Sequence and inactivation of the pss gene of Escherichia coli. Phosphatidylethanolamine may not be essential for cell viability

Phosphatidylethanolamine is the only zwitterionic phospholipid in Escherichia coli and accounts for 70-80% of the total glycerophospholipids of this organism. To investigate the function of phosphatidylethanolamine in E. coli, we constructed an inactivated allele (pss93::kan) of the gene encoding the phosphatidylserine synthase which catalyzes the committed step to the synthesis of phosphatidylethanolamine. Growth of this mutant was dependent on a plasmid-borne copy of the wild type gene. After curing the mutant of the wild type gene, growth stopped when the content of phosphatidylethanolamine reached 30% of the total phospholipid. Divalent metal ions at millimolar concentrations suppressed the growth phenotype of the mutant in the following order of efficiency: Ca2+ greater than Mg2+ greater than Sr2+. Although phosphatidylserine synthase activity was not detectable, phosphatidylethanolamine was still present at 0.007% of the total phospholipid after growth for many generations in rich medium containing 20 mM Mg2+. The remainder of the phospholipid was primarily phosphatidylglycerol and cardiolipin with no other unique phosphate-containing chloroform-soluble material present. The phospholipid to protein ratio and the fatty acid composition were very similar to the parental strain. The broad divalent metal ion auxotrophy brought about by the lack of phosphatidylethanolamine suggests a primarily structural role for this phospholipid in E. coli.     

Yeast mutant defective in phosphatidylcholine synthesis

The Saccharomyces cerevisiae opi3-3 mutant was shown to be defective in the synthesis of phosphatidylcholine via methylation of phosphatidylethanolamine. The opi3-3 mutant was isolated on the basis of an inositol excretion phenotype and was not auxotrophic for choline. Inositol, but not choline, stimulated growth of the mutant. The opi3-3 mutation was recessive and was genetically linked to the ino4 locus. When grown in the absence of exogenous choline, the opi3-3 mutant had a phospholipid composition consisting of 2 to 3% phosphatidylcholine compared with 40 to 50% in wild-type strains. In addition, the mutant accumulated elevated amounts of two intermediates, phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The incorporation of label from [methyl-14C]methionine into phosphatidylcholine was reduced 80 to 90% in the mutant compared with wild-type strains. However, label was recovered in the intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The mutant is believed to be defective in the third and possibly the second methylation reaction in the formation of phosphatidylcholine from phosphatidylethanolamine. The first methylation reaction appeared to be occurring at normal or even elevated levels. Based upon incorporation of choline into phosphatidylcholine, it is concluded that the opi3-3 mutant has no defect in the synthesis of phosphatidylcholine from exogenous choline. Furthermore, phosphatidylcholine represents over 25% of the phospholipid composition of the mutant when it is grown in the presence of exogenous choline.     

Membrane phospholipid composition of Escherichia coli affects secretion of periplasmic alkaline phosphatase into the medium

Secretion of alkaline phosphatase (PhoA) encoded by a gene constituent of plasmids has been studied in Escherichia coli strains with controlled synthesis of anionic phospholipids (phosphatidylglycerol and cardiolipin, strain HDL11) and zwitterionic phospholipid (phosphatidylethanolamine, strain AD93). Changing the phospholipid composition of the membrane of these strains leads to an increase in secretion of PhoA, which is usually localized in the periplasm, into the culture medium. This correlates with a higher secretion of exopolysaccharides and lower content of lipopolysaccharide in the outer membrane. The results show the possibility of coupling protein secretion into the medium with biogenesis of cell envelope components in which phospholipids are involved.     

Cardiolipin Accumulation in the Inner and Outer Membranes of Escherichia coli Mutants Defective in Phosphatidylserine Synthetase

