Cellular incorporation of poly-beta-hydroxybutyrate into plasma membranes of Escherichia coli and Azotobacter vinelandii alters native membrane structure

Under growth-limiting conditions or conditions which mediate genetic transformation, Escherichia coli and Azotobacter vinelandii incorporate poly-beta-hydroxybutyrate into their plasma membranes. Genetic transformation competence of both bacteria increased in proportion to the concentration of membrane poly-beta-hydroxybutyrate. The effects of this lipid polymer on membrane structure were investigated by freeze-fracture electron microscopy. Before poly-beta-hydroxybutyrate incorporation, freeze-fracture revealed a typical mosaic of particles and pits on both concave and convex surfaces of the plasma membrane. As the cells incorporated the lipid polymer into the membrane, transformability developed and small semiregular plaques which possessed shallow particles were seen. These plaques grew in size and frequency as the membrane poly-beta-hydroxybutyrate concentrations and transformability increased.     

Improved electro-transformation of highly DNA-restrictive corynebacteria with DNA extracted from starved Escherichia coli

Abstract Differences of up to 33000-fold in electro-transformability of highly DNA restrictive corynebacteria are observed in the DNA of a shuttle plasmid extracted from Escherichia coli hosts propagated in different nutritional conditions. Growth of the host in minimal medium increases plasmid transformability, whereas growth on rich media decreases it. In the E. coli DH5a host, the starvation-dependent increase in DNA transformability is reverted by supplementing with methionine, an obligate S-adenosyl-methionine (SAM) precursor. This suggests that an E. coli nutritionally modulated SAM-dependent DNA-methyltransferase may be involved in this phenomenon.     

D-(-)-poly-beta-hydroxybutyrate in membranes of genetically competent bacteria.

D-(-)-Poly-beta-hydroxybutyrate is a constituent of the membranes and the cytoplasms of genetically competent Azotobacter vinelandii, Bacillus subtilis, and Haemophilus influenzae cells. Within each species the concentration of D-(-)-poly-beta-hydroxybutyrate in the membranes and cytoplasm correlates with transformability. Fluorescence analysis of the thermotropic lipid phase transitions in A. vinelandii and B. subtilis cells indicates that D-(-)-poly-beta-hydroxybutyrate forms an organized gel structure in the membranes which is very labile. The concentration of organized D-(-)-poly-beta-hydroxybutyrate in the membranes, which can be estimated from the intensity of its phase transition, can be used to assess the competence of a culture.     

Investigations into the polymorphism of lipid A from lipopolysaccharides of Escherichia coli and Salmonella minnesota by Fourier-transform infrared spectroscopy

The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5 degrees and 58 degrees C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41 degrees, 46 degrees, 44 degrees and 47 degrees C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4'-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52 degrees C for E. coli lipid A). The analysis of greater than CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22 degrees, 30 degrees, 27 degrees and 25.5 degrees C (first transition) and at about 34 degrees, 42 degrees, 38.5 degrees and 40.5 degrees C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: Up to about 30 degrees C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of greater than CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli.

The lipid phase transition of Escherichia coli phospholipids containing cyclopropane fatty acids was compared with the otherwise homologous phospholipids lacking cyclopropane fatty acids. The phase transitions (determined by scanning calorimetry) of the two preparations were essentially identical. Infection of E. coli with phage T3 inhibited cyclopropane fatty acid formation over 98%, whereas infection with mutants which lack the phage coded S-adenosylmethionine cleavage enzyme had no effect on cyclopropane fatty acid synthesis. These data indicate that S-adenosylmethionine is the methylene in cyclopropane fatty acid synthesis.     

Multiple Regulatory Events in the Development of Competence for Genetic Transformation in Haemophilus influenzae

Cyclic adenosine 3',5'-monophosphate derepresses enzyme synthesis but does not yield high levels of transformability or detectable levels of competence-related envelope polypeptides. Additional regulatory events must trigger complete expression of competence.     

