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Biomedical subjects

M Bott

Publications and source records attributed to M Bott.

At least 37 records · Page 2Linked to original sources
PubMed1994

Purification of two active fusion proteins of the Na(+)-dependent citrate carrier of Klebsiella pneumoniae.

The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified by means of bioengineerical methods. By fusing the biotin acceptor domain of the alpha-subunit of the oxaloacetate decarboxylase of K. pneumoniae to the C-terminus of CitS, purification of the carrier was achieved by use of a monomeric avidin-Sepharose column. Additionally, we were able to purify a CitS-protein with an N-terminal histidine-tag by immobilized metal chelate affinity chromatography (with Ni2(+)-nitrilotriacetic acid-(NTA-) resin). Both purified fusion proteins showed citrate transport activity after reconstitution into liposomes by the freeze/thaw/sonication procedure.

Bacterial Proteins↗
PubMed1994

Klebsiella pneumoniae genes for citrate lyase and citrate lyase ligase: localization, sequencing, and expression.

In the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium-dependent citrate carrier (citS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probably form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the gamma-, beta-, and alpha-subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated M(r) 38,476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC-expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5-phosphoribosyl-dephospho-acetyl-coenzyme A which is covalently bound to serine-14 of the acyl carrier protein (gamma-subunit). CitC was thus identified as acetate:SH-citrate lyase ligase. The function of the gene product derived from citG (M(r) 32,645) has not yet been identified. Expression of the citCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl-coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo-citrate lyase.

Acetyl Coenzyme A↗
PubMed1993

Bradyrhizobium japonicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochrome aa3 and development of symbiosis.

We report the discovery of a bacterial gene, tlpA, that codes for a hitherto unknown type of thioredoxin-like protein. The gene was found in the course of studying a Tn5 insertion mutant of the soybean root nodule symbiont Bradyrhizobium japonicum. The TlpA protein shared up to 31% amino acid sequence identity with various eukaryotic and prokaryotic thioredoxins and protein disulfide isomerases, and possessed a characteristic active-site sequence, Trp-Cys-Val-Pro-Cys. In contrast to all members of the thioredoxin family known to date, TlpA was shown to be anchored to the cytoplasmic membrane by means of an N-terminal transmembrane domain, while the active site-containing part of the protein faced the periplasm. The tlpA mutant had a pleiotropic phenotype in that it was defective in the development of a nitrogen fixing endosymbiosis and exhibited a strongly decreased oxidase activity, as compared with the wild-type. Holocytochrome aa3 was spectroscopically undetectable in the mutant, whereas the apoprotein of subunit one (CoxA) of this oxidase was still synthesized and incorporated into the cytoplasmic membrane. Since cytochrome aa3 is not a prerequisite for the development of symbiosis, the results suggest that TlpA is involved in at least two independent cellular processes, one of which is an essential periplasmic step in the maturation of cytochrome aa3.

Alkaline Phosphatase↗
PubMed1993

Formation of several bacterial c-type cytochromes requires a novel membrane-anchored protein that faces the periplasm.

We report here the discovery of a novel bacterial gene (cycH) whose product is involved in the biogenesis of most of the cellular cytochromes c. The cycH gene was detected in the course of characterizing a cytochrome oxidase-deficient Bradyrhizobium japonicum Tn5 mutant (strain COX3) in which the transposon insertion disrupted cycH. All of the c-type cytochromes detectable in aerobically grown B. japonicum wild-type cells were absent in the COX3 mutant, with the exception of cytochrome c1. A secondary phenotypic effect was the spectroscopic absence of the aa3-type cytochrome c oxidase. The nucleotide sequence of the cloned wild-type cycH gene predicted a membrane-bound 369-amino-acid protein with an M(r) of 39727. Results from studies on its membrane topology suggested that approximately 110 N-terminal amino acids are involved in anchoring the protein in the membrane, whereas the remaining two-thirds of the protein are exposed to the periplasm. We postulate that the CycH protein plays an essential role in an as yet unidentified periplasmic step in the biogenesis of holocytochromes c, except that of cytochrome c1.

Aerobiosis↗
PubMed1993

Situational supports and job stress: a correlational study.

This correlational study provides information for creating a nursing work environment that supports coping with stress. Questionnaire-generated data were collected from registered staff nurses (N = 538) in two urban, acute care hospitals. Results of multiple regression analysis indicated that situational support variables (autonomy, control over practice, group cohesion, substantive exchange, manager consideration) correlated inversely with job stress. Predictor variables and demographics explained 37% of the variance in situational job stress [F(13, 442) = 19.74, P < .001] and 28% of the variance in personal job stress [F(13, 442) = 13.03, P < .001]. The variables may serve as situational supports for nurses coping with job stress.

