Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.     

Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions.

Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichia coli at 0 degrees C in the presence of divalent cations. During incubation at 42 degrees C, 0.1 to 1% of this DNA became resistant to deoxyribonuclease. Deoxyribonuclease-resistant DNA binding and the ability to produce transformants became saturated when transformation mixtures contained 1 to 2 micrograms of plasmid NTP16 DNA and about 5 X 10(8) viable cells. Under optimum conditions, between 1 and 2 molecule equivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonuclease resistant. Despite this, only 0.1 to 1% of viable cells became transformed by saturating amounts of the plasmid. The results suggest that transport of DNA across the inner membrane is a limiting step in transformation. After transformation the bulk of labeled plasmid DNA remained associated with outer membranes. However, in vitro assays indicated that plasmid DNA would bind equally well to preparations of inner or outer membranes provided divalent cations were present to preparations of inner or outer membranes provided divalent cations were present. Divalent cations promoted differing levels of binding to isolated inner and outer membranes in the order Ca2+ much greater than Ba2+ greater than Sr2+ greater than Mg2+. This parallels their relative efficiencies in promoting transformation. Binding of plasmid DNA was greatly reduced when outer membranes were treated with trypsin; this suggests that protein components may be required for the binding or transport of DNA (or both) during transformation.     

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5.

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkyline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.     

Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium.

The reversibility and specificity of phospholipid translocation between the inner and outer membrane of Salmonella typhimurium has been investigated by incorporating exogenous lipids from phospholipid vesicles into the outer membrane of intact cells. Translocation of newly incorporated phospholipids to the inner membrane was demonstrated by decarboxylation of vesicle-derived phosphatidylserine and by recovery of vesicle constituents in both inner and outer membrane fractions. All Salmonella phospholipids tested, as well as phosphatidylcholine and cholesteryl oleate were effectively translocated to the inner membrane. However, no translocation of vesicle-derived lipopolysaccharide or an incomplete biosynthetic precursor of lipid A could be detected. Translocation of phospholipids and cholesteryl ester was rapid and extensive, and appeared to lead to equilibration of the lipids between the two membranes. The mechanism of intermembrane translocation has not been established, but the results are suggestive of diffusional flow across zones of adhesion between the inner and outer membranes.     

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkaline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose-containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.     

Conjugative junctions in RP4-mediated mating of Escherichia coli

The physical association of bacteria during conjugation mediated by the IncPalpha plasmid RP4 was investigated. Escherichia coli mating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (trbC), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.     

Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates.

To determine whether certain outer membrane proteins are associated with growth of Bacteroides thetaiotaomicron on polysaccharides, we developed a procedure for separating outer membranes from inner membranes by sucrose density centrifugation. Cell extracts in 10% (wt/vol) sucrose-10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4) were separated into two fractions on a two-step (37 and 70% [wt/vol]) sucrose gradient. These fractions were further resolved into outer membranes (p = 1.21 g/cm3) and inner membranes (p = 1.14 g/cm3) on sucrose gradients. About 20 to 26% of the total 3-hydroxy fatty acids from lipopolysaccharide and 2 to 3% of the total cellular succinate dehydrogenase activity were recovered in the outer membrane preparation. The inner membrane preparation contained 22 to 49% of the total succinate dehydrogenase activity and 2 to 3% of the total 3-hydroxy fatty acids from lipopolysaccharide. Outer membranes contained a lower concentration of protein (0.34 mg/mg [dry weight]) than did the inner membranes (0.68 mg/mg [dry weight]). Molecular weights of inner membrane polypeptides ranged from 11,000 to 133,000. The most prominent polypeptides had molecular weights ranging from 11,000 to 26,000. In contrast, the molecular weights of outer membrane polypeptides ranged from 17,000 to 117,000. The most prominent polypeptides had molecular weights ranging from 42,000 to 117,000. There were several polypeptides in the outer membranes of bacteria grown on polysaccharides (chondroitin sulfate, arabinogalactan, or polygalacturonic acid) which were not detected or were not as prominent in outer membranes of bacteria grown on monosaccharide components of these polysaccharides.     

Effect of variations in lipopolysaccharide on the fluidity of the outer membrane of Escherichia coli.

The lipid hydrocarbon chains in the outer membrane of gram-negative bacteria appear from previous experiments to be less mobile than in the cytoplasmic membrane. To determine whether lipopolysaccharide, a unique outer membrane component, is a cause of this restricted mobility, outer membranes differing in the amount of lipopolysaccharide, and the length of the polysaccharide side chain, were prepared from Escherichia coli J5. Cytoplasmic membranes were prepared for comparison. The probes, 5- and 12-doxylstearate, were introduced into these membranes, electron spin resonance spectra were analyzed, and the order parameter (S) and empirical motion parameter (tau0) were calculated. Outer membrane preparations containing long chain lipopolysaccharide were much less fluid by these criteria than were preparations containing short chain lipopolysaccharide. Removing about 40% of the lipopolysaccharide from the former preparations greatly increased their fluidity. The lipid in the cytoplasmic membrane preparations was more fluid than in the outer membrane and cytoplasmic membranes were similar to each other regardless of the composition of the outer membrane. These results indicate that lipopolysaccharide, and especially the polysaccharide portion, directly or indirectly causes the restricted mobility of the lipid hydrocarbon chains observed in the outer membrane.     

