Microbial assimilation of hydrocarbons: cellular distribution of fatty acids

The distribution of cellular fatty acids in defined lipid classes was analyzed in Micrococcus cerificans after growth on specified hydrocarbons. Neutral lipid, phospholipid, and cell residue fatty acids were qualitatively and quantitatively determined for M. cerificans grown on nutrient broth, tetradecane (C(14)), pentadecane (C(15)), hexadecane (C(16)), and heptadecane (C(17)), respectively. Percentage of total cellular fatty acid localized in defined lipid classes from cells grown on the above growth substrates was (i) neutral lipid-11.8, 1.81, 7.74, 23.1, and 2%; (ii) phospholipid-74.5, 65, 66.43, 62.1, and 86%; (iii) cell residue lipid-13.5, 33.29, 25.82, 14.78, and 11.9%. Phospholipid fatty acid chain length directly reflected the carbon number of the alkane substrate, with 40, 84, 98, and 77% of the fatty acids being 14, 15, 16, and 17 carbons when cells were grown on C(14), C(15), C(16), and C(17)n-alkanes, respectively. The bound lipids of the cell residue after chloroform-methanol extraction were characterized by 2-hydroxydodecanoic and 2-hydroxytetradecanoic acids plus a broad spectrum of fatty acids ranging from C(10) to C(17) chain length. An increase in total unsaturated fatty acid localized in the phospholipids was noted from cells grown on alkanes greater than 15 carbons long. An extracellular accumulation of free fatty acid (FFA) was demonstrated in hexadecane-grown cultures that was not apparent in non-hydrocarbon-grown cultures. Identification of extracellular FFA demonstrated direct derivation from hexadecane oxidation. Studies supporting inhibition of de novo fatty acid biosynthesis in relationship to extracellular FFA and hexadecane oxidation are described. The ability to alter the fatty acid composition of membrane polar lipids in a predictable manner by the alkane carbon source provides an excellent model system for the investigation of membrane structure-function relationships in M. cerificans.     

Analysis of Dictyostelium discoideum plasma membrane fluidity by electron spin resonance.

Dictyostelium discoideum grown axenically in media containing polyunsaturated fatty acids exhibited normal growth rates but impaired differentiation (Weeks, G. (1976) Biochim. Biophys. Acta 450, 21--32). Since cell-cell contact is vital for differentiation but unnecessary for growth we have examined the isolated plasma membranes of these cells. The lipids of the plasma membranes of cells grown in the presence of polyunsaturated fatty acids contain considerable quantities of these acids, but the total phospholipid and sterol contents of the plasma membrane are close to normal. Electron spin resonance studies using 5-doxyl-stearic acid as the spin probe reveal two things. Firstly, there are no detectable characteristic transition temperatures in the plasma membranes of D. discoideum. Secondly, the plasma membranes of cell grown in the presence of polyunsaturated fatty acids have essentially the same fluidity as that of the control cells. The possible significance of this result to impaired cell-cell interaction is discussed.     

Plasma membranes from Candida tropicalis grown on glucose or hexadecane. I. Isolation, identification and purification.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes. After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation. Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences. The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg2+-dependent ATPase was determine. By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.     

Lipids and fatty acids of a moderately halophilic bacterium, No. 101.

The extractable and bound lipids of a moderately halophilic gram-negative rod, strain No. 101 (wild type) grown in a medium containing 2 M NaC1, were examined. The extractable lipids were separated into at least 8 components by using thin-layer chromatography. The major phospholipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phosphoglycolipid in the whole cells, cell envelopes and outer membrane preparations, commonly. Judging from mild alkaline hydrolysis and exzymatic treatment with phospholipase A2, C and D, the unidentified phosphoglycolipid possessing Pi, glycerol, fatty acids and glucose in a molar ratio of 1 : 2 : 2 : 1, appeared likely to be a glucosyl derivative of phosphatidylglycerol. No glucuronic acid containing lipid was detected. The exractable lipid composition varied greatly with the concentrations of NaC1 in the medium and the stages of bacterial growth. The most characteristic phosphoglycolipid in this organism increased up to 25% of the total phospholipids with the addition of 1% glucose in the medium. The major fatty acids of the extractable lipids were C16:0, C16:1, C18:0, C18:1 and cyclopropanoic C17 and C19 acids and these compositions were very similar for each phospholipid. The cyclopropanoic fatty acids predominated as growth proceeded. The fatty acids of the bound lipids comprised a high concentration of 3-hydroxydodecanoic acid. The esterified fatty acids of the lipopolysaccharide molecule seemed to contain a wide variety of hydroxy and non-hydroxy shorter chain fatty acids, while the amide-linked fatty acids consisted almost entirely of 3-hydroxydodecanoic acid.     

Changes in (Na+ + K+)-ATPase activity of Ehrlich ascites tumor cells produced by alteration of membrane fatty acid composition.

The fatty acid composition of plasma membrane derived from Ehrlich ascites tumor cells was altered in vivo by changing the dietary lipid of the tumor-bearing mice. The activity of (sodium + potassium)-adenosinetriphosphatase ((Na+ + K+ATPase), in partially purified plasma membranes, was measured ass a function of temperature. Arrhenius plots of the data were biphasic. Striking differences, dependent on the membrane fatty acid composition, were observed in the transition temperature and in the energies of activation below the transition temperature. The transition temperatures for the (Na+ + K+)-ATPase of plasma membrane derived from tumor cells grown in mice fed a regular chow diet containing a mixture of fatty acids (PMC), a 16% sunflower oil diet (PMSU), or a 4% tristearin diet (PMTS) were 20, 21, and 13.5 degrees C, respectively...     