Mutants of Escherichia coli defective in phosphatidylserine synthetase (pss) make less phosphatidylethanolamine than normal cells, and they are temperature sensitive for growth. We have isolated a new mutant, designated RA2021, which is better than previously available strains in that the residual phosphatidylethanolamine level approaches 25% after 4 h at 42 degrees C. The total amount of phospholipid normalized to the density of the culture is about the same in RA2021 (pss-21) as in the isogenic wild-type RA2000 (pss(+)). Consequently, there is a net accumulation of polyglycerophosphatides in the mutant, particularly of cardiolipin. The addition of 10 to 20 mM MgCl(2) to a culture of RA2021 prolongs growth under nonpermissive conditions and prevents loss of cell viability, but it does not eliminate the temperature-sensitive phenotype. Divalent cations, like Mg(2+), do not correct the phospholipid composition of the mutant, but may act indirectly by balancing the negative charges of phosphatidylglycerol and cardiolipin. To determine the effects of the pss mutation on membrane composition, we have examined the subcellular distribution of the polyglycerophosphatides that accumulate in these strains. All of the excess anionic lipids of RA2021 are associated with the envelope fraction and are distributed equally between the inner and outer membranes. The protein compositions of the isolated membranes do not differ significantly in the mutant and wild type. The fatty acid composition of RA2021 is almost the same as wild type at 30 degrees C, but there is more palmitic and cyclopropane fatty acid at 42 degrees C. These results demonstrate that the modification of the polar lipid composition observed in pss mutants affects both membranes and that cardiolipin, which is not ordinarily present in large quantities, can accumulate in the outer membrane when it is overproduced by the cell. The altered polar headgroup composition of the outer membrane in pss mutants may account, in part, for their hypersensitivity to the aminoglycoside antibiotics.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

Composition and synthesis of cellular lipids in Neurospora crassa during cellular differentiation.

The synthesis of cellular lipids of Neurospora crassa was measured during growth on low (2% sucrose)- and high (15% glucose)-carbohydrate supplementation. The amount of lipid per dry weight of cells does not change during the germination and early logarithmic growth periods, but the percentage of phospholipid in the lipid does increase, reaching a maximal value of 90% at 4 to 5 h after inoculation, at which time the phospholipid content of the cells is approximately 60 mumol/g (dry weight). The content of the anionic phospholipids, as a percentage of the lipid fraction, is relatively constant during the growth period, but the contents of the zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine change in a reciprocal fashion. During the first 8 h of growth, phosphatidylcholine falls from 53% of the phospholipid to 43%, whereas phosphatidylethanolamine rises from 29 to 38%. The total of these two phospholipids is approximately 83% during the growth period studied. The synthesis of cellular phospholipids, measured either by [32P]H3PO4 or [14C]glucose incorporation, reached maximal levels between 3 and 5 h of growth. The effect of the high-carbohydrate supplement on cellular lipids was minimal. Inclusion of 15% glucose decreased the labeling of phospholipid by [32P]H3PO4, but did not affect lipid composition. This observation is in contrast to the effects of high glucose on mitochondrial phospholipid synthesis.     

Effects of phospholipid composition on MinD-membrane interactions in vitro and in vivo

The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic alpha-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15693-15698). To investigate whether E. coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E. coli that lacks phosphatidylethanolamine. In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content. In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2. Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes. The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes. The degree of acyl chain unsaturation contributed positively to binding strength. These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.     

Effect of doxorubicin on the order of the acyl chains of anionic and zwitterionic phospholipids in liquid-crystalline mixed model membranes: absence of drug-induced segregation of lipids into extended domains.

We investigated the effect of the antineoplastic drug doxorubicin on the order of the acyl chains in liquid-crystalline mixed bilayers consisting of dioleoylphosphatidylserine (DOPS) or -phosphatidic acid (DOPA), and dioleoylphosphatidylcholine (DOPC) or -phosphatidylethanolamine (DOPE). Previous 2H-NMR studies on bilayers consisting of a single species of di[11,11-2H2]oleoyl-labeled phospholipid showed that doxorubicin does not affect the acyl chain order of pure zwitterionic phospholipid but dramatically decreases the order of anionic phospholipid [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 1096, 67-80]. In the present work, we studied mixed bilayers in which alternatively the anionic or the zwitterionic phospholipid component was 2H-labeled so as to monitor its individual acyl chain order. Doxorubicin decreased the order parameter of the mixed anionic and zwitterionic lipids by approximately the same amount and did not induce a clear segregation of the lipid components into extended, separate domains. The drug had a comparable disordering effect on mixed bilayers of unlabeled cardiolipin and 2H-labeled zwitterionic phospholipid, indicating the absence of extensive segregation also in that case. Upon addition of doxorubicin to bilayers consisting of 67 mol% DOPE and 33 mol% anionic phospholipid, a significant part of the lipid adopted the inverted hexagonal (HII) phase at 25 degrees C. This bilayer destabilization, which occurred only in mixtures of anionic phospholipid and sufficient amounts of DOPE, might be of physiological importance. Even upon formation of extended HII-phase domains, lipid segregation was not clearly detectable, since the relative distribution of 2H-labeled anionic phospholipid and [2H]DOPE between the bilayer phase and HII phase was very similar. Our findings argue against a role of extensive anionic/zwitterionic lipid segregation in the mechanism of action and toxicity of doxorubicin.     