Isolation and partial characterization of outer and inner membranes from encapsulated Haemophilus influenzae type b.

A method has been developed to separate the cell envelope of encapsulated (type b) Haemophilus influenzae into its outer and inner membrane components with procedures that avoided two problems encountered in fractionation of this envelope: (i) the tendency of the outer and inner membranes to hybridize and (ii) the tendency of the apparently fragile inner membrane to fragment into difficulty sedimentable units. Log phage cells, whose lipids were radioactively labeled, were lysed by passage through a French press. The lysate was applied to a discontinuous sucrose gradient, and envelope-rich material was collected by centrifugation onto a cushion of dense sucrose under carefully controlled conditions. This material was then further fractionated by isopycnic centrifugation in a sucrose gradient to yield four membrane fractions which were partially characterized. On the basis of their radioactivity, buoyant density, ultrastructure, polypeptide composition, and content of phospholipid, protein, lipopolysaccharide, and succinic dehydrogenase, these fractions were identified as follows: fraction 1, outer membrane vesicles with very little inner membrane contamination (less than 4%); fraction 2, outer membrane vesicles containing entrapped inner membrane; fraction 3, a protein-rich fraction of inner membrane; fraction 4, a protein-poor fraction of inner membrane. Fractions 3 and 4 contained about 25% outer membrane contamination.     

Outer and inner membrane preparations of extreme thermophile and their physico-chemical properties

Outer and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation. In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis. When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar. The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature. A similar phenomenon was observed with the TEMPO parameter of liposomal membranes. The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth. The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes. The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its biological function.     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Comparison of methods used to separate the inner and outer membranes of cell envelopes of Campylobacter spp

The outer membrane of Campylobacter coli, C. jejuni and C. fetus cell envelopes appeared as three fractions after sucrose gradient centrifugation. Each outer membrane fraction was contaminated with succinate dehydrogenase activity from the cytoplasmic membrane fraction. Similarly the inner membrane fraction was contaminated with 2-ketodeoxyoctonate and outer membrane proteins including the porin(s). The separation of these two membranes was not facilitated by variations in lysozyme treatment, cell age, presence or absence of flagella, or longer lipopolysaccharide chain length. Sodium lauroyl sarcosinate extraction resulted in an outer membrane fraction which contained some inner membrane contamination and produced multiple bands upon sucrose gradient centrifugation. Triton X-100 extraction removed the inner membrane from the outer membrane and Triton X-100/EDTA treatment extracted lipopolysaccharide-rich regions of the outer membrane which contained almost exclusively the Campylobacter porin(s). These data indicated that the inner and outer membranes of the Campylobacter cell envelope were very difficult to separate, possibly because of extensive fusions between these two membranes.     

Microelectrode studies of the effect of lanthanum on the electrical potential and resistance of outer and inner cell membranes of isolated frog skin

Microelectrodes were used to investigate the effect of 0.5 mM mucosal lanthanum (La3+) on the intracellular potential and the resistance of outer and inner isolated frog skin (Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potential Vsco (mean value = -65.4 +/- 3.2 mV, inside negative) hyperpolarized to -108.7 +/- 2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potential Vo or the inner membrane potential Vi. The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La3+ to the outer medium increased the short-circuit current to 190% of the control value. Vsco concomitantly changed to -28 +/- 3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La3+ is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La3+ on short-circuit current has been observed. It is ascribed to the penetration of La3+ into the sodium channels.     

Apparent variability in expression of the Bla+ phenotype of plasmid RP1 inPseudomonas acidovorans (RP1) strains

Expression of the Bla⁺ phenotype of the incompatibility group P-1 plasmid RP1 appears to be a variable phenomenon inPseudomonas acidovorans strains, although all plasmid-bearing strains examined synthesize the RP1-encoded TEM 2 β-lactamase. There is also evidence suggesting that plasmid-encoded alterations to the outer membrane could be affecting the degree of resistance observed to β-lactam drugs in at least some strains.     

Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes.

The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.     

Architecture of the outer membrane of Escherichia coli K12. I. Action of phospholipases A2 and C on wild type strains and outer membrane mutants.

