PubMed Health⌕ Search

Biomedical subjects

R K Poole

Publications and source records attributed to R K Poole.

At least 55 records · Page 3Linked to original sources
PubMed1997

Transcriptional regulation of the cydDC operon, encoding a heterodimeric ABC transporter required for assembly of cytochromes c and bd in Escherichia coli K-12: regulation by oxygen and alternative electron acceptors.

The expression of the cydDC operon was investigated by using a chromosomal phi(cydD-lacZ) transcriptional fusion and primer extension analysis. A single transcriptional start site was found for cydD located 68 bp upstream of the translational start site, and Northern blot analysis confirmed that cydDC is transcribed as a polycistronic message independently of the upstream gene trxB. cydDC was highly expressed under aerobic growth conditions and during anaerobic growth with alternative electron acceptors. Aerobic expression was independent of ArcA and Fnr, but induction of cydDC by nitrate and nitrite was dependent on NarL and Fnr.

ATP-Binding Cassette Transporters↗
PubMed1996

The respiratory chain of Helicobacter pylori: identification of cytochromes and the effects of oxygen on cytochrome and menaquinone levels.

The quinone and cytochrome components of the respiratory chain of the microaerophilic bacterium Helicobacter pylori have been investigated. The major isoprenoid quinone was menaquinone-6, with traces of menaquinone-4; no methyl-substituted or unusual menaquinone species were found. Cell yield was highest after growth at 10% (v/v) oxygen and menaquinone levels (per dry cell mass) were maximal at 5-10% (v/v) oxygen. Helicobacter pylori cells and membranes contained b- and c-type cytochromes, but not terminal oxidases of the a- or d-types, as judged by reduced minus oxidised difference spectra. Spectra consistent with the presence of a CO-binding terminal oxidase of the cytochrome b- or o-type were obtained. The soluble fraction from disrupted cells also contained cytochrome c. There were no significant qualitative differences in the cytochrome complements of cells grown at oxygen concentrations in the range 2-15% (v/v) but putative oxidases were highest in cells grown at 5-10% (v/v) oxygen.

Cytochromes↗
PubMed1996

The flavohaemoglobin (HMP) of Escherichia coli generates superoxide in vitro and causes oxidative stress in vivo.

Purified flavohaemoglobin (HMP) of Escherichia coli reduces Fe(III) in a superoxide dismutase (SOD)-sensitive reaction, demonstrating superoxide anion generation during aerobic NADH oxidation. In vivo, sodA-lacZ fusion activity was increased 3-fold by introducing plasmid pPL341, containing the hmp gene, or by growth with paraquat. The effects were additive and SOXS-dependent. Thus HMP activity causes oxidative stress in vivo. Activities of sodA-lacZ and hmp-lacZ fusions were stimulated in a himA mutant, demonstrating repression of both promoters by integration host factor (IHF), but the effects of pPL341 on sodA-lacZ activity were not due to titration of IHF by the hmp promoter.

Aerobiosis↗
PubMed1996

The cytochrome bc1 complex but not CycM is necessary for symbiotic nitrogen fixation by Rhizobium leguminosarum.

Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, two mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules. Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes aa3, but increased levels of cytochrome d. Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate- and NADH-dependent respiration. NADH oxidation by the mutants was insensitive to the respiratory inhibitor antimycin A, that targets the cytochrome bc1 complex. Molecular analysis of the mutants revealed that they were affected in the cytochrome bc1 complex. One of the mutants contained Tn5 in a gene homologous to that encoding cytochrome c1, and in the other the Tn5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc1 complex. Haem staining revealed that haem proteins of M(r)31,000 and M(r)23,000 were absent from membranes from the mutants whereas an additional soluble c-type cytochrome protein of M(r)23,000 was present. We conclude that the larger of these two haem proteins corresponds to cytochrome c1 and, in its absence, the protein of M(r)23,000 does not remain associated with the membrane. Formation of this M(r)23,000 component was specifically blocked in a third respiratory-defective mutant which contained Tn5 in a region of DNA showing homology to a Bradyrhizobium Japonicum gene previously shown to encode the membrane-bound c-type cytochrome CycM. Although the cytochrome bc1 complex is essential for symbiotic nitrogen fixation, the other membrane-bound c-type cytochrome (CycM) is not.