Alteration of phospholipid composition by combined defects in phosphatidylserine and cardiolipin synthases and physiological consequences in Escherichia coli.

Escherichia coli K-12 derivatives with a common genetic background carrying, either alone or in combination, the pss-1 allele coding for a temperature-sensitive phosphatidylserine synthase (A. Ohta and I. Shibuya, J. Bacteriol. 132:434-443, 1977) and cls- for a defective cardiolipin synthase (G. Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) were constructed. The phospholipid polar headgroup compositions of these strains were significantly different from each other depending on their genotypes and growth temperature, whereas other membrane characteristics such as the total phospholipid content, fatty acid composition, membrane protein profile, and lipopolysaccharide content were practically the same, suggesting that the phenotypes of these strains were the direct consequences of abnormalities in membrane phospholipid composition. The cls pss-1 double mutation caused an unusual accumulation of phosphatidylglycerol with an extremely low content of cardiolipin. The cls mutation alone was found to give a growth defect, and its introduction into a pss-1 mutant resulted in an enhanced temperature sensitivity of growth. Addition to a broth medium of a proper concentration of sucrose, NaCl, Mg2+, or Ca2+ allowed the growth of a pss-1 mutant at otherwise nonpermissive temperature, but a pss-1 cls double mutant required the combined addition of sucrose or NaCl and MgCl2 for full growth at 42 degrees C. The possible mechanisms for these physiological consequences of the mutations are discussed on a molecular basis. The remedial effects of culture supplements allowed the pss-1 mutants to grow at 42 degrees C resulting in enhanced abnormalities of membrane phospholipid composition.     

Effects of temperature and sodium chloride concentration on the phospholipid and fatty acid compositions of a halotolerant Planococcus sp.

The phospholipid headgroup composition and fatty acid composition of a gram-positive halotolerant Planococcus sp. (strain A4a) were examined as a function of growth temperature (5 to 35 degrees C) and NaCl content (0 to 1.5 M) of the growth medium. When the growth temperature was decreased, the relative amount of mono-unsaturated branched-chain fatty acids increased. When Planococcus sp. strain A4a was grown in media containing high NaCl concentrations, the relative amount of the major fatty acid, Ca15:0, increased. The relative amount of anionic phospholipid also increased when the NaCl concentration of the growth medium was increased. The increase in anionic phospholipid content resulted from a decrease in the relative mole percent content of phosphatidylethanolamine and an increase in the relative mole percent content of cardiolipin.     

Diabetes affects phospholipid content in the nuclei of the rat liver.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on the content of different phospholipids and the incorporation of blood-borne 14C-palmitic acid into the phospholipid moieties in the rat liver nuclei. Diabetes was produced by intravenous administration of streptozotocin, and determinations were carried out two and seven days thereafter. Phospholipids were extracted from isolated nuclei and separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. Following that, they were quantified and radioactivity was measured. It was found that, in comparison to non-diabetic controls, two-day diabetes reduced the total content of phospholipids in the nuclei by 9.6%. The content of phospholipids in the nuclei by 9.6%. The content of phosphatidylcholine and phosphatidylserine was reduced and the content of the remaining phospholipids was stable. The specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin, based on radioactivity incorporated from 14C-palmitic acid, was elevated. Seven-day diabetes resulted in a reduction of the total phospholipid content in the nuclei by 39.4%. This was accounted for by a reduction in the content of each phospholipid fraction with the exception of cardiolipin. The specific activity of each phospholipid fraction, was elevated in this group. It is concluded that insulin is involved in the regulation of the nuclear phospholipid content.     

