Structure--function relationships of Micrococcus lysodeikticus membranes: a bacterial membrane model system.
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Adenosine Triphosphatases↗
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Bacterial membrane proteins.
Bacterial membranes have diverse functions, depending on whether they are specialized membranes or cytoplasmic membranes possessing transport, mitochondrial activities and biosynthetic functions for assembly of membranes, walls and capsules. In contrast to plasma membranes which serve as major biochemical organelles of both Gram-positive and Gram-negative bacteria, the outer membranes of the latter group confer barrier functions on the cells, providing a variety of selective channels. Although prokaryotic cells lack the array of membranous organelles characteristic of eukaryotic cells, bacteria with specific physiological and genetic capabilities form specialized membrane systems such as the bacteriorhodopsin purple membrane, chromatophore membranes of phototrophs, and forespore membranes essential to bacterial endospore formation. Unravelling the structure, function and proteins of these membranes presents a formidable biochemical, immunochemical and structural challenge.
Bacterial Outer Membrane Proteins↗
Energetics and molecular biology of active transport in bacterial membrane vesicles.
Bacterial membrane vesicles retain the same sidedness as the membrane in the intact cell and catalyze active transport of many solutes by a respiration-dependent mechanism that does not involve the generation of utilization of ATP or other high-energy phosphate compounds. In E. coli vesicles, most of these transport systems are coupled to an electrochemical gradient of protons (deltamuH+, interior negative and alkaline) generated primarily by the oxidation of D-lactate or reduced phenazine methosulfate via a membrane-bound respiratory chain. Oxygen or, under appropriate conditions, fumarate or nitrate can function as terminal electron acceptors, and the site at which deltamuH+ is generated is located before cytochrome b1 in the respiratory chain. Certain (N-dansyl)aminoalkyl-beta-D-galactopyranosides (Dns-gal) and N(2-nitro-4-azidophenyl)aminoalkyl 1-thio-beta-D-galactopyranosides (APG) are competitive inhibitors of lactose transport but are not transported themselves. Various fluorescence techniques, direct binding assays, and photoinactivation studies demonstrate that the great bulk of the lac carrier protein (ca. 95%) does not bind ligand in the absence of energy-coupling. Upon generation of a deltamuH+ (interior negative and alkaline), binding of Dns-gal and APG-dependent photoinactivation are observed. The data indicate that energy is coupled to the initial step in the transport process, and suggest that the lac carrier protein may be negatively charged.
Bacteria↗
Determination of the membrane potential in bacterial membrane vesicles from the accumulation of N-methyldeptropine.
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Biological Transport, Active↗
The biogenesis and assembly of bacterial membrane proteins.
Bacterial proteins in the inner and outer membranes differ dramatically in their architecture. Although both types of proteins are transported across the inner membrane through a common pore, recent studies have identified distinct factors that target them to transport sites and catalyze proper folding.
Bacterial Outer Membrane Proteins↗
Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes.
The peptidoglycan layers surrounding bacterial membranes are essential for bacterial cell survival and provide an important target for antibiotics. Many antibiotics have mechanisms of action that involve binding to Lipid II, the prenyl chain-linked donor of the peptidoglycan building blocks. One of these antibiotics, the pore-forming peptide nisin uses Lipid II as a receptor molecule to increase its antimicrobial efficacy dramatically. Nisin is the first example of a targeted membrane-permeabilizing peptide antibiotic. However, it was not known whether Lipid II functions only as a receptor to recruit nisin to bacterial membranes, thus increasing its specificity for bacterial cells, or whether it also plays a role in pore formation. We have developed a new method to produce large amounts of Lipid II and variants thereof so that we can address the role of the lipid-linked disaccharide in the activity of nisin. We show here that Lipid II is not only the receptor for nisin but an intrinsic component of the pore formed by nisin, and we present a new model for the pore complex that includes Lipid II.
Membrane Lipids↗
Membrane adhesion in photosynthetic bacterial membranes. Light harvesting complex I (LHI) appears to be the main adhesion factor.
