Article

Outer Membrane Proteins of Smooth and Rough Strains of Proteus mirabilis

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  • Hebrew University of Jerusalem, The Faculty of Medicine
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Abstract

The outer membranes of the smooth Proteus mirabilis S1959 strain and its rough R13, R110, R51 and R45 mutants were isolated by sonication of the cells and sucrose density gradient centrifugation. The outer membrane of the rough strains had a lower density than that of their parent smooth strain, but the protein-to-phospholipid ratios were the same. The electrophoretic patterns of outer membrane polypeptides of the S and R strains in sodium dodecylsulfate/polyacrylamide gels were identical, with two major polypeptide bands, C1 and C2 (Mr 39,000 and 38,000) predominating. The C1 polypeptide band was a heat-modifiable polypeptide, which migrated as a band at Mr 33,000 when membranes were solubilized at 37 degrees C or 50 degrees C, and at Mr 39,000 when solubilization was at 100 degrees C. Susceptibility of outer membrane polypeptides to proteolytic digestion was found to be higher in isolated outer membrane preparations of the rough strains than in the smooth strain, suggesting that the availability of the polypeptide chains to proteolytic activity depends on the length of the polysaccharide chains of the outer membrane lipopolysaccharide.

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... So-called nbsB mutants of E. coli, sensitive to a number of hydrophobic agents, were shown to produce LPS molecules variably deficient in their deep core portion, due to a leaky rfaD mutation (45,46). Deep rough mutants of Proteus mirabilis were also found to be hypersensitive to various hydrophobic agents (256), and a rough mutant of P. aeruginosa, hypersensitive to penicillin G and some detergents, but not to most dyes, has been reported (131). Other' hydrophobic agents to which the deep rough mutants become sensitive include fatty acids (273), phenol (266), polycyclic hydrocarbons (4), more hydrophobic derivatives of tetracycline (137), and anionic (257, 266, 284) as well as cationic (306) detergents. ...
... The differences in the lipopolysaccharide structure of smooth forms of Sh. flexneri found by several authors [10,12] are therefore not associated with the differences in the OMP composition. Even rough mutants of some enterobacteria have been found to contain almost the same set of OMP as their smooth parent strains [13][14][15]. ...
... 7 and 8 B). Pronase is well known not to cross cell membranes and has been a useful tool for revealing membrane protein topology and cannot cross the bilayer (see, for example, Bezanilla and Armstrong, 1977;Arias and Kyte, 1979;Rottem et al., 1979;Wagner and Kelly, 1979). Because pronase alone has no effect on either the nonactin-or the alamethicin-induced conductance, its effect in the presence of annexin XII must be mediated by digestion of annexin XII on one side of the membrane and by pronase on the other side. ...
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Annexins are proteins that bind lipids in the presence of calcium. Though multiple functions have been proposed for annexins, there is no general agreement on what annexins do or how they do it. We have used the well-studied conductance probes nonactin, alamethicin, and tetraphenylborate to investigate how annexins alter the functional properties of planar lipid bilayers. We found that annexin XII reduces the nonactin-induced conductance to ∼30% of its original value. Both negative lipid and ∼30 μM Ca2+ are required for the conductance reduction. The mutant annexin XIIs, E105K and E105K/K68A, do not reduce the nonactin conductance even though both bind to the membrane just as wild-type does. Thus, subtle changes in the interaction of annexins with the membrane seem to be important. Annexin V also reduces nonactin conductance in nearly the same manner as annexin XII. Pronase in the absence of annexin had no effect on the nonactin conductance. But when added to the side of the bilayer opposite that to which annexin was added, pronase increased the nonactin-induced conductance toward its pre-annexin value. Annexins also dramatically alter the conductance induced by a radically different probe, alamethicin. When added to the same side of the bilayer as alamethicin, annexin has virtually no effect, but when added trans to the alamethicin, annexin dramatically reduces the asymmetry of the I-V curve and greatly slows the kinetics of one branch of the curve without altering those of the other. Annexin also reduces the rate at which the hydrophobic anion, tetraphenylborate, crosses the bilayer. These results suggest that annexin greatly reduces the ability of small molecules to cross the membrane without altering the surface potential and that at least some fraction of the active annexin is accessible to pronase digestion from the opposite side of the membrane.
... This results in overall changes in cell surface properties. For example, LPS mutants which have lost the O antigen component have increased affinity for hydrophobic probes (14,23,25,29,31,32). EDTA treatment of cells has been shown to cause partial release of LPS (4,5,15,30), resulting in cells that are more susceptible to the action of hydrophobic antibiotics (15,16). ...