Efficacy of antifreeze protein types in protecting liposome membrane integrity depends on phospholipid class

Antifreeze proteins have been reported to be capable of maintaining the membrane integrity of cold sensitive mammalian cells when exposed to hypothermic temperatures. However the mechanism(s) whereby these proteins exert this protective effect is unknown. The present study used liposomes as a model system to examine the nature of the interactions between four antifreeze (glyco)protein types (AFP I, II, III and AFGP) and albumin, with lipid membranes. Fluorescein isothiocyanate labelling indicated that all of the proteins bound to the three liposome types (dielaidoylphosphatidylcholine (DEPC), dielaidoylphosphatidylethanolamine (DEPE) and dielaidoylphosphatidylglycerol (DEPG)). AFGP was found to be highly effective at preventing leakage from all three liposome compositions as they were cooled through their phase transition temperatures. This was not the case for the other proteins. All four antifreeze types prevented zwitterionic DEPC liposomes from leaking as they were cooled through their phase transition temperature. However, albumin was equally as effective, indicating that this capacity was not unique to antifreeze proteins. All of the proteins, except AFGP, induced the negatively charged DEPG liposomes to leak prior to cooling, and were less effective than AFGP in preventing phase transition leakage from DEPE liposomes. It is proposed that many proteins, including antifreeze proteins, can protect zwitterionic liposomes, such as DEPC, by binding to the lipid bilayer thereby maintaining the ordered structure of the membrane during phase transition. However, when the membrane contains a negatively charged polar group, such as with DEPE and DEPG, proteins, although bound to them, may not be able to maintain sufficient membrane organization to prevent leakage during phase transition or, they may gain entry into the lipid bilayer, disrupt the structure and induce leakage. These results imply that the efficacy of antifreeze proteins in the cold protection of mammalian cells will not only depend on protein structure, but also on the lipid composition of the cell membrane.     

Lipid biosynthesis and its coordination with cell cycle progression

The activation of cell cycle regulators at the G1/S boundary has been linked to the cellular protein synthesis rate. It is conceivable that regulatory mechanisms are required to allow cells to coordinate the synthesis of other macromolecules with cell cycle progression. The availability of highly synchronized cells and flow cytometric methods facilitates investigation of the dynamics of lipid synthesis in the entire cell cycle of the heterotrophic dinoflagellate Crypthecodinium cohnii. Flow cytograms of Nile red-stained cells revealed a stepwise increase in the polar lipid content and a continuous increase in neutral lipid content in the dinoflagellate cell cycle. A cell cycle delay at early G1, but not G2/M, was observed upon inhibition of lipid synthesis. However, lipid synthesis continued during cell cycle arrest at the G1/S transition. A cell cycle delay was not observed when inhibitors of cellulose synthesis and fatty acid synthesis were added after the late G1 phase of the cell cycle. This implicates a commitment point that monitors the synthesis of fatty acids at the late G1 phase of the dinoflagellate cell cycle. Reduction of the glucose concentration in the medium down-regulated the G1 cell size with a concomitant forward shift of the commitment point. Inhibition of lipid synthesis up-regulated cellulose synthesis and resulted in an increase in cellulosic contents, while an inhibition of cellulose synthesis had no effects on lipid synthesis. Fatty acid synthesis and cellulose synthesis are apparently coupled to the cell cycle via independent pathways.     

Macromolecular Synthesis in Bacillus subtilis During Development of the Competent State

Rates of deoxyribonucleic acid, ribonucleic acid, and protein synthesis were examined in purified competent cells of Bacillus subtilis during the development of the transformable state. To become competent, a cell must depart from the normal course of vegetative growth and pass through a precompetent phase beginning as early as 90 to 180 min before the appearance of transformability. While in the precompetent state, the cell decreases its rate of deoxyribonucleic acid synthesis and lowers its ratio of ribonucleic acid synthesis to protein synthesis. This altered pattern of synthesis eventually leads to a decreased buoyant density of precompetent cells. Once a cell has become both precompetent and low in density, it can be converted to a competent (transformable) cell. The early alterations in macromolecular synthesis were found in two competence regimens, one utilizing a nutritional step-down and one free of such a shift. The data imply that the precompetent state is a generalized characteristic of the B. subtilis transformation system and is not specific to the procedure used to allow competence development. Since precompetence-specific events occur very early in a competence regimen, we conclude that the induction of precompetence is unrelated to sporulation or a nutritional shift.     