Amino Acid Sequence↗
PubMed1996

Nisin stimulates oxygen consumption by Staphylococcus aureus and Escherichia coli.

Nisin stimulated oxygen consumption by nongrowing, glucose-metabolizing Staphylococcus aureus and Escherichia coli cells, indicating a protonophore mode of action. A similar stimulation in E. coli cells osmotically stressed to disrupt the outer cell membrane confirmed the cytoplasmic membrane as the site of nisin action and showed that nisin uptake was not prevented by the outer membrane.

Escherichia coli↗
PubMed1996

Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12.

Escherichia coli possesses a soluble flavohemoglobin, with an unknown function, encoded by the hmp gene. A monolysogen containing an hmp-lacZ operon fusion was constructed to determine how the hmp promoter is regulated in response to heme ligands (O2, NO) or the presence of anaerobically utilized electron acceptors (nitrate, nitrite). Expression of the phi (hmp-lacZ)1 fusion was similar during aerobic growth in minimal medium containing glucose, glycerol, maltose, or sorbitol as a carbon source. Mutations in cya (encoding adenylate cyclase) or changes in medium pH between 5 and 9 were without effect on aerobic expression. Levels of aerobic and anaerobic expression in glucose-containing minimal media were similar; both were unaffected by an arcA mutation. Anaerobic, but not aerobic, expression of phi (hmp-lacZ)1 was stimulated three- to four-fold by an fnr mutation; an apparent Fnr-binding site is present in the hmp promoter. Iron depletion of rich broth medium by the chelator 2'2'-dipyridyl (0.1 mM) enhanced hmp expression 40-fold under anaerobic conditions, tentatively attributed to effects on Fnr. At a higher chelator concentration (0.4 mM), hmp expression was also stimulated aerobically. Anaerobic expression was stimulated 6-fold by the presence of nitrate and 25-fold by the presence of nitrite. Induction by nitrate or nitrite was unaffected by narL and/or narP mutations, demonstrating regulation of hmp by these ions via mechanisms alternative to those implicated in the regulation of other respiratory genes. Nitric oxide (10 to 20 microM) stimulated aerobic phi (hmp-lacZ)1 activity by up to 19-fold; soxS and soxR mutations only slightly reduced the NO effect. We conclude that hmp expression is negatively regulated by Fnr under anaerobic conditions and that additional regulatory mechanisms are involved in the responses to oxygen, nitrogen compounds, and iron availability. Hmp is implicated in reactions with small nitrogen compounds.

Aerobiosis↗
PubMed1995

Inhibition of the cytochrome bd-terminated NADH oxidase system in Escherichia coli K-12 by divalent metal cations.

Co(II), Zn(II) and Cd(II) ions inhibited NADH oxidase activity in membranes prepared from two cytochrome bo'-deficient mutants of Escherichia coli K-12 with the following order of potency: Zn(II) > Cd(II) >> Co(II). The degree of inhibition exhibited by these metal ions was not diminished in membranes which contained elevated levels of the cytochrome bd complex, suggesting that the most sensitive site precedes this complex in the aerobic respiratory chain. For each of the metal ions studied, inhibition was determined to be of the non-competitive type. Based upon the efficacy with which EDTA alleviated inhibition, Co(II), Zn(II) and Cd(II) ions are proposed to inhibit NADH oxidase activity by binding to at least two sites in the respiratory chain with significantly different affinities.

Binding Sites↗
PubMed1995

Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori.

Oxygen uptake by Helicobacter pylori cells and membranes was determined. Cells from stirred broth cultures or agar plates, suspended in buffer, possessed a variable and apparently endogenous respiration which could be sustained for several hours. In contrast, oxygen consumption by cells from statically incubated broth cultures, in the absence of added substrate, was transient or undetectable. These latter cells, however, oxidised ethanol, fumarate, glucose, D-lactate, pyruvate and succinate, though glucose-oxidising ability declined rapidly. The Kms for D-lactate, pyruvate and succinate metabolism were low (< or = 20 microM) and oxygen uptake was approximately 1.5, 2 and 2 mol per mol substrate respectively, indicating metabolism beyond acetate plus CO2 and implying the presence of tricarboxylic acid cycle activity. Cell membranes oxidised fumarate, D-lactate, NADH, NADPH and succinate. NADPH oxidation was six times more rapid than that of NADH. Rates of oxygen uptake by cells suspended in buffer with metabolisable substrate were < 20% of those for cells suspended in a brain heart infusion medium. Uninoculated medium consumed significant quantities of oxygen.