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... So-called nbsB mutants of E. coli, sensitive to a number of hydrophobic agents, were shown to produce LPS molecules variably deficient in their deep core portion, due to a leaky rfaD mutation (45,46). Deep rough mutants of Proteus mirabilis were also found to be hypersensitive to various hydrophobic agents (256), and a rough mutant of P. aeruginosa, hypersensitive to penicillin G and some detergents, but not to most dyes, has been reported (131). ...
Chapter
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Chapter
This chapter discusses the assay of inorganic phosphate, total phosphate, and phosphatases. The phosphomolybdate complex is reduced by ascorbic acid. The method is about seven times as sensitive as the Fiske–SubbaRow procedure and involves less pipetting. One can easily determine 0.01 micromole of phosphate. Pyrophosphate breaks down about 5% in the method and compounds such as glucose 1-phosphate also break down somewhat, so that the method is not very satisfactory for determining inorganic phosphate if labile phosphate esters are present in large excess. The sample of organic phosphate and a drop of magnesium nitrate solution in a small test tube are taken to dryness by shaking the tube in flame. The ashing procedure is rapid and is good for various biological materials and phosphate esters such as nucleic acid, carbohydrate phosphate esters, viruses, and phospholipids. The assay method of phosphatases for inorganic phosphate can be used as an assay for phosphatases hydrolyzing stable phosphate esters such as glucose-6-phosphate, ribose-5-phosphate, and histidinol phosphate. The enzyme incubation can be stopped with the one ascorbic-molybdate solution thus avoiding an extra pipetting.
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1.1. The solubilization and fractionation of the outer cytoplasmic membrane components of Proteus mirabilis were studied as a first step towards the reconstitution of the membranes from their solubilized components.2.2. Membrane phospholipids were specifically labeled by growing the organisms with radioactive oleic acid. The lipopolysaccharide component of the outer membrane was effectively labeled by adding radioactive galactose to the medium. Less than 5% of the label derived from galactose was found in the outer membrane phospholipids and none in the protein.3.3. Sodium dodecyl sulfate effectively solubilized all three components of the outer membrane while sodium deoxycholate and Triton X-100 solubilized almost all the phospholipid, but only about half of the protein and lipopolysaccharide. The cytoplasmic membranes of P. mirabilis were much more susceptible to solubilization by the three detergents tested. Treatment of the outer membrane with aqueous butan-l-ol separated the phospholipid into the butanol phase, while most of the protein and lipopolysaccharide was found in the aqueous phase.4.4. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate separated the lipopolysaccharide into two bands. Both bands were stained with the periodic acid-Schiff reagent, but only the slower migrating one stained with Coomassie blue. Filtration of the solubilized outer membranes through a Sephadex G-200 column containing sodium dodecyl sulfate separated the two lipopolysaccharide types. Our results support and supplement those of Gmeiner (Eur. J. Biochem. (1975) 58, 621–626) in showing that P. mirabilis produces two lipopolysaccharide types, one having long O-antigenic side chains, rich in lysine and amino sugars, while the other one is poorer in 0-antigenic chain components and more hydrophobic in character.
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The peptidoglycan-associated protein fractions of various strains of Escherichia coli and of other gramnegative rod-shaped bacteria were isolated and compared. Peptidoglycan-associated proteins are always outer membrane proteins. All E. coli strains tested contain at least one peptidoglycan-associated protein.Procedures are described for the isolation of the two peptidoglycan-associated proteins b and c of E. coli K12.All nine Enterobacteriaceae tested contain one or more peptidoglycan-associated proteins. Two gramnegative rod-shaped bacteria that do not belong to the family of Enterobacteriaceae do not contain peptidoglycan-associated protein.It is suggested that peptidoglycan-associated proteins form, or are part of, hydrophilic channels through the outer membrane.
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1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.
Article
The supramolecular structure of the outer membrane of Salmonella typhimurium that produces an Rc-type lipopolysaccharide was studied by adding spin-labeled fatty acid probes to membranes as well as model bilayers. Lipopolysaccharide of this organism apparently formed a bilayer structure in 0.2 M NaCl/0.01 M MgCl2, and the electron spin resonance spectra suggested that the motion of the segments of hydrocarbon chains near the carboxyl end was quite restricted even at high temperature; this is presumably due to the anchoring of more than a dozen fatty acid residues to a single backbone structure. In the presence of Mg2+, we could produce lipoplysaccharide-phospholipid mixed bilayers contining up to 50% (by weight) lipoplysaccharide. Their spectra showed no sign of major heterogeneity, and the maximum hyperfine splitting values were considerably larger than in phospholipid-only liposomes; these results suggest that the two components are finely interspersed and that the mobility of phospholipid hydrocarbons is severely restricted by the hydrocarbon chains of lipopolysaccharide. In spite of the presence of lipoplysaccharide in an amount equal to or exceeding that of phospholipids, the outer membrane produced spectra remarkably similar to those of the inner membrane, which does not contain lipoplysaccharide, and there was little sign of immobilization by lipopolysaccharides. Signals corresponding to the pure lipoplysaccharide phase were not detected, either. These results suggest that the phospholipids and lipopolysaccharides are segregated into separate domains in the outer membrane, and the fatty acid probes enter almost exclusively into the phospholipid domains. This conclusion was fully corroborated by determining, through the exchange broadening of line width, the total area of the domains that accommodated the spin label probes.
