scholarly journals Methane synthesis by membrane vesicles and a cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum

1984 ◽  
Vol 221 (1) ◽  
pp. 61-69 ◽  
Author(s):  
F D Sauer ◽  
S Mahadevan ◽  
J D Erfle
Keyword(s):  
Cell Wall ◽  
Membrane Vesicles ◽  
Methanobacterium Thermoautotrophicum ◽  
Small Vesicle ◽  
Heat Stable ◽  
Absolute Requirement ◽  
Carbonyl Cyanide ◽  
Total Cell Population ◽  
Oxygen Sensitive ◽  
Osmotic Lysis

Methanobacterium thermoautotrophicum when grown on ordinary culture medium has a tough cell wall which is lysozyme-resistant and difficult to disrupt by physical means. The cell wall, however, can be weakened by the addition of D-sorbitol to the growth medium and the organisms form protoplasts after lysozyme addition. This technique allowed the isolation of two types of intracellular small vesicles: (a) isolated by disruption of the total cell population (lysozyme-sensitive and lysozyme-resistant cells) by ultrafrequency sound and (b) isolated by osmotic lysis of protoplasts. For the first time, a small vesicle fraction isolated as in (a) was capable of synthesizing methane from CO2 and H2 without cytoplasm. There was, however, an absolute requirement for a small, heat-stable, oxygen-sensitive cofactor which was isolated from the cytoplasm. Methane synthesis with this vesicle fraction was inhibited by the detergent deoxycholate, and by the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Mg2+-ATPase appeared to be located on the outer or cytoplasmic surface of the small vesicle fraction isolated as in (b). The results were consistent with a previously made suggestion [Sauer, Erfle & Mahadevan (1981) J. Biol. Chem. 256, 9843-9848] that the interior of the small intracellular vesicles becomes acid during methane synthesis.

Bacterial Peptidoglycan Stapling with Functionalized D-Amino Acids

2019 ◽  
Author(s):  
Sylvia L. Rivera ◽  
Akbar Espaillat ◽  
Arjun K. Aditham ◽  
Peyton Shieh ◽  
Chris Muriel-Mundo ◽  
...  
Keyword(s):  
Cell Wall ◽  
Clinical Success ◽  
Bacterial Species ◽  
Enzymatic Reaction ◽  
Amino Acid Derivative ◽  
Wall Structure ◽  
Live Bacteria ◽  
Cross Links ◽  
Osmotic Lysis ◽  
Bacterial Peptidoglycan

Transpeptidation reinforces the structure of cell wall peptidoglycan, an extracellular heteropolymer that protects bacteria from osmotic lysis. The clinical success of transpeptidase-inhibiting β-lactam antibiotics illustrates the essentiality of these cross-linkages for cell wall integrity, but the presence of multiple, seemingly redundant transpeptidases in many bacterial species makes it challenging to determine cross-link function precisely. Here we present a technique to covalently link peptide strands by chemical rather than enzymatic reaction. We employ bio-compatible click chemistry to induce triazole formation between azido- and alkynyl-D-alanine residues that are metabolically installed in the cell walls of Gram-positive and Gram-negative bacteria. Synthetic triazole cross-links can be visualized by substituting azido-D-alanine with azidocoumarin-D-alanine, an amino acid derivative that undergoes fluorescent enhancement upon reaction with terminal alkynes. Cell wall stapling protects the model bacterium Escherichia coli from β-lactam treatment. Chemical control of cell wall structure in live bacteria can provide functional insights that are orthogonal to those obtained by genetics.<br>

Growth and Division of the Peptidoglycan Matrix

2021 ◽  
Vol 75 (1) ◽  
Author(s):  
Patricia D.A. Rohs ◽  
Thomas G. Bernhardt
Keyword(s):  
Cell Wall ◽  
Bacterial Infections ◽  
Model Organisms ◽  
Annual Review ◽  
Publication Date ◽  
Drug Resistant ◽  
Peptidoglycan Cell Wall ◽  
Integral Role ◽  
Osmotic Lysis ◽  
Peptidoglycan Layer

Most bacteria are surrounded by a peptidoglycan cell wall that defines their shape and protects them from osmotic lysis. The expansion and division of this structure therefore plays an integral role in bacterial growth and division. Additionally, the biogenesis of the peptidoglycan layer is the target of many of our most effective antibiotics. Thus, a better understanding of how the cell wall is built will enable the development of new therapies to combat the rise of drug-resistant bacterial infections. This review covers recent advances in defining the mechanisms involved in assembling the peptidoglycan layer with an emphasis on discoveries related to the function and regulation of the cell elongation and division machineries in the model organisms Escherichia coli and Bacillus subtilis. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Glycine transport into plasma-membrane vesicles derived from rat brain synaptosomes