A confirmation of the phase behavior of Escherichia coli cytoplasmic membrane lipids by X-ray diffraction.

The lipid fatty acid composition of the cytoplasmic membranes of Escherichia coli can be varied by growing an unsaturated fatty acid auxotroph in the presence of different fatty acid supplements. Electron spin resonance (ESR) studies of spin-label partitioning into the cytoplasmic membranes of different lipid fatty acid compositions as a function of temperature have been interpreted as indicating a broad order-to-disorder transition in the membrane lipids, the end points of the transition depending upon the fatty acid composition. We have utilized x-ray diffraction to confirm the ESR studies for three different fatty acid supplements (oleic, elaidic, and bromostearic). We found that the characteristic end-point temperatures detected by ESR were indeed the end-point temperatures of a broad order-to-disorder transition of the cytoplasmic membrane lipids. In addition, Patterson functions calculated from lamellar x-ray diffraction from partially oriented cytoplasmic membranes indicate a decrease in average membrane thickness upon fatty acid chain melting.     

A high-conductance mode of a poly-3-hydroxybutyrate/calcium/polyphosphate channel isolated from competent Escherichia coli cells

Reconstitution into planar lipid bilayers of a poly-3-hydroxybutyrate/calcium/polyphosphate (PHB/Ca(2+)/polyP) complex from Escherichia coli membranes yields cationic-selective, 100 pS channels (Das, S., Lengweiler, U.D., Seebach, D. and Reusch, R.N. (1997) Proof for a non-proteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate. Proc. Natl. Acad. Sci. USA 94, 9075-9079). Here, we report that this complex can also form larger, weakly selective pores, with a maximal conductance ranging from 250pS to 1nS in different experiments (symmetric 150mM KCl). Single channels were inhibited by lanthanum (IC(50)=42+/-4microM, means+/-S.E.M.) with an unusually high Hill coefficient (8.4+/-1.2). Transition to low-conductance states (<250pS) was favored by increased membrane polarization (/V/ >or=50mV). High conductance states (>250pS) may reflect conformations important for genetic transformability, or "competence", of the bacterial cells, which requires the presence of the PHB/Ca(2+)/polyP complex in the membrane.     

Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.

The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of lateral stress in lipid layer containing nonbilayer phase preferring lipids by frequency-domain fluorescence spectroscopy.

Frequency-resolved fluorescence measurements have been performed to quantitate the lateral stress of the lipid layer containing nonbilayer phase preferring dioleoylphosphatidylethanolamine (DOPE). On the basis of a new rotational diffusion model, the wobbling diffusion constant (Dw), the curvature-related hopping diffusion constant (DH), and the two local orientational order parameters ([P2] and [P4]) of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC) in fully hydrated DOPE and DOPE/dioleoylphosphatidylcholine (DOPC) mixtures were calculated from the frequency-domain anisotropy data. The values of [P2], [P4], and DH for DOPE were found to increase significantly at approximately 12 degrees C, the known lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition temperature of DOPE. Similar features as well as a decline of Dw were detected in the DOPE/DOPC mixtures as the DOPE content was increased from 85% to 90% at 23 degrees C, corresponding to the known lyotropic phase transition of the DOPE/DOPC. In contrast, for DOPC (0-40 degrees C) and DOPE/DOPC (0-100% DOPE at 3 degrees C), which remained in the L alpha phase, these changes were not detected. The most probable local orientation of DPH-PC in the DOPE/DOPC mixtures shifted progressively toward the normal of the lipid/water interface as the content of DOPE increased. We concluded that the curvature-related lateral stress in the lipid layer increases with the content of the nonbilayer phase preferring lipids.     