We have reconstituted pigment-protein complexes isolated from Rhodopseudomonas palustris photosynthetic membranes into phospholipid liposomes. The various complexes were tested for their ability to promote adhesion of the liposome membrane in the presence and absence of Mg2+ ions. Samples containing a reaction center (RC)/light-harvesting I (LHI) complex appeared to stack in a manner resembling control thylakoids in 2 and 5 mM Mg2+. We also tested for the effects of Mg2+ on detergent extractability of pigment-protein complexes from intact membranes. Mg2+ sharply reduced the amount of LHI solubilized from membranes, while having little effect on the extractability of the light harvesting II complex (LHII) and the RC. Based on these results we suggest that LHI is the principal adhesion factor of R. palustris thylakoids.
Adhesiveness↗
[Spin-probe study of structural transitions in biological membranes: temperature changes in bacterial membranes].
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Bacterial Proteins↗
Translocation of DNA across bacterial membranes.
DNA translocation across bacterial membranes occurs during the biological processes of infection by bacteriophages, conjugative DNA transfer of plasmids, T-DNA transfer, and genetic transformation. The mechanism of DNA translocation in these systems is not fully understood, but during the last few years extensive data about genes and gene products involved in the translocation processes have accumulated. One reason for the increasing interest in this topic is the discussion about horizontal gene transfer and transkingdom sex. Analyses of genes and gene products involved in DNA transfer suggest that DNA is transferred through a protein channel spanning the bacterial envelope. No common model exists for DNA translocation during phage infection. Perhaps various mechanisms are necessary as a result of the different morphologies of bacteriophages. The DNA translocation processes during conjugation, T-DNA transfer, and transformation are more consistent and may even be compared to the excretion of some proteins. On the basis of analogies and homologies between the proteins involved in DNA translocation and protein secretion, a common basic model for these processes is presented.
Bacteria↗
Incorporation of beef heart cytochrome c oxidase as a proton-motive force-generating mechanism in bacterial membrane vesicles.
Membrane vesicles derived from the strictly fermentative lactic acid bacterium Streptococcus cremoris have been fused with proteoliposomes containing the beef heart mitochondrial cytochrome c oxidase by means of a freeze/thaw-sonication technique. Evidence that fusion has taken place was obtained by freeze-etch electron microscopy, showing a less-dense intramembranous particle distribution in the fused membranes than in the bacterial membranes, and by sucrose gradient centrifugation, indicating a buoyant density of the majority of the membranes after fusion that was between the buoyant densities of the starting membrane preparations. In the fused membranes, 55-60% of the cytochrome c oxidase molecules are oriented with the cytochrome c binding site at the outer surface of the membrane. With the electron-donor system ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c, a high proton-motive force (greater than 130 mV), inside negative and alkaline, can be generated in the fused membrane, and this proton-motive force can drive secondary transport of several amino acids. The procedure described can be used for incorporating a proton-motive force-generating system in isolated membrane vesicles from bacterial or eukaryotic origin that lack a suitable primary proton pump.
Animals↗
[Effect of levomycetin (chloramphenicol) on the biosynthesis of membrane proteins, structure and some functions of Micrococcus lysodeikticus bacterial membranes].
Levomycetin (chloroamphenicol), an inhibitor of protein synthesis, caused drastic changes in the molecular organization of bacterial membranes being introduced into the cultural medium of Micrococcus lysodeikiticus (50-100 mkg/ml. Isolated membranes of levomycetin-treated cells are enriched with lipids as compared with the control, they are more labile and they lose the X protein component and a considerable part of dehydrogenases, which results in the significant inhibition of all the respiration chain. At the same time the content of cytochrome alpha was increased by 17%, and the activity of malate dehydrogenase (as estimated in cell lysates) was two-fold increased in levomycetin-treated cell as compared with the control. It means that biosynthesis of some membrane proteins can be twice more resistant to the action of the antibiotic than the biosynthesis of other proteins, the total protein biosynthesis being significantly depressed (the incorporation of labelled leucine in membrane proteins is inhibited by 78%, and in cytoplasmic proteins-by 83%). The ratio of the respiration rate of intact levomycetin-treated cells to the protein content or to the cell bulk was similar to that of the control culture, which testifies the stability of membranes in vivo even under considerable "fattening". The loss of dehydrogenases and the X protein from the membranes apparently takes place at the moment of the cell rupture during the lysis in hypotonic medium. The ratio of the respiration rate of intact levomycetin-treated cells to the content of cytochromes a, b, c was 1.15-1.55 times as low as that of control cells which is probably due to the tendency of respiration components to the independent translocation in the membrane and is the result of the decrease in the concentration of the respiration components under the less than fattening greater than of the membrane.