Cell Membrane↗
PubMed1995

Distribution of the flavohaemoglobin, HMP, between periplasm and cytoplasm in Escherichia coli.

The subcellular distribution of the soluble flavohaemoglobin (HMP) of Escherichia coli has been determined. Cells over-expressing HMP from the cloned hmp gene on a multicopy plasmid were fractionated by osmotic shock and lysozyme treatment. Spectral analysis of subcellular fractions showed the CO-binding haemoprotein to be cytoplasmic. However, Western blotting using antibody raised to purified HMP revealed approximately 30% of the protein to be periplasmic in the over-expressing strain. Western analysis also revealed substantial levels of periplasmic HMP in a strain expressing only chromosomally encoded protein but none in an hmp mutant. The results are discussed in relation to protein function and the similar distribution reported for Vitreoscilla globin.

Amino Acid Sequence↗
PubMed1995

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.

Amino Acid Sequence↗
PubMed1995

The oxygen affinity of cytochrome bo' in Escherichia coli determined by the deoxygenation of oxyleghemoglobin and oxymyoglobin: Km values for oxygen are in the submicromolar range.

Apparent oxygen affinities for Escherichia coli cells and membranes containing a terminal oxidase with only one type of ligand-binding heme, cytochrome o', were measured with oxyleghemoglobin and oxymyoglobin as sensitive oxygen reporters. Two Km values (0.15 to 0.35 microM and 0.016 to 0.085 microM) were detected, well below values determined for the purified oxidase by insensitive electrode methods.

Cytochrome a Group↗
PubMed1994

Mutagenesis of a gene encoding a cytochrome o-like terminal oxidase of Azotobacter vinelandii: a cytochrome o mutant is aero-tolerant during nitrogen fixation.

The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo-type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A. vinelandii by homologous recombination. The mutant contained no spectrally detectable cytochrome o. However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd) was 11-fold higher than in the wild-type strain. Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o. Cytochrome o-deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd, which is required for respiratory protection of oxygen-labile nitrogenase.

Amino Acid Sequence↗
PubMed1994

The cydD gene product, component of a heterodimeric ABC transporter, is required for assembly of periplasmic cytochrome c and of cytochrome bd in Escherichia coli.

The cydD gene of Escherichia coli encodes a protein which, together with the CydC protein, probably constitutes a heterodimeric, ABC-family membrane transporter, necessary for biosynthesis of the cytochrome bd quinol oxidase. Here, we demonstrate that a cydD mutant also fails to synthesise periplasmic c-type cytochrome(s), suggesting that the transporter exports haem or some other component involved in assembly of cytochromes that are found in, or exposed to, the periplasm. The CydDC system appears to be the first example of a transporter required for periplasmic cytochrome assembly processes requiring more than one type of haem. A mutant defective in trxB (adjacent to the cydDC operon, and encoding thioredoxin reductase) was unaffected in cytochrome c or bd assembly.

Adenosine Triphosphatases↗
PubMed1994

Reactions of the Escherichia coli flavohaemoglobin (Hmp) with oxygen and reduced nicotinamide adenine dinucleotide: evidence for oxygen switching of flavin oxidoreduction and a mechanism for oxygen sensing.

The soluble flavohaemoglobin (Hmp) of Escherichia coli contains haem B and FAD in a single 44 kDa polypeptide, and shows NADH oxidase activity. The oxidized protein reacted rapidly with NADH in the presence of O2 to form an oxygenated species while the flavin remained largely oxidized. Spectral and kinetic analyses revealed rapid biphasic reduction and oxygenation of high-spin haem with apparent relaxation times of 6 and 64 ms at pH 8 and 25 degrees c, suggestive of a significant physiological role for the protein. This was followed by a monophasic reduction of the flavin with a relaxation time of 92 ms. On exhaustion of oxygen, the oxygenated haem was converted into the deoxy form biphasically with relaxation times of 43 and 170 s, followed by extensive reduction of the flavin with corresponding relaxation times of 70 and 256 s. Based on these observations, we propose that Hmp could act as an oxygen sensor in E. coli by combining with intracellular oxygen, thus limiting flavin reduction in the aerobic steady state. Lowering of the oxygen concentration causes dissociation of the oxy species and sustained flavin reduction. Because Hmp can reduce Fe(III), such a mechanism might control, for example, flavin-mediated Fe(III) reduction required for activation of the anaerobic gene regulator, Fnr.