Structure and phase behavior of hydrated mixtures of L-dipalmitoylphosphatidylcholine and palmitic acid. Correlations between structural rearrangements, specific volume changes and endothermic events

Several new features of the phase diagram of L-dipalmitoylphosphatidylcholine (DPPC)/palmitic acid mixtures in excess water were established by means of static and time-resolved X-ray diffraction, densitometry and differential scanning calorimetry (DSC). At low temperatures, palmitic acid has a biphasic effect on the lamellar subgel phases: at concentrations below 5-6 mol%, it prevents formation of the DPPC subgel phase (Lc), while at higher contents (between about 40 and 90 mol%) another subgel phase (Lccom) is formed as a result of lipid co-crystallization at 1 DPPC: 2 palmitic acid stoichiometry. A crystalline palmitic acid phase separates from Lccom above 70-80 mol% of fatty acid. The Lccomphase transforms into a lamellar gel phase (L beta) in an endothermic transition centered at 38 degrees C. At high temperatures, the mixtures form hexagonal liquid-crystalline phase (HII) in the region of 60-70 mol% and an isotropic phase (I) at 90-100 mol% of palmitic acid. No coexistence of HII phase with the fluid lamellar phase of DPPC was observed at intermediate compositions (20 and 50 mol% of palmitic acid) but rather formation of a complex phase with non-periodic geometry characterized by molten chains and a broad, continuous small-angle scattering band. No evidence for fluid phase coexistence was found also at compositions between HII and I phases. The L beta--HII transition at 60-70 mol% of palmitic acids is readily reversible and two-state in both heating and cooling modes. It is characterized by the coexistence of initial and final phases with no detectable intermediates by time-resolved and static X-ray diffraction. The crystalline-isotropic transition in palmitic acid is two-state only in heating direction. On cooling, it is characterized by strong undercooling and gradually relaxing lamellar crystalline structures. The slowly reversible Lccom--L beta transition proceeds continuously through intermediate states. Although clearly discernible by both DSC and X-ray diffraction, it is not accompanied by specific volume changes.     

relA gene control of the synthesis of lipid A fatty acyl moieties.

The incorporation of [14C]acetate into the fatty acid moieties of lipid A was measured during amino acid starvation of rel+ and relA strains of Escherichia coli K-12. The synthesis of the beta-hydroxymyristate and other fatty acid moieties was inhibited two- to fourfold in rel+ strains, whereas no inhibition was observed in relA strains. The fatty acid compositions of the phospholipids synthesized after amino acid starvation or rel+ and relA strains were also determined.     

Hyperproduction of Poly-beta-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD

The transformation of Azotobacter vinelandii UW with A. vinelandii 113 DNA resulted in the formation of rifampin-resistant colonies, 13% of which also inherited a previously unrecognized mutation in the respiratory NADH oxidase. These transformants produced colonies with a white-sectored phenotype after prolonged incubation. Cells from these sectors were separated and purified by streaking and were named UWD. The dense white phenotype was due to the production of a large amount of poly-beta-hydroxybutyrate during the exponential growth of strain UWD. The polymer accounted for 65 or 75% of the cell dry weight after 24 h of incubation of cultures containing glucose and either ammonium acetate or N(2), respectively, as the nitrogen source. Under the same conditions, strain UW cells contained 22 to 25% poly-beta-hydroxybutyrate, but O(2)-limited growth was required for these optimal production values. Polymer production was not dependent on O(2) limitation in strain UWD, but the efficiency of conversion of glucose to poly-beta-hydroxybutyrate was enhanced in O(2)-limited cultures. Conversion efficiencies were >0.25 and 0.33 mg of poly-beta-hydroxybutyrate per mg of glucose consumed under vigorous- and low-aeration conditions, respectively, compared with an efficiency of 0.05 achieved by strain UW. Strain UWD, therefore, appeared to from poly-beta-hydroxybutyrate under novel conditions, which may be useful in designing new methods for the industrial production of biodegradable plastics.     