Bacterial Proteins↗
In vitro system for high-throughput screening of random peptide libraries for antimicrobial peptides that recognize bacterial membranes.
Antibacterial peptides have been isolated from a wide range of species. Some of these peptides act on microbial membranes, disrupting their barrier function. With the increasing development of antibiotic resistance by bacteria, these antibacterial peptides, which have a new mode of action, have attracted interest as antibacterial agents. To date, however, few effective high-throughput approaches have been developed for designing and screening peptides that act selectively on microbial membranes. In vitro display techniques are powerful tools to select biologically functional peptides from peptide libraries. Here, we used the ribosome display system to form peptide-ribosome-mRNA complexes in vitro from nucleotides encoding a peptide library, as well as immobilized model membranes, to select specific sequences that recognize bacterial membranes. This combination of ribosome display and immobilized model membranes was effective as an in vitro high-throughput screening system and enabled us to identify motif sequences (ALR, KVL) that selectively recognized the bacterial membrane. Owing to host toxicity, it was not possible to enrich any sequence expected to show antimicrobial activity using another in vitro system, e.g. phage display. The synthetic peptides designed from these enriched motifs acted selectively on the bacterial model membrane and showed antibacterial activity. Moreover, the motif sequence conferred selectivity onto native peptides lacking selectivity, and decreased mammalian cell toxicity of native peptides without decreasing their antibacterial activity.
Amino Acid Sequence↗
Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.
Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations.
Anesthetics, Intravenous↗
Transport studies in bacterial membrane vesicles.
The use of bacterial membrane vesicles as an experimental system for the study of active transport has been discussed. Vesicles are prepared from osmotically sensitized bacteria, and consist of osmotically intact, membranebound sacs without internal structure. They retain litle or no cytoplasm. Under appropriate conditions, these vesicles catalyze the transport of a variety of solutes at rates which are comparable, in many cases, to those of intact cells. Two general types of transport systems have been elucidated in the vesicle system: (i) group translocation systems which catalyze vectorial covalent reactions; and (ii) respirationlinked transport systems that catalyze the active transport of a whole range of metabolites against an electrochemical or osmotic gradient. In E. coli membrane vesicles, the respiration-linked transport systems are coupled primarily to the oxidation of (D)-lactate to pyruvate, catalyzed by a flavin-linked, membrane-bound (D)-lactate dehydrogenase which has been purified to homogeneity. Electrons derived from (D)-lactate or certain artificial electron donors are transferred to oxygen by means of a membrane-bound respiratory chain, and respiration is coupled to active transport within a segment of the respiratory chain between the primary dehydrogenase and cytochrome. b(l). The great majority of the individual membrane vesicles in the population catalyze active transport, and the generation or hydtolysis of ATP is not involved. Under anaerobic conditions, fumarate or nitrate can be utilized in place of oxygen as terminal electron acceptors. With the exception that (D)-lactate is not always the most effective electron donor for active transport, vesicles prepared from a number of other organisms catalyze transport in a similar manner. Fluorescent dansylgalactosides are useful molecular probes of active transport in the vesicle system. These compounds are competitive inhibitors of beta-galactoside transport, but are not transported themselves. Fluorescence studies indicate that the lac carrier protein constitutes approximately 3 to 6 percent of the total membrane protein, and that it is not accessible to the external medium unless the membrane is "energized." Thus, energy is coupled to one of the initial steps in the transport process. Studies with a photoaffinity-labeled galactoside provide independent support for this conclusion. When membrane vesicles prepared from a (D)-lactate dehydrogenase mutant of E. coli are treated with (D)-lactate dehydrogenase, the enzyme binds to the vesicles and they regain the capacity to catalyze (D)-lactate oxidation and (D)-lactate-dependent active transport. The maximal specific transport activity obtained in the reconstituted system is similar in magnitude to that of wildtype vesicles. Titration studies with dansylgalactoside demonstrate that there is at least a seven- to eightfold excess of lac carrier protein relative to (D)-lactate dehydrogenase. Evidence is presented indicating that the enzyme is bound to the inner surface of native membrane vesicles and to the outer surface of reconstituted vesicles, and that the flavin coenzyme moiety is critically involved in binding. Possible mechanisms of respirationlinked active transport are discussed.