Adaptation, Psychological↗
PubMed1992

Genes for a second terminal oxidase in Bradyrhizobium japonicum.

Bradyrhizobium japonicum possesses a mitochondria-like respiratory chain terminating with an aa3-type cytochrome c oxidase. The gene for subunit I of this enzyme (coxA) had been identified and cloned previously via heterologous hybridization using a Paracoccus denitrificans DNA probe. In the course of these studies, another B. japonicum DNA region was discovered which apparently encoded a second terminal oxidase that was different from cytochrome aa3 but also belonged to the superfamily of heme/copper oxidases. Nucleotide sequence analysis revealed a cluster of at least four genes, coxMNOP, organized most probably in an operon. The predicted coxM gene product shared significant similarity with subunit II of cytochrome c oxidases from other organisms: in particular, all of the proposed CuA ligands were conserved as well as three of the four acidic amino acid residues that might be involved in the binding of cytochrome c. The coxN gene encoded a polypeptide with about 40% sequence identity with subunit I representatives including the previously found CoxA protein: the six presumed histidine ligands of the prosthetic groups (two hemes and CuB) were strictly conserved. A remarkable feature of the DNA sequence was the presence of two genes, coxO and coxP, whose products were both homologous to subunit III proteins. A B. japonicum coxN mutant strain was created by marker exchange mutagenesis which, however, exhibited no obvious defects in free-living, aerobic growth or in root nodule symbiosis with soybean. This shows that the coxMNOP genes are not essential for respiration in the N2 fixing bacteroid.

Amino Acid Sequence↗
PubMed1992

The adenosine analogue N6-L-phenylisopropyladenosine inhibits catecholamine secretion from bovine adrenal medulla cells by inhibiting calcium influx.

We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine-induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6-L-phenylisopropyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium, or by elevated external K+. The half-maximal effect on 1,1-dimethyl-4-phenylpiperazinium-induced secretion occurred at approximately 5 x 10(-5) M. The inhibition is immediate and reversible. Fura-2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium-evoked secretion from digitonin-permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage-sensitive calcium channel.

Adrenal Medulla↗
PubMed1991

The Bradyrhizobium japonicum cycM gene encodes a membrane-anchored homolog of mitochondrial cytochrome c.

Mitochondrial cytochrome c is a water-soluble protein in the intermembrane space which catalyzes electron transfer from the cytochrome bc1 complex to the terminal oxidase cytochrome aa3. In Bradyrhizobium japonicum, a gene (cycM) which apparently encodes a membrane-anchored homolog of mitochondrial cytochrome c was discovered. The apoprotein deduced from the nucleotide sequence of the cycM gene consists of 184 amino acids with a calculated Mr of 19,098 and an isoelectric point of 8.35. At the N-terminal end (positions 9 to 31), there was a strongly hydrophobic domain which, by forming a transmembrane helix, could serve first as a transport signal and then as a membrane anchor. The rest of the protein was hydrophilic and, starting at position 72, shared about 50% sequence identity with mitochondrial cytochrome c. The heme-binding-site motif Cys-Gly-Ala-Cys-His was located at positions 84 to 88. A B. japonicum cycM insertion mutant (COX122) exhibited an oxidase-negative phenotype and apparently lacked cytochrome aa3 in addition to the CycM protein. The wild-type phenotype with respect to all characteristics tested was restored by providing the cycM gene in trans. The data supported the conclusion that the assembly of cytochrome aa3 depended on the prior incorporation of the CycM protein in the cytoplasmic membrane.

Amino Acid Sequence↗
PubMed1990

Genetic analysis of the cytochrome c-aa3 branch of the Bradyrhizobium japonicum respiratory chain.

Further genetic evidence is provided here that Bradyrhizobium japonicum possesses a mitochondria-like electron-transport pathway: 2[H]----UQ----bc1----c----aa3----O2. Two Tn5-induced mutants, COX122 and COX132, having cytochrome c oxidase-negative phenotypes, were obtained and characterized. Mutant COX122 was defective in a novel gene, named cycM, which was responsible for the synthesis of a c-type cytochrome with an Mr of 20,000 (20K). This 20K cytochrome c appeared to catalyse electron transport from the cytochrome bc1 complex to the aa3-type terminal oxidase and, unlike mitochondrial cytochrome c, was membrane-bound in B. japonicum. The Tn5 insertion of mutant COX132 was localized in coxA, the structural gene for subunit I of cytochrome aa3. This finding also led to the cloning and sequencing of the corresponding wild-type coxA gene that encoded a 541-amino-acid protein with a predicted Mr of 59,247. The CoxA protein shared about 60% sequence identity with the cytochrome aa3 subunit I of mitochondria. The B. japonicum cycM and coxA mutants were able to fix nitrogen in symbiosis with soybean (Fix+). In contrast, mutants described previously which lacked the bc1 complex did not develop into endosymbiotic bacteroids and were thus Fix-. The data suggest that a symbiosis-specific respiratory chain exists in B. japonicum in which the electrons branch off at the bc1 complex.