1981 ◽  
Vol 198 (3) ◽  
pp. 535-541 ◽  
Author(s):  
F Mayor ◽  
J G Marvizón ◽  
M C Aragón ◽  
C Gimenez ◽  
F Valdivieso
Keyword(s):  
Rat Brain ◽  
Membrane Vesicles ◽  
Uptake System ◽  
Plasma Membrane Vesicles ◽  
Glycine Uptake ◽  
High Affinity ◽  
Carbonyl Cyanide ◽  
Brain Synaptosomes ◽  
Glycine Transport ◽  
Sole Energy

1. Transport of glycine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. 2. The uptake of glycine is strictly dependent on the presence of Na+ and Cl- in the medium, and the process can be driven either by an Na+ gradient (out greater than in) or by a C1- gradient (out greater than in) when the other essential ion is present. 3. The uptake of glycine is stimulated by a membrane potential (interior negative), as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions of different permeabilities. 4. The kinetic analysis shows that glycine is accumulated by two systems with different affinities. 5. The presence of ouabain, an inhibitor (Na+ + K+)-activated ATPase, does not affect glycine transport. 6. The existence of a high-affinity, Na+-dependent glycine-uptake system in membrane vesicles derived from rat brain suggests that this amino acid may have a transmitter role in some areas of the rat brain.

scholarly journals Binding of Adenosine Diphosphate (ADP) to Isolated Human Platelet Membranes

1977 ◽  
Author(s):  
J. Lips ◽  
J. J. Sixma
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Membrane Vesicles ◽  
Adenosine Diphosphate ◽  
Ph Optimum ◽  
High Affinity ◽  
Absolute Requirement ◽  
Millipore Filtration

Human platelet plasma membranes were isolated according to the glycerol loading technique of Barber and Jamieson. The binding of 14C ADP was studied with Millipore filtration in a Ca2+ and Mg2+ containing buffer at pH 7,4. At least two types of binding sites were found: A high affinity system with a maximum binding of 160 pMoles/mg protein and an association constant of 1,1 χ 106 M-1; and alow affinity system with a maximum binding of about 4500 pMoles/mg protein and an association constant of 0,6 χ 1θ4 M-1. The binding according to the high affinity system showed little temperature dependency (Q10 = 1,10). The pH optimum was at 7,3. Ca2+ ions were an absolute requirement for binding.Nucleoside diphosphokinase (NDPK) was found in the membrane vesicles. Evidence that this enzyme was not responsible for ADP binding was obtained. The enzyme is Mg2+ dependent and is inhibited by AMP, in contrast to ADP binding. The Q10 was 1,44.ADP binding was inhibited by ATP, IDP and β/γ-imidoadenosine triphosphate.

scholarly journals Plant expression of NifD protein variants resistant to mitochondrial degradation

2020 ◽  
Vol 117 (37) ◽  
pp. 23165-23173 ◽  
Author(s):  
Robert S. Allen ◽  
Christina M. Gregg ◽  
Shoko Okada ◽  
Amratha Menon ◽  
Dawar Hussain ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Design ◽  
Nitrogenase Activity ◽  
Plant Mitochondria ◽  
Single Amino Acid ◽  
Synthetic Proteins ◽  
Amino Acid Variant ◽  
Absolute Requirement ◽  
Oxygen Sensitive ◽  
Protein Variants

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)–linker–NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)–linker–NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

scholarly journals Methane production by the membranous fraction of Methanobacterium thermoautotrophicum

1980 ◽  
Vol 190 (1) ◽  
pp. 177-182 ◽  
Author(s):  
F D Sauer ◽  
J D Erfle ◽  
S Mahadevan
Keyword(s):  
Methane Production ◽  
High Speed ◽  
High Pressures ◽  
Reductase Activity ◽  
Membrane Vesicles ◽  
Methanobacterium Thermoautotrophicum ◽  
Cell Extract ◽  
Supernatant Fraction ◽  
Coenzyme M ◽  
Intact Membrane

Intact membrane vesicles are required to synthesize methane from CO2 and H2 by disrupted preparations of Methanobacterium thermoautotrophicum cells. When membrane vesicles were removed by high-speed centrifugation at 226 600 g, the remaining supernatant fraction no longer synthesized methane. Alternatively, if vesicle structure was disrupted by passage through a Ribi cell fractionator at very high pressures (345 MPa), the bacterial cell extract, with all the particulate fraction in it, did not synthesize methane. Methyl-coenzyme M, a new coenzyme first described by McBride & Wolfe [(1971) Biochemistry 10, 2317–2324], was shown to stimulate methane production from CO2 and H2, as previously reported, but the methyl group of the coenzyme did not appear to be a precursor of methane in this reaction. No methyl-coenzyme M reductase activity was detected in the cytoplasmic fraction of M. thermoautotrophicum cells.

