Molecular organization in bacterial cell membranes II. Reevaluation and identification of some chemical components of Micrococcus lysodeikticus membranes

Standard membranes of Micrococcus lysodeikticus were prepared by protoplastlysis in the presence of 50 mM Mg²⁺ and by repeated washings in the absence of this cation. Different washing procedures with EDTA-30 mM Tris (pH 7.5) and low-ionic-strength Tris buffers (pH 7.5) yielded three distinct depleted membranes. RNA and carbohydrates were reevaluated in all these membranes after extraction and/or partial fractionation of the membrane complexes. Values higher than 10% of membrane dry weight have been found for both components in the standard membranes. Figures between 6–12% for carbohydrates and 0.8–16% for RNA were found in the three depleted membranes. The protein: lipid ratio of depleted membranes is lower than that of the standard membrane.The existence of RNA has been confirmed by polyacrylamide gel electrophoresis and by sensitivity to ribonuclease (EC 2.7.7.16). Different patterns of proteins, RNAs and carbohydrate-containing material were revealed by gel electrophoresis in sodium dodecyl sulphate of each type of M. lysodeikticus membrane. These results confirm and extend previous findings obtained with a depleted membrane of M. lysodeikticus (Estrugo, S. F., Larraga, V., Corrales, M. A.; Duch, C. and Munõz, E. (1972) Biochim. Biophys. Acta 255, 960–973). They suggest the existence of specific interactions between membrane components which are broken down and/or altered by different membrane treatments. They also suggest the likely significance in bacterial membranes of components other than lipids and proteins.     

Action of the steroid glycoside deltonin on the bacterial membranes of Micrococcus lysodeikticus

A slight detergent-like effect of steroid glycoside deltonine from Dioscorea deltoidea on the bacterial membranes of Micrococcus lysodeikticus was detected which resulted in the breaking of the osmotic barrier of protoplasts and in the loss from the membranes of small fragments containing the dehydrogenases of the respiratory chain but without cytochromes. These small fragments still retained the membrane structure.     

Mannose incorporation and lectin recognition of pronase-sensitive components in Micrococcus lysodeikticus (M. luteus) membranes

Summary Isolated cytoplasmic membranes from Micrococcus lysodeikticus were able to incorporate [¹⁴C]mannose from GDP-[¹⁴C]mannose. Labelled mannose remained in the membrane fraction after its repeated washing and lipid extraction. Sodium dodecyl sulfate gel electrophoresis in 12% acrylamide showed a set of bands with molecular weights ranging from 230 000 to 19 000 which stained for protein and carbohydrate, and incorporated [¹⁴C]mannose. Some of these bands reacted with different lectins (concanavalin A, wheat germ agglutinin and ricin).Furthermore, the mannose was incorporated via a glycosylation pathway similar to that followed in eukaryotic system as shown by the preliminary identification of a lipid intermediate transfering the sugar to proteins and by the differential sensitivity to bacitracin and tunicamycin.These complex membrane components were sensitive to digestion with pronase. All the results presented suggest their glycoprotein nature.     

Affinity chromatography of succinate dehydrogenase from the membranes of Micrococcus lysodeikticus.

Isolated plasma membranes of Micrococcus lysodeikticus were subjected to extraction with n-butanol in a two-phase system. Succinate dehydrogenase obtained in the soluble aqueous phase after high-speed centrifugation was resolved by separation on calcium phosphate gel and affinity chromatography. The affinity ligand used was oxaloacetate and elution from the column was achieved with 0.5 M succinate. In the final product there was an eleven-fold reduction in the 32P-lipid to protein ratio and a fourteen-fold increase in specific activity relative to the high speed supernatant fraction following n-butanol extraction.     

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.     

Solubility characteristics of Micrococcus lysodeikticus membrane components in detergents and chaotropic salts analyzed by immunoelectrophoresis.

In order to evalute the effectiveness and selectivity of various reagents in the solubilization of bacterial membranes, membranes of Micrococcus lysodeikticus were treated with detergents and chaotropic agents. The composition of the extracts so obtained was analyzed by rocket and two-dimensional immunoelectrophoretic techniques. Recoveery of succinate-, malate-, and reduced nicotinamide adenine dinucleotide- (NADH) dehydrogenases, ATPase, succinylated lipomannan and cytochromes in the extracts was measured. Treatment with a variety of non-denaturing detergents produced extracts that were generally qualitatively uniform although quantitative differences were observed. The degree of extraction of various components was correlated with the hydrophile-lipophile balance. Several chaotropic agents were also evaluated as reagents for membrane solubilization. These agents were less effective in extraction of bulk protein, but produced extracts enriched in some membrane components.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus.

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated. Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [14C]mannose from GDP-[14C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [14C]mannosyl-1-phosphorylundecaprenol. In contrast, this is the major mannolipid synthesized from GDP-[14C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents. Both membrane preparations possessed the ability to catalyse the transfer of [14C]mannose from purified [14C]mannosyl-1-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [14C]mannosyl units from 14C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.