Intramolecular excimer formation of pyrene-labeled lipids in lamellar and inverted hexagonal phases of lipid mixtures containing unsaturated phosphatidylethanolamine

The rates of intramolecular excimer formation of di(1'-pyrenemyristoyl)phosphatidylcholine (dipyPC) in dioleoylphosphatidylethanolamine (DOPE), egg PE/diolein (DG) and dilinoleoyl-PE (DLPE)/1-palmitoyl-2-oleoyl-PC (POPC) were studied at different temperatures and lipid compositions. Both the excimer-to-monomer intensity ratio and the excimer association rate constant were employed to quantify the rate of excimer formation. The latter was calculated from the measured monomer fluorescence lifetime of dipyPC. We observed that the rate of excimer formation was sensitive to either the temperature-induced or lipid composition-induced lamellar-to-inverted hexagonal phase transition of the above lipid systems. As the lipids entered the inverted hexagonal phase, the rate of excimer formation increased at the temperature-induced phase transition for DOPE, but decreased at the composition-induced phase transition for both TPE/DG and DLPE/POPC systems by increasing the DG% and decreasing the PC%, respectively. We conclude that the rate of intramolecular excimer formation of dipyPC in the non-lamellar phase is influenced both by the intra-lipid free volume of the hydrocarbon region and the intra-rotational dynamics of the two lipid acyl chains.     

Cyclosporin A inhibits initiation but not progression of human T cell proliferation triggered by phorbol esters and calcium ionophores

Cyclosporin A (CsA) is a potent inhibitor of T lymphocyte proliferation induced by Ag and mitogens. In an attempt to further delineate the mechanism of action of CsA, we have examined its effects on T cell proliferation induced by the combination of the phorbol ester, phorbol 12,13-dibutyrate (PDB), and the calcium ionophore, ionomycin. T cells were rendered competent as the result of a 30-min initial incubation with both drugs, after which the drugs were washed out. Competence is defined as the ability to subsequently proliferate in response to exogenously added IL-2 or PDB in the second phase of the culture, but not to synthesize IL-2 or proliferate without these additions. Addition of CsA (1 microgram/ml) to the cells in the initial, competence-inducing 30-min incubation with PDB/ionomycin abrogated their subsequent response to IL-2 or PDB. In contrast, addition of CsA to cells after they had been treated for 30 min with PDB/ionomycin and then washed did not affect their responses to subsequent addition of either IL-2 or PDB. Treatment with CsA during induction of competence prevented the expression of the 55-kDa IL-2R gene during competence induction and inhibited IL-2 gene expression and IL-2 production in response to PDB in the second phase. These results indicate that the effects of CsA are limited to the initiation (competence induction) period of T cell activation, that CsA apparently affects expression of more than one gene, and in competent cells, CsA does not affect their ability to progress to DNA synthesis.     

The effect of microenvironment and formation of intra-molecular links on the tertiary structure of cytochrome P-450 LM2

The effect of intramolecular cross-links formation in isolated cytochrome P-450 LM2 on its reactivation after incorporation into the liposome lipid bilayer was studied. Treatment with bifunctional reagents results in the inactivation of the solubilized haemoprotein. The degree of the enzyme immobilization determines the degree of inhibition of p-nitroanisol demethylation and aniline hydroxylation. Whereas the complete inhibition of oxidation of type II substrate turnover needs two intramolecular cross-links, that of type I substrates necessitates at least seven cross-links. The incorporation of modified and native enzymes into the membrane lipid bilayer at temperatures above the phase transition point results in the enzyme activation. However, in case of the preimmobilized enzyme the activation does not reach the maximal values. Both stabilized and liposome-incorporated cytochrome P-450 can fully be reactivated via the cross-link disulfide bond reduction. No such effect is observed at temperatures below the phase transition point.