oxygen-sensitive derivative Oxys-11 (11) were obtained from H. I. Adler (Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN). E. coli So1452, a strain with an insertion mutation in the gene for cytidine deaminase, was a gift from B. Mygind (Institute for Biological Chemistry, University of Copenhagen, Denmark).Preparation of Bacterial Extracts. Five-liter cultures of AB1157 or Oxys-11 were grown anaerobically in Luria broth containing 1 g of glucose per liter under continuous bubbling with 4% C02/96% N2 in a Microferm New Brunswick fermentor with constant pH control. When the bacteria had reached midlogarithmic phase (OD = 1.0 at 640 nm) they were harvested by centrifugation in twelve 0.5-liter bottles in cold Sorvall centrifuges, resuspended under a flow of argon in a small volume of an anaerobic 50 mM Tris buffer (pH 7.5), and transferred under argon to four preweighed capped Beckman no. 355603 thick-walled polycarbonate tubes. After centrifugation and removal of the supernatant solution, the weighed bacteria (1-2 g per tube) were stored under liquid...

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Mutations in Escherichia coli that effect sensitivity to oxygen

Abstract: Fifteen oxygen-sensitive (Oxys)

Cited by 28 publications

References 39 publications

A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.

A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.

Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

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