A sodium-hydrogen exchange system in isolated apical membrane from LLC-PK1 epithelia

1986 ◽  
Vol 251 (6) ◽  
pp. F1003-F1008
Author(s):  
A. Moran ◽  
J. Biber ◽  
H. Murer
Keyword(s):  
Hydrogen Exchange ◽  
Brush Border Membrane ◽  
Cultured Cells ◽  
Apical Membrane ◽  
Short Circuit ◽  
Membrane Vesicles ◽  
Ph Gradients ◽  
Noncompetitive Inhibition ◽  
Carbonyl Cyanide ◽  
Orange Fluorescence

We have monitored transmembrane pH gradients using acridine orange fluorescence quenching and traced Na+ flux to study the properties of Na+-H+ exchange in apical membrane vesicles isolated from LLC-PK1 epithelia. The membranes have low conductance for Na+, H+, and K+ ions. An outwardly directed K+ gradient in the presence of valinomycin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone produced intravesicular acidification. This pH gradient was collapsed by addition of extravesicular Na+ or Li+ ions but not by tetramethylammonium. Amiloride (10(-4) M) inhibited the effect of both Na+ and Li+ . An outwardly directed Na+ gradient stimulated H+ influx, which was also inhibited by 10(-4) M amiloride. Membrane short-circuit conditions affected neither Na+ nor H+ flux, consistent with transport mediated by an electroneutral process. The interaction of amiloride and sodium is consistent with noncompetitive inhibition with Ki = 100+/- 10 microM for amiloride and an apparent Km for Na+ of approximately 20 mM. This finding is in agreement with previous studies of intact LLC-PK1 epithelia but differs from observations in brush-border membrane vesicles isolated from kidney proximal tubule in which competitive and mixed inhibition have been reported. These observed differences can be reconciled if two types of Na+-H+ exchange systems exist along the nephron, one with competitive and the other with noncompetitive inhibition, and if only the latter is expressed in the homogeneous cultured cells.

Orientation of cell wall β-glucan synthases in plasma membrane vesicles

Plant Science ◽  
1985 ◽  
Vol 41 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Dieter Haass ◽  
Gertrud Hackspacher ◽  
Gerhard Franz
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles

scholarly journals Antilisterial Activity of Peptide AS-48 and Study of Changes Induced in the Cell Envelope Properties of an AS-48-Adapted Strain ofListeria monocytogenes

1999 ◽  
Vol 65 (2) ◽  
pp. 618-625 ◽  
Author(s):  
Fátima Mendoza ◽  
Mercedes Maqueda ◽  
Antonio Gálvez ◽  
Manuel Martínez-Bueno ◽  
Eva Valdivia
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Intracytoplasmic Membrane ◽  
Highly Active ◽  
Bactericidal Effects ◽  
Cytoplasmic Content ◽  
Oflisteria Monocytogenes

ABSTRACT The peptide AS-48 is highly active on all Listeriaspecies. It has a bactericidal and bacteriolytic mode of action onListeria monocytogenes CECT 4032, causing depletion of the membrane electrical potential and pH gradient. The producer strainEnterococcus faecalis A-48-32, releases sufficient amounts of AS-48 into the growth medium to suppress L. monocytogenes in cocultures at enterococcus-to-listeria ratios above 1 at 37°C or above 10 at 15°C. As the temperature decreases, the bactericidal effects of AS-48 are less pronounced, but at 2.5 μg/ml it still can inhibit the growth of listeria at 6°C. AS-48 is highly active on liquid cultures, although concentrations above 0.2 μg/ml are required to avoid adaptation of listeria. AS-48-adapted cells can be selected at low (but still inhibitory) concentrations, and they can be inhibited completely by AS-48 at 0.5 μg/ml. The adaptation is lost gradually upon repeated subcultivation. AS48ad cells are cross-resistant to nisin and show an increased resistance to muramidases. Their fatty acid composition is modified: they show a much higher proportion of branched fatty acids as well as a higher C15:0 An-to-C17:0 An ratio. Resistance to AS-48 is also maintained by protoplasts from AS48ad cells. Electron microscopy observations show that the cell wall of AS48ad cells is thicker and less dense. The structure of wild-type cells is severely modified after AS-48 treatment: the cell wall and the cytoplasmic membrane are disorganized, and the cytoplasmic content is lost. Intracytoplasmic membrane vesicles are also observed when the wild-type strain is treated with high AS-48 concentrations.

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