Membrane phospholipid synthesis in Escherichia coli: alteration by glycerol and physiological consequences in a pss mutant

Escherichia coli mutants harboring the pss-1 allele (coding for a temperature-sensitive phosphatidylserine synthase) are temperature sensitive for growth and synthesize less phosphatidylethanolamine at higher temperatures, giving rise to abnormal membrane phospholipid compositions. To obtain information concerning the determinant for the phospholipid polar headgroup composition and the lethal factor in the defective membranes, we have examined the effect of increased supply of sn-glycerol 3-phosphate on the phospholipid synthesis and the growth ability of a pss-1 mutant. For this purpose, a pair of E. coli K-12 derivatives isogenic except for the pss-1 allele was constructed from strain BB26-36 to harbor the mutations related to glycerol metabolism (glpD3, glpR2, glpKi, and phoA8). Pulse- and uniform-labeling of phospholipids with 32P at 42 degrees C in a synthetic medium with (0.2%) or without glycerol showed that glycerol further lowered the temperature-sensitive formation of phosphatidylethanolamine, removed the phosphatidate and CDP-diacylglycerol accumulated in the absence of glycerol, and resulted in an increase in cardiolipin content in the pss-1 mutant. The phospholipid synthesis and contents in the pss+ strain were not significantly affected by glycerol. Glycerol in the medium markedly enhanced the growth defect of the pss-1 mutant, which was remediable by sucrose. The results indicate that the intracellular pool of sn-glycerol 3-phosphate is the limiting factor for acidic phospholipid synthesis in the pss-1 mutant, and cardiolipin unusually accumulated is injurious to the functional E. coli membranes. Possible determinants for the phospholipid composition of the wild-type E. coli cells are also discussed on the basis of the present observations.     

The effects of altered levels of phosphatidylcholine and phosphatidylethanolamine on fatty acid desaturase activity and sterol metabolism during temperature acclimation in a choline auxotroph of Neurospora crassa

Experiments were conducted to determine the effects of choline deprivation on levels of phospholipid fatty acids in a choline auxotroph (chol-1; chol-2) of Neurospora crassa with respect to high (37 degrees C) and low (15 degrees C) growth temperatures and during acclimation following a shift from high to low temperature conditions. Although grossly altered levels of phosphatidylethanolamine and phosphatidylcholine were observed at both temperatures, phospholipid fatty acid levels remained virtually identical to those found in a phenotypically wild-type and maximally supplemented chol-1; chol-2 strains grown under the same conditions. Deprivation of choline from supplemented cultures of the mutant followed by a shift from high to low growth temperatures did not significantly affect the level of fatty acid desaturation with respect to control cultures. Free sterols did not significantly affect the level of fatty acid desaturation with respect to control cultures. Free sterols were reduced, however, and sterol ester levels were elevated in choline-deprived cultures, suggesting that sterol interconversions may be closely tied to aspects of phospholipid biosynthesis. These experiments suggest that although major modifications in membrane fluidity may be brought about by thermally induced changes in fatty acid desaturase activity, it seems probable that additional cellular mechanisms may be involved if fluidity is under precise control.     

The interaction of cardiolipin with rat liver carbamoyl phosphate synthetase I

A selective interaction of rat liver carbamoyl phosphate synthetase I with cardiolipin, and other anionic phospholipids, has been demonstrated. The enzymatic activity of the synthetase is inhibited by cardiolipin and, to a lesser extent, by phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. This group of anionic phospholipids also induced a conformational change in the synthetase, yielding a species with increased exposure of the linkages between independently folded domains of the enzyme, as determined by limited proteolysis under nondenaturing conditions. The interaction of cardiolipin with carbamoyl phosphate synthetase I was a fairly slow process, with complex kinetics, and was apparently irreversible. The inclusion of Mg2+ or of MgATP in the incubation mixture prevented the cardiolipin effects. The zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine had negligible effects on the structure and activity of the synthetase. This interaction between cardiolipin and carbamoyl phosphate synthetase I potentially constitutes one of the mechanisms by which the synthetase forms its loose association with the inner mitochondrial membrane. Multiple mechanisms, including synthetase conformational changes, cardiolipin phase changes, and ATP/ADP binding site involvement, are possibly involved in the phospholipid/synthetase interaction and the resulting potential regulatory mechanism(s) for urea cycle activity.     

Membrane phospholipid asymmetry in Bacillus amyloliquefaciens.