Bacterial Proteins↗
PubMed1994

Oxygen reactions with bacterial oxidases and globins: binding, reduction and regulation.

Oxygen is favoured as terminal electron acceptor in aerobic and facultative microorganisms because of its appropriate physical state, satisfactory solubility and its desirable combinations of kinetic and thermodynamic properties. Oxygen is generally reduced by four electrons to yield oxygen, but there are important biological consequences of, and roles for, the partial reduction to superoxide and peroxide. Complex and multiple regulatory networks ensure (i) the utilization of oxygen in preference to other oxidants, (ii) the synthesis of oxygen-consuming enzymes with appropriate properties (particularly affinity for the ligand), and (iii) appropriate cellular protection in the event of oxidative stress. This contribution reviews the terminal respiratory oxidases of selected Gram-negative bacteria and microbial haemoglobin-like proteins. Recent studies of the cytochrome bd-type oxidases of Escherichia coli and Azotobacter vinelandii suggest that, despite probable similarity at the amino acid level, the reactivities of these oxidases with oxygen are strikingly different. The respiratory protection afforded to nitrogenase in the obligately aerobic diazotroph A. vinelandii by the cytochrome bd complex appears to be accompanied by, and may be the result of, a low affinity for oxygen and a high Vmax. The poorly characterized cytochrome o-containing oxidase in this bacterium is not required for respiratory protection. In E. coli, the cytochrome bd-type oxidase has a remarkably high affinity for oxygen, consistent with the view that this is an oxygen-scavenging oxidase utilized under microaerobic conditions. The demonstration of substrate (i.e. oxygen) inhibition in this complex suggests a mechanism whereby wasteful electron flux through a non-proton-pumping oxidase is avoided at higher dissolved oxygen tensions. The demonstration of two ligand-binding sites (haems d and b595) in oxidases of this type suggests plausible mechanisms for this phenomenon. In E. coli, assembly of the cytochrome bd-type oxidase (and of periplasmic cytochromes b and c) requires the presence of an ABC transporter, which may serve to export haem or some "assembly factor" to the periplasm. There is at least one additional oxygen-consuming protein in E. coli-the flavohaemoglobin encoded by the hmp gene. Globin-like proteins are also widely distributed in other bacteria, fungi and protozoa, but most have unknown functions. The function of HMP and the related chimaeric flavohaemoglobins in other bacteria and yeast is unknown; one of several possibilities for HMP is that its relatively low affinity for oxygen during turnover with NADH as substrate could enable it to function as a sensor of failing (or rising) cytoplasmic oxygen concentrations.

Bacteria↗
PubMed1994

The high-spin cytochrome o' component of the cytochrome bo-type quinol oxidase in membranes from Escherichia coli: formation of the primary oxygenated species at low temperatures is characterized by a slow 'on' rate and low dissociation constant.

Cytochromes b and o in membrane vesicles from aerobically grown Escherichia coli were readily reduced by succinate; one cytochrome, which we propose should be called cytochrome o', reacted with CO in the Fe(II) state to give a photodissociable CO adduct. The photodissociation spectrum (photolysed minus pre-photolysis) at sub-zero temperatures had a relatively high gamma/alpha absorbance ratio, indicating a high-spin haem, which, in the reduced state, probably contributes little to the sharp alpha absorbance of the oxidase complex in membranes. Reaction with oxygen of the unliganded high-spin haem between -132 degrees C and -95 degrees C following photolytic activation gave a product that is identified as the oxygenated form, being spectrally similar to, but not identical with, the CO adduct. In membranes, the forward velocity constant at -95 degrees C was 61 M-1s-1, and the dissociation constant was 1.6 x 10(-5) M O2, as it is in intact cells. These data clearly distinguish the oxygen-trapping strategy of the cytochrome o' in this oxidase from that of cytochrome a3 and also suggest that the presence of the soluble flavohaemoglobin (Hmp) in intact cells is without effect on such measurements of the primary oxygen reaction. In view of recent findings that this oxidase complex contains predominantly one mole of haem O and one of haem B, a revised nomenclature for the oxidase complex is proposed, namely, cytochrome bo'.

Carbon Monoxide↗