Article
Proteins exposed on the outer surface of the outer membrane of Salmonella typhimurium were identified by reacting intact cells with a covalent labeling reagent. Since the outer membrane permitted the free diffusion of small hydrophilic molecules, we used a macromolecular reagent, CNBr-activated dextran, as the non-penetrating labeling agent. We also used a mutant producing a lipopolysaccharide with a very short (i.e. hexasaccharide) carbohydrate chain, in order to avoid steric hindrance by the carbohydrates on membrane surface. Results showed that out of the four "major" proteins of molecular weight around 35 000, three were exposed, and that at least six other proteins were also exposed on cell surface. Only two or three outer membrane proteins consistently did not react with the reagent in intact cells.
Article
Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine with cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes. Three major outer membrane proteins of apparent molecular weights 39000, 36000 and 17000 were isolated using acetic acid and sodium deoxycholate solution as solvents and avoiding the strongly denaturing sodium dodecyl sulfate. The isolated proteins were assayed for their ability to form hydrophilic pores in reconstituted membranes. The trypsin-sensitive 39000-Mr protein and the peptidoglycan-associated 36000-Mr protein were equally effective in this function whereas the 17000-Mr protein mediated little penetration of low molecular weight solute. The 39000-Mr and 36000-Mr proteins also protected reconstituted membrane vesicles from disruption by detergent while 17000-Mr protein was ineffective in this regard.
Article
Lipopolysaccharides of qualitatively identical but quantitatively different sugar composition were extracted from Proteus mirabilis strain 1959. The lipopolysaccharide with the higher percentage of typical O-specific constituents was subjected to partial acid hydrolysis. An oligosaccharide B22 was separated by paper chromatography and electrophoresis. It was found to be composed of equimolar amounts of D-galacturonic acid, D-galactosamine and L-lysine. Dinitrophenylation of the oligosaccharide as well as of the genuine lipopolysaccharide afforded xi-dinitrophenyl-L-lysine after acid hydrolysis, showing that lysine was linked to the disaccharide via its alpha-amino group. Further studies including the Morgan-Elson and Elson-Morgan reactions, NaBH4-reduction, hydrazinolysis and periodate oxidation revealed the structure of oligosaccharide B22 as D-galacturonyl-(1 leads to 4)-D-galactosamine with lysine attached to the carboxylic group of galacturonic acid via its alpha-amino group. Judged from its high inhibition capacity this oligosaccharide has to be considered as an essential part of the serological determinant of Proteus mirabilis 1959. The frequent occurrence of lysine and galacturonic acid in Proteus mirabilis O-serogroups and their possible significance for the respective serological specificities are discussed.
Article
Major outer membrane proteins of Escherichia coli K-12 with apparent molecular weights ranging from 30,000 to 40,000 were resolved into four distinct bands by electrophoresis on an improved urea-sodium dodecyl sulfate (SDS)-polyacrylamide gel containing a high concentration of N, N′-methylenebisacrylamide. The electrophoretic mobilities of three of these proteins, O-8, O-9, and O-10, changed when they were heated in SDS solution. Proteins O-8, O-9, and O-10 were purified to near homogeneity without heating in SDS solution. Electrophoretic profiles of the purified proteins changed depending on the solubilization temperature in SDS solution. Infrared and CD spectra of these proteins revealed that they were extremely rich in β-structured polypeptide, which is stable in SDS solution at room temperature. On the other hand, CD spectra typical of α-helix structure were obtained when the proteins were heated in SDS solution, indicating that a gross conformational change occurred in the protein molecules upon heating in SDS solution. The conformational change was confirmed by the abnormal profiles of Ferguson plots in gel electrophoretic analysis. It was concluded that conformational changes in the protein molecules are responsible for the heat modifiability of these proteins in SDS gel electrophoresis.