Amino Acid Sequence↗
PubMed1989

Proton translocation coupled to the oxidation of carbon monoxide to CO2 and H2 in Methanosarcina barkeri.

Cell suspensions of acetate-grown Methanosarcina barkeri mediate the conversion of CO and H2O to CO2 and H2. The reaction is coupled with the phosphorylation of ADP. Evidence is presented that CO oxidation by the cells is associated with the transient acidification of the suspension medium. Up to 2 mol vectorial protons were measured/mol CO oxidized when the transmembrane electrical gradient was kept low by the addition of valinomycin (20 nmol/mg protein) and KCl (200 mM) or of KSCN (50 mM). No transient acidification was observed in the presence of the protonophore tetrachlorosalicylanilide which stimulated rather than inhibited CO oxidation. Proton extrusion remained unaltered when the proton-translocating ATPase was specifically inhibited by dicyclohexylcarbodiimide. The latter finding indicates that proton translocation is associated with CO conversion to CO2 and H2 rather than with ATP hydrolysis in the cells. The data substantiate that the coupling of CO oxidation with ADP phosphorylation in M. barkeri occurs via a chemiosmotic mechanism.

Archaea↗
PubMed1987

Proton-motive-force-driven formation of CO from CO2 and H2 in methanogenic bacteria.

Cell suspensions of methanogenic bacteria (Methanosarcina barkeri, Methanospirillum hungatei, Methano-brevibacter arboriphilus, and Methanobacterium thermoautotrophicum) were found to form CO from CO2 and H2 according to the reaction: CO2 + H2----CO + H2O; delta G0 = +20 kJ/mol. Up to 15,000 ppm CO in the gas phase were reached which is significantly higher than the equilibrium concentration calculated from delta G0 (95 ppm under the experimental conditions). This indicated that CO2 reduction with H2 to CO is energy-driven and indeed the cells only generated CO when forming CH4. The coupling of the two reactions was studied in more detail with acetate-grown cells of M. barkeri using methanogenic substrates. The effects of the protonophore tetrachlorosalicylanilide (TCS) and of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide (cHxN)2C were determined. TCS completely inhibited CO formation from CO2 and H2 without affecting methanogenesis from CH3OH and H2. In the presence of the protonophore the proton motive force delta p and the intracellular ATP concentration were very low. (cHxN)2C, which partially inhibited methanogenesis from CH3OH and H2, had no effect on CO2 reduction to CO. In the presence of (cHxN)2C delta p was high and the intracellular ATP content was low. These findings suggest that the endergonic formation of CO from CO2 and H2 is coupled to the exergonic formation of CH4 from CH3OH and H2 via the proton motive force and not via ATP. CO formation was not stimulated by the addition of sodium ions.

Adenosine Triphosphate↗
PubMed1986

Coupling of carbon monoxide oxidation to CO2 and H2 with the phosphorylation of ADP in acetate-grown Methanosarcina barkeri.

Cell suspensions of Methanosarcina barkeri, grown on acetate, catalyzed the conversion of carbon monoxide and H2O to CO2 and H2 in stoichiometric amounts when methane formation was inhibited by bromoethanesulfonate. The specific activity was 80-120 nmol min-1 mg protein-1 at 5% CO in the gas phase. CO oxidation was coupled with the phosphorylation of ADP as indicated by a rapid increase of the intracellular ATP level upon start of the reaction. At least 0.1 mol ATP was formed/mol CO consumed. The onset of CO oxidation was also accompanied by an increase of the proton motive force (delta p) from 100 mV to 150 mV (inside negative). Addition of the uncoupler tetrachlorosalicylanilide to CO-metabolizing cells led to a rapid decrease of the ATP level and of delta p, and to an increase of the CO oxidation rate up to 70%. In the presence of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide the phosphorylation of ADP was inhibited and CO oxidation slowed down, whereas delta p was almost unaffected. Inhibition of CO oxidation under these conditions was relieved by the addition of the protonophore tetrachlorosalicylanilide. The results indicate that in acetate-grown M. barkeri the free-energy change associated with the formation of CO2 and H2 from CO and H2O (delta G degrees = -20 kJ/mol) can be used to drive the phosphorylation of ADP and that the coupling proceeds via a chemiosmotic mechanism. A possible role of the carbon monoxide oxidation reaction as an energy-conserving site in acetate fermentation to CH4 and CO2 is discussed.

Adenosine Diphosphate↗