scholarly journals Oxidative phosphorylation in Escherichia coli K12. An uncoupled mutant with altered membrane structure

1974 ◽  
Vol 138 (2) ◽  
pp. 211-215 ◽  
Author(s):  
G. B. Cox ◽  
F. Gibson ◽  
L. McCann
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Membrane Structure ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Mineral Salts ◽  
Normal Cells ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Membrane Bound

1. A new mutant strain (AN228) of Escherichia coli K12, unable to couple phosphorylation to electron transport, has been isolated. The mutant allele (unc-405), in strain AN228, was found to map near the uncA and uncB genes at about minute 74 on the E. coli genome. 2. A transductant strain (AN285) carrying the unc-405 allele is similar to the uncA and uncB mutants described previously in that it is unable to grow on succinate, gives a low aerobic yield on limiting concentrations of glucose, has a normal rate of electron transport, is unable to couple phosphorylation to electron transport, and lacks ATP-dependent transhydrogenase activity. 3. Strain AN285 (unc-405) is similar to an uncA mutant, but different from an uncB mutant, in that it is unable to grow anaerobically in a glucose–mineral-salts medium, and membrane preparations do not have Mg2+-stimulated adenosine triphosphatase activity. 4. Strain AN285 (unc-405) does not form an aggregate analogous to the membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate found in normal cells. In this respect it differs from strain AN249 (uncA−), which forms an inactive membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate.

scholarly journals A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele

1977 ◽  
Vol 164 (1) ◽  
pp. 193-198 ◽  
Author(s):  
F Gibson ◽  
G B Cox ◽  
J A Downie ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Fluorescence Quenching ◽  
Mutant Strain ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Magnesium Ion ◽  
New Mutation ◽  
Similar Phenotype

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase. A method was developed to incorporate mutant unc alleles into plasmids. Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa. Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements. The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424.

scholarly journals The energy-linked transhydrogenase reaction in respiratory mutants of Escherichia coli K 12

1971 ◽  
Vol 125 (2) ◽  
pp. 489-493 ◽  
Author(s):  
G. B. Cox ◽  
N. A. Newton ◽  
J. D. Butlin ◽  
F. Gibson
Keyword(s):  
Escherichia Coli ◽  
Energy Source ◽  
Electron Transport ◽  
Electron Transport Chain ◽  
Adenosine Triphosphatase ◽  
E Coli ◽  
Transport Chain ◽  
Mutant Strains ◽  
K 12 ◽  
A Site

1. Energy-linked and non-energy-linked transhydrogenase activities were assayed in membrane preparations from normal Escherichia coli K 12 and from various mutant strains. 2. The energy-linked transhydrogenase, which uses ATP as energy source, was dependent for activity on the presence of a functional Mg2++Ca2+-stimulated adenosine triphosphatase. 3. Neither of the quinones formed by E. coli, namely ubiquinone-8 and menaquinone-8, was required for normal ATP-dependent energy-linked transhydrogenase activity. 4. The energy-linked transhydrogenase was inhibited by piericidin A at a site unrelated to the sites of inhibition of the electron-transport chain by piericidin A.

scholarly journals Energy coupling to active transport in anaerobically grown mutants of Escherichia Coli K12

1976 ◽  
Vol 154 (3) ◽  
pp. 731-734 ◽  
Author(s):  
S J. Gutowski ◽  
H Rosenberg
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Active Transport ◽  
Adenosine Triphosphatase ◽  
Energy Coupling ◽  
High Energy ◽  
Escherichia Coli K12 ◽  
Anaerobic Electron Transport ◽  
Glutamine Uptake

1. Anaerobic uptake of proline requires either the presence of a coupled Mg2+-stimulated adenosine triphosphatase or anaerobic electron transport. 2. Anaerobic uptake of glutamine does not require anaerobic electron transport even in the absence of a coupled Mg+2-stimulated adenosine triphosphatase. 3. These results support previous suggestions [Berger (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1514––1518; Berger & Heppel (1974) J. Biol. Chem. 249, 7747-7755; Kobayashi, Kin & Anraku (1974) J. Biochem. (Tokyo) 76, 251-261] that two distinct mechanisms of energy coupling to active transport exist in Escherichia coli in that energization of anaerobic proline uptake requires the ‘high-energy membrane state’, whereas the energization of anaerobic glutamine uptake does not.

scholarly journals Synthesis of alternative membrane-bound redox carriers during aerobic growth of Escherichia coli in the presence of potassium cyanide

1975 ◽  
Vol 148 (2) ◽  
pp. 349-352 ◽  
Author(s):  
J R Ashcroft ◽  
B A Haddock
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Carbon Source ◽  
Potassium Cyanide ◽  
Growth Conditions ◽  
Aerobic Growth ◽  
E Coli ◽  
Low Concentrations ◽  
Electron Transport Chains ◽  
Membrane Bound