Phospholipids in whole cells of wild type Escherichia coli K12 are not degraded by exogenous phospholipases, whereas those of isolated outer membranes are completely degraded. It is concluded that the resistance of phospholipids in whole cells is due to shielding by one or more other outer membrane components. The nature of the shielding component(s) was investigated by testing the sensitivity of whole cells of a number of outer membrane mutants. Mutants lacking both major outer membrane proteins b and d or the heptose-bound glucose of their lipopolysaccharide, are sensitive to exogenous exogenous phospholipases. Moreover, cells of a mutant which lacks protein d can be sensitized by pretreatment of the cells with EDTA. From these results and from data on the chemical composition of the outer membranes, it is concluded that proteins b and d, the heptose-bound glucose of lipopolysaccharide and divalent cations are responsible for the inaccessibility of phospholipids to to exogenous phospholipases.     

Architecture of the outer membrane of Escherichia coli K12. I. Action of phospholipases A2 and C on wild type strains and outer membrane mutants

Phospholipids in whole cells of wild type Escherichia coli K12 are not degraded by exogenous phospholipases, whereas those of isolated outer membranes are completely degraded. It is concluded that the resistance of phospholipids in whole cells is due to shielding by one or more other outer membrane components. The nature of the shielding component(s) was investigated by testing the sensitivity of whole cells of a number of outer membrane mutants. Mutants lacking both major outer membrane proteins b and d or the heptose-bound glucose of their lipopolysaccharide, are sensitive to exogenous phospholipases. Moreover, cells of a mutant which lacks protein d can be sensitized by pretreatment of the cells with EDTA. From these results and from data on the chemical composition of the outer membranes, it is concluded that proteins b and d, the heptose-bound glucose of lipopolysaccharide and divalent cations are responsible for the inaccessibility of phospholipids to exogenous phospholipases.     

Fine structure of the cell envelope layers of Flexibacter polymorphus.

Electron microscopy of the filamentous gliding marine bacterium Flexibacter polymorphus demonstrated that the cell envelope consists of an electron-dense intermediate layer located between two unit-type membranes: an outer membrane, presumably of lipopolysaccharide, and an inner cytoplasmic membrane. Separation of living filaments into single cells by lysozyme suggests that a peptidoglycan moiety, possibly corresponding to the intermediate layer, might be situated between the two membranes. Cell division proceeds by invagination of the cytoplasmic membrane and intermediate layer forming a transverse septum. Cells generally fail to separate after the division process, so that a common outer membrane encloses all of the cells in a single filament. There is a continuous layer of macromolecular cup-shaped elements ('goblets') attached to the outermost surface of the lipopolysaccharide membrane. Tangential thin sections, as well as negatively stained preparations of envelope fragments (produced by sonication of autolyzed cells), showed that the goblets are arranged in a close-packed hexagonal array. The presence of electron-dense structures located between the outer and inner membranes, and exhibiting the same periodicity as the goblets, suggests that some part of the goblets penetrates the outer membrane and extends across the periplasmic space to the dense intermediate layer or cytoplasmic membrane. Spontaneous autolysis in aging cultures is accompanied by the formation and release into the culture medium of large numbers of outer membrane vesicles coated with globlets. A tentative reconstruction of the envelope of F. polymorphus, based on the fine-structural data, is presented.     

Disruption of Escherichia coli outer membranes by EM 49. A new membrane active peptide.

A new peptide antibiotic, EM 49, is shown to disrupt the structure of Escherichia coli outer membranes and release outer membrane fragments into the surrounding media. Evidence supporting this conclusion indludes EM 49 stimulated release of outer membrane phospholipids, lipopolysaccharide, and membrane fragments having a phospholipid and polypeptide composition similar to outer membranes. The density of the membrane fragments released by EM 49 was 1.22 g/cm3, which was identical to isolated outer membranes. Approximately 10 to 15% of the E. coli lipopolysaccharide was released upon treatment with EM 49. Both scanning and transmission electron microscopy revealed that the antibiotic caused the formation of numerous protrusions or blebs on the surface of E. coli with apparent release of membrane vesicles from the cells. Direct interaction between EM 49 and outer membranes was demonstrated using outer membranes labeled with the fluorescent dye diphenylhexatriene. Treatment of the fluorescent-labeled outer membranes with EM 49 increased fluorescence intensity and decreased polarization, indicating that the peptide perturbed outer-membrane structure. In addition, strong interactions between EM 49 and purified E. coli phospholipids were detected using the Hummel and Dreyer technique. Association constants between the peptide and phospholipids were approximately 10(5) M-1. A model for the disruptive effect of EM 49 on outer-membrane structure is proposed in which the fatty acid chain of the antibiotic is inserted into the hydrophobic core of the membrane. This orientation would allow the polycationic, peptide portion of the antibiotic to disrupt the antibiotic to disrupt the normal electrostatic interactions between divalent cations and components of the outer membrane. Evidence supporting this conclusion includes specific protection of E. coli from EM 49 by Mg2+ and Ca2+ and inhibition of EM 49 stimulated phospholipid release by these cations. Disruption of the antibiotic to penetrate to the inner membrane, which is probably the primary killing site of EM 49.