The phospholipid distribution in the membrane of Bacillus amyloliquefaciens was studied by using phospholipase C (B. cereus), phospholipase A2 (Crotalus), and the nonpenetrating chemical probe trinitrobenzenesulfonic acid. After treatment of intact protoplasts of B. amyloliquefaciens with either phospholipase, about 70% of total membrane phospholipid was hydrolyzed; specifically, about 90, 90, and 30% of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, respectively. Under these conditions, protoplasts remained intact and sealed. However, when protoplasts that were permeabilized by cold-shock treatment were incubated with either of the phospholipases, up to 80% of cardiolipin was hydrolyzed and phosphatidylglycerol and phosphatidylethanolamine were hydrolyzed virtually to completion. In intact cells, 92% of the phosphatidylethanolamine could be labeled with trinitrobenzenesulfonic acid under conditions in which the reagent did not penetrate the membrane to any significant extent. These results indicate that 70% of total phospholipid of this bacillus exists in the outer half of the bilayer. The distribution of phosphatidylethanolamine in this bilayer is highly asymmetric with it being located predominantly in the outer half. The results with phospholipases suggest that the distributions of cardiolipin and phosphatidylglycerol are also asymmetric but independent confirmation of this is required.     

Pulmonary surfactant protein SP-B is significantly more immunoreactive in anionic than in zwitterionic bilayers

Binding of polyclonal and monoclonal antibodies, quantitated by enzyme-linked immunosorbent assay, to porcine SP-B reconstituted in different phospholipid bilayers has been used to assess differences in protein structure due to lipid-protein interactions. SP-B bound significantly more antibodies when it was reconstituted in bilayers made of anionic phospholipids (phosphatidic acid, cardiolipin, phosphatidylglycerol, phosphatidylinositol or phosphatidylserine) than in zwitterionic bilayers (phosphatidylcholine, phosphatidylcholine/cholesterol, or phosphatidylethanolamine) or in fatty acid micelles (made of salts of palmitic or stearic acids). These differences in immunoreactivity can be important in the development of quantitation methods for SP-B in clinical samples based on immunological techniques.     

Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs.

The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane.     

Role of anionic lipid in bacterial membranes

The major phospholipids of Bacillus stearothermophilus are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Under the growth conditions used in this study the concentration of anionic lipid (PG + CL) was determined by the pH of the culture medium. Cells grown in a complex medium at pH 5.8, 7.0, and 8.0 contained 17, 29 and 36 nmol of anionic (PG + CL) lipid/mg cell (dry weight). The concentration of the zwitterionic lipid phosphatidylethanolamine (PE) was 17-20 nmol/mg cell (dry weight) under all conditions. Analysis of isolated membrane preparations suggested that the amount of anionic lipid per unit area of membrane increased as the pH of the growth medium was increased. Membranes from cells grown at pH 5.8 and 8.0 contained 130 and 320 nmol anionic lipid/mg membrane protein, respectively. Phosphatidylethanolamine appeared to be localized on the inner membrane surface in cells grown under all conditions. Increasing the ionic strength of the culture medium by the addition of NaCl or KCl had little effect on growth at pH 5.8 but inhibited growth at pH 7 and 8. It was concluded that anionic phospholipid plays an important physiological role in maintaining an acidic pH at the outer membrane surface.     

The modification of the membrane of Oceanomonas baumannii when subjected to both osmotic and organic solvent stress

Oceanomonas baumanniioff a novel halotolerant bacterium which was isolated from the estuary of the river Wear (Sunderland, UK). When grown in tryptone soya broth it can tolerate high levels of phenol, which is not utilised as a carbon source in this medium. However, the level of tolerance was reduced from 35 mM to 3 mM phenol as salinity increased from 1% to 12% NaCl (w/v). Increasing salinity up to 12% NaCl also decreased the growth rate 8-fold and caused modifications to the cytoplasmic membrane particularly anionic phosphatidylglycerol levels, which doubled at the expense of zwitterionic phosphatidylethanolamine. In addition, changes in the phospholipid fatty acid composition were noted, cis-vaccenic acid decreased significantly at higher salinities. Intracellular solute levels also increased with increasing salinity and there was an accumulation of the compatible solutes ectoine, glycine betaine and glutamate. The addition of phenol to osmotically compromised cultures led to a further modification of the cytoplasmic membrane phospholipid composition, in particular, that the decrease in zwitterionic phosphatidylethanolamine and the increase of anionic phospholipid species was much less pronounced. A further decrease in unsaturation, particularly in the proportion of cis-vaccenic acid, and the mean chain length of the fatty acids suggested that this response was important in maintaining membrane integrity in the presence of phenol.