Aerobic growth of Escherichia coli with an oxidizable substrate as carbon source in the presence of low concentrations of KCN leads to the synthesis and integration into the membrane of menaquinone and cytochromes b558, a1 and d in addition to the redox carriers normally present under aerobic growth conditions, namely ubiquinone and cytochromes b562, b556 and o. The results are discussed with reference to other phenotypic and genotypic modifications to the electron-transport chains of E. coli.

scholarly journals Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1973 ◽  
Vol 132 (4) ◽  
pp. 689-695 ◽  
Author(s):  
G. B. Cox ◽  
F. Gibson ◽  
L. M. McCann ◽  
J. D. Butlin ◽  
F. L. Crane
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Calcium Ion ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Gel Filtration ◽  
Magnesium Ion ◽  
Adenosine Triphosphatase Activity ◽  
Escherichia Coli K12

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg2+,Ca2+-stimulated adenosine triphosphatase. 2. The Mg2+,Ca2+-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg2+,Ca2+-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg2+,Ca2+-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg2+,Ca2+-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA-) could not be re-activated by the purified Mg2+,Ca2+-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg2+,Ca2+-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA-).

scholarly journals Metabolite transport in mutants of Escherichia coli K12 defective in electron transport and coupled phosphorylation

1975 ◽  
Vol 146 (2) ◽  
pp. 417-423 ◽  
Author(s):  
H Rosenberg ◽  
G B Cox ◽  
J D Butlin ◽  
S J Gutowski
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Active Transport ◽  
Adenosine Triphosphatase ◽  
Minimal Medium ◽  
Sole Source ◽  
Whole Cells ◽  
Escherichia Coli K12 ◽  
Mutant Strains ◽  
Uptake Medium

1. The uptakes of Pi and serine by whole cells of mutant strains of Escherichia coli K12, grown under both aerobic and anaerobic conditions, were studied. 2. Uptake by aerobic cells was low in a ubiquinone-less mutant but normal in two mutant strains unable to couple phosphorylation to electron transport. 3. One of these uncoupled strains, carrying the unc-405 allele, does not form a membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate, and it is concluded that the Mg2+-stimulated adenosine triphosphatase does not serve a structural role in the aerobic active transport of Pi or serine. 4. The other uncoupled strain, in which aerobic uptake is unaffected, carries a mutation in the uncB gene, thus distinguishing this gene from the etc gene, previously shown to be concerned with the coupling of electron transport to active transport. 5. The uptakes of Pi and serine by anaerobic cells were normal in the ubiquinone-less mutant, but defective in both the uncoupled strains. 6. The uptake of Pi and serine by anaerobic cells of the uncB mutant could be increased by the addition of fumarate to the uptake medium. The unc-405 mutant, however, required the addition of fumarate for growth and for uptake. 7. The uncB mutant, unlike the unc-405 mutant, is able to grow anaerobically in a minimal medium with glucose as sole source of carbon. Similarly a strain carrying a mutation in the frd gene, which is the structural gene for the enzyme fumarate reductase, is able to grow anaerobically in a glucose-minimal medium. However, a mutant strain carrying mutations in both the uncB and frd genes resembles the unc-405 mutant in not being able to grow under these conditions.

scholarly journals Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the F1 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12

1978 ◽  
Vol 170 (3) ◽  
pp. 593-598 ◽  
Author(s):  
G B Cox ◽  
J A Downie ◽  
F Gibson ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Fluorescence Quenching ◽  
Oxidative Phosphorylation ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
The Other ◽  
Genetic Complementation ◽  
Magnesium Ion ◽  
Adenosine Triphosphatase Activity

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.

scholarly journals Properties of membranes from mutant strains of Escherichia coli in which the β-subunit of the adenosine triphosphatase is abnormal

1979 ◽  
Vol 180 (1) ◽  
pp. 111-118 ◽  
Author(s):  
A E Senior ◽  
D R Fayle ◽  
J A Downie ◽  
F Gibson ◽  
G B Cox
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Beta Subunit ◽  
Beta Subunits ◽  
F1 Atpase ◽  
Mutant Strains ◽  
Membrane Bound ◽  
Phenotypic Variations

Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.

scholarly journals Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli

2002 ◽  
Vol 184 (13) ◽  
pp. 3630-3639 ◽  
Author(s):  
David E. Nelson ◽  
Anindya S. Ghosh ◽  
Avery L. Paulson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Site Directed Mutagenesis ◽  
Outer Face ◽  
Penicillin Binding Protein ◽  
Membrane Anchor ◽  
E Coli ◽  
Membrane Bound ◽  
Carboxy Terminal ◽  
Membrane Anchoring ◽  
Β Sheet

ABSTRACT Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar dd-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other dd-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal β-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the dd-carboxypeptidase enzymatic core.

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