Article
1. The crude envelope preparation obtained by sonication of Proteus mirabilis cells in the presence of lysozyme was separated into outer and cytoplasmic membrane fractions by sucrose density gradient centrifugation. The outer membrane fraction accounted for about two thirds of the dry weight of the envelope preparation. 2. In thin sections, the outer and cytoplasmic membrane fractions were shown to consist of vesicles bounded by a single trilaminar membrane, but those of the outer membrane were considerably smaller and were frequently open, forming C-shaped structures. The cytoplasmic membrane vesicles were cleaved by freeze fracturing to expose fracture faces studded with particles, while the outer membrane fragments resisted cleavage. 3. The outer membrane fraction consisted of protein (similar to 40%), lipopolysaccharide (similar to 36%) and lipid (similar to 18%) and had a density of about 1.22 g/cm3. The cytoplasmic membrane fraction consisted mostly of protein (similar to 56%) and lipid (similar to 38%), had a density of about 1.16 g/cm3, and contained almost all the NADH oxidase, succinate and D-lactate dehydrogenase activities of the crude envelope preparation. 4. Electrophoresis in polyacrylamide gels containing sodium dodecylsulfate revealed over 20 polypeptide bands in the cytoplasmic membrane fraction and only 6-7 in the outer membrane fraction. The outer membrane electrophorogram was dominated by a major band (mol. wt 40 000) which was resolved into two bands when electrophoresed in an acidic gel system. Amino acid analysis revealed a higher content of polar amino acids in the protein moiety of the outer membrane.
Article
Four distinct Proteus mirabilis strains were extracted by the phenol/water procedure. After ultracentrifugation of the dialyzed water phase, the pelleted lipopolysaccharide was purified and analyzed. The sugar composition of this lipopolysaccharide fraction I was similar for all four strains, containing only small amounts of strain-specific constituents. A second lipopolysaccharide fraction was isolated from the supernatant above (termed L1 fraction) after removal of nucleic acids. DEAE-cellulose chromatography indicated that this material is not a polysaccharide but rather a water-soluble lipopolysaccharide containing strain-specific constituents such as uronic acids, amino acids, amino sugars, neutral sugars, ethanolamine and phosphate, depending on the strain from which lipopolysaccharide II was isolated.
Article
Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.
Article
Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.
Article
1.1. The proteins of the outer membrane of Escherichia coli were characterized by polyacrylamide gel electrophoresis and their molecular weights determined.2.2. A buffer system is described which permits reproducible resolution of three proteins which do not resolve on polyacrylamide gel electrophoresis using conventional phosphate-sodium dodecyl sulfate buffer systems.3.3. Pronase digestion of spheroplast membranes, Triton-treated cell envelopes and isolated outer membranes of E. coli showed that only two major proteins were accessible to the enzyme in spheroplast membranes, while Triton treatment revealed several others to pronase attack. Several other proteins were not attacked by pronase under the conditions employed. An asymmetric arrangement of proteins within the membrane is suggested.4.4. Pronase digestion of reformed outer membrane gave a result similar to that obtained for the original outer membrane preparation. It is suggested that reaggregation of the components of the solubilized outer membrane has occurred in a specific manner with the proteins organized as in the native membrane.
Article
The major envelope protein from Escherichia coli has been purified by differential heat extraction in dodecyl sulfate and subsequently freed of the detergent. The polypeptide is homogeneous and has a mass of 36,500 daltons. Homogeneity is based on four criteria, three of which are independent of its behavior in detergents. Its molecular weight was established by three methods independent of dodecyl sulfate binding and agrees with that derived from the mobility of the major band observed in standard dodecyl sulfate gel electrophoretic analysis of unfractionated cell envelopes after treatment at 100°. The mass of the protein is accounted for entirely, or nearly entirely, by the mass of its constituent amino acids. These results imply that dodecyl sulfate is bound in amounts corresponding to those found in most polypeptides. The protein was also isolated in association with the rigid layer of the cell by extraction of cell envelopes in 2 % dodecyl sulfate at 60°. This complex is composed of about 65 % envelope protein, the remaining mass being accounted for largely by the peptidoglycan-lipoprotein structure. In this form the protein is completely resistant to trypsin, but upon dissociation it is quickly degraded to small fragments. Unlike the dissociated polypeptide, the complexed form of the protein does not bind dodecyl sulfate tightly even upon prolonged exposure to high excess at 60°. A large fraction of the polypeptide exists as β-structure, as determined by circular dichroism and infrared spectroscopy. The 105 copies of this polypeptide per cell are arranged in a lattice structure with hexagonal symmetry and a periodicity of 7.5 nm on the outer face of the peptidoglycan. The regular array observed appears to be closely related to its quaternary structure in vivo. All strains of E. coli tested contain this protein.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
A method is described which is specific for the extraction of lipopolysaccharides from R form bacteria. The extraction mixture which is monophasic, consists of aqueous phenol, chloroform and petroleum ether. R form lipopolysaccharides (glycolipids). due to their lipophilic nature, are completely soluble in the mixture. S and T form lipopolysaccharides as well as proteins, nucleic acids, and polysaccharides, are insoluble, and they are excluded from the extracts. The method is mild, as it can be carried out at below 10°. The yields are generally higher than those obtained by phenol-water extraction, and the products are usually water-soluble. Lipopolysaccharides have been successfully extracted from all R form bacteria so far attempted.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
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