Anaerobiosis↗
Naturally processed dermcidin-derived peptides do not permeabilize bacterial membranes and kill microorganisms irrespective of their charge.
Dermcidin (DCD) is a recently described antimicrobial peptide, which is constitutively expressed in eccrine sweat glands and transported via sweat to the epidermal surface. By postsecretory proteolytic processing in sweat the dermcidin protein gives rise to several truncated DCD peptides which differ in length and net charge. In order to understand the mechanism of antimicrobial activity, we analyzed the spectrum of activity of several naturally processed dermcidin-derived peptides, the secondary structure in different solvents, and the ability of these peptides to interact with or permeabilize the bacterial membrane. Interestingly, although all naturally processed DCD peptides can adopt an alpha-helical conformation in solvents, they have a diverse and partially overlapping spectrum of activity against gram-positive and gram-negative bacteria. This indicates that the net charge and the secondary structure of the peptides are not important for the toxic activity. Furthermore, using carboxyfluorescein-loaded liposomes, membrane permeability studies and electron microscopy we investigated whether DCD peptides are able to permeabilize bacterial membranes. The data convincingly show that irrespective of charge the different DCD peptides are not able to permeabilize bacterial membranes. However, bacterial mutants lacking specific cell envelope modifications exhibited different susceptibilities to killing by DCD peptides than wild-type bacterial strains. Finally, immunoelectron microscopy studies indicated that DCD peptides are able to bind to the bacterial surface; however, signs of membrane perturbation were not observed. These studies indicate that DCD peptides do not exert their activity by permeabilizing bacterial membranes.
Amino Acid Sequence↗
Effects of the membrane action of tetralin on the functional and structural properties of artificial and bacterial membranes.
Tetralin is toxic to bacterial cells at concentrations below 100 mumol/liter. To assess the inhibitory action of tetralin on bacterial membranes, a membrane model system, consisting of proteoliposomes in which beef heart cytochrome c oxidase was reconstituted as the proton motive force-generating mechanism, and several gram-positive and gram-negative bacteria were studied. Because of its hydrophobicity, tetralin partitioned into lipid membranes preferentially (lipid/buffer partition coefficient of tetralin is approximately 1,100). The excessive accumulation of tetralin caused expansion of the membrane and impairment of different membrane functions. Studies with proteoliposomes and intact cells indicated that tetralin makes the membrane permeable for ions (protons) and inhibits the respiratory enzymes, which leads to a partial dissipation of the pH gradient and electrical potential. The effect of tetralin on the components of the proton motive force as well as disruption of protein-lipid interaction(s) could lead to impairment of various metabolic functions and to low growth rates. The data offer an explanation for the difficulty in isolating and cultivating microorganisms in media containing tetralin or other lipophilic compounds.
Arthrobacter↗
Selective recognition of bacterial membranes by zinc(II)-coordination complexes.
Two fluorophore-dipicolylamine-Zn2+ conjugates are shown by epifluorescence microscopy to stain the membranes of bacterial cells in preference to mammalian cells.
Amines↗
Effect of cultivation medium on some physicochemical parameters of outer bacterial membrane.
The changes of surface charge and hydrophobicity of the outer bacterial membrane in relation to utilization of n-hexadecane were studied. For this spectrophotometric study adsorption of methylene blue and transport of gentian violet were used. The decrease in the negative charge of the bacterial strains Pseudomonas putida CCM 3423, P. aeruginosa, and P. fluorescens CCM 2115, depended on the type of growth medium. The decrease of surface charge was in the order: meat extract peptone broth > mineral medium with glucose > mineral medium with n-hexadecane. The highest permeability of the bacterial membrane for gentian violet was determined in the case of P. fluorescens grown in meat extract peptone broth. This effect can be explained by a greater hydrophobicity of the bacterial surface for this strain. In other strains a lower permeability was observed. P. fluorescens showed a greater adherence to hexadecane.
Cell Membrane↗