Glycolipids and fatty acids of two dog kidney cell lines.

Glycolipid and fatty acid compositions were studied in whole cells and plasma membranes from two dog kidney cell lines (Madin-Darby and SV40-transformed cells) grown in monolayer and suspension cultures. Glycolipids, which account for 5% or less of the total lipids in dog kidney cells, were substantially increased in plasma membranes relative to whole cells. Sialoglycolipids more complex than a Tay-Sachs-like ganglioside were not found in any whole-cell or plasma-membrane preparation of this study. Dog kidney cells transformed by SV40 virus contained primarily a less complex sialoglycolipid, haematoside. Neutral glycolipids comprised 26-43% of the total glycolipid content in Madin-Darby preparations, whereas in transformed cells and membranes neutral glycolipids constituted only 1-22% of the total glycolipid content. Ceramide trihexoside was found in Madin-Darby cultures, but not in transformed cultures. The values for short-chain fatty acids from neutral glycolipids and for saturated fatty acids were generally higher than the values for these fatty acids in calf serum.     

Activity and substrate specificity of the alcohol dehydrogenases of n-alkane oxidizing yeasts

The activity and substrate specificity of alcohol dehydrogenases (ADH) in the fractions of cytosol and membrane particles were compared in the yeasts Torulopsis candida, Candida lipolytica and Candida tropicalis grown in media with glucose and hexadecane. In all studied yeast cultures growing in the medium with hexadecane, NAD-dependent ADH specifically dehydrogenating only medium and higher alcohols are induced in the membrane structures of the cells. Soluble ADH are found in the cytosol of the cultures grown either on glucose or on hexadecane. These ADH oxidize all alcohols with the carbon chain length from C2 to C16. As was found by electrophoresis in polyacrylamide gel, the number of ADH molecular forms in the cytosol fraction of the cultures depends on the carbon growth substrate being used and the peculiarities of yeast culture.     

Membranes of Tetrahymena: III. The effect of temperature on membrane core structures and fatty acid composition of Tetrahymena cells

Membrane core structures as revealed by the freeze-etch electron microscopy and the fatty acid composition measured by gas-liquid chromatography have been analyzed in Tetrahymena cells exposed to low temperature for varying periods.When cells were grown to mid-log phase at the optimal growth temperature of 28 °C and then chilled to 10 °C, cell division was inhibited. However, within 16 h the cells adapted to the low temperature.Chilling effected drastic structural alterations in the cores of different membrane types (membranes of the pellicula, the alveolar sacs, the endoplasmic reticulum and the nuclei). In all cases, there was a segregation of smooth faces from particle-rich faces in the fracture planes. However, the native membrane state, i.e. like that of cells grown at 28 °C, reappeared when the cells adapted to the low temperature.The total lipids of Tetrahymena cells contained primarily even-numbered fatty acids ranging from C₁₂ to C₁₈, but we also detected appreciable amounts of C₂₀ acids; this has not been reported before. During the initial phase of chilling, when cell division is inhibited, about 50% of the saturated fatty acids were replaced by unsaturated fatty acids, primarily monoenoic, dienoic and trienoic acids.We conclude that the structural recovery of the membranes in chilled Tetrahymena cells is accomplished by a desaturation of membrane fatty acids. This is discussed with respect to membrane “fluidity”.     

Effects of moderately enhanced levels of ozone on the acyl lipid composition and dynamical properties of plasma membranes isolated from garden pea (Pisum sativum)

Plasma membranes were isolated from leaves of 16-day-old garden pea, Pisum sativum L., that had been grown in the absence or presence of 65 nl l⁻¹ ozone for 4 days prior to membrane isolation. Plasma membranes from ozone-fumigated plants contained significantly more acyl lipids per protein than those from leaves of plants grown in filtered air on a molar/weight ratio. The ratio between the major acyl lipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), also increased due to the ozone fumigation, while the fatty acid unsaturation level was unaltered in total plasma membrane acyl lipids, as well as in PC and PE. The amount of free sterols per protein was unaltered, but the percentage of campesterol increased, concomitant with a decrease in stigmasterol. The dynamical properties of the isolated plasma membranes were assessed using Laurdan fluorescence spectroscopy, which monitors water penetration and mobility at the hydrophilic-hydrophobic interface of the membrane. At 0°C, the molecular mobility was slightly lower in plasma membranes from ozone-fumigated plants than in plasma membranes from control plants, possibly reflecting the increased PE/PC, campesterol/stigmasterol and lipid/protein ratios, and suggesting that ozone-fumigated pea plants may be more susceptible to freezing injuries.     

Lipids of rabies virus and BHK-21 cell membranes.

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.     

The response of plasma membrane lipid composition in callus of the halophyte Spartina patens (Poaceae) to salinity stress

Callus cultures of the salt marsh grass Spartina patens were examined to determine changes and consistencies in membrane lipid composition in response to salt. Major membrane lipid classes remained stable at all salinity levels (0, 170, 340 mmol/L). However, the membrane protein to lipid ratio decreased significantly in response to elevated NaCl. Callus plasma membrane (PM) consisted predominantly of sterols, about 60% (mol%) of the total lipids. Glycolipid was the second largest lipid class, making up about 20% (mol%) of the total. With increasing salinity, the relative percentage of sitosterol decreased, while that of campesterol increased. The phospholipid species detected were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). When callus was grown at 340 mmol/L NaCl, PC increased significantly. PI and PS were also significantly elevated in salinity treatments. Only 24-32% of the PM fatty acids were common plant membrane fatty acids, C16, C18, C20, and C22, while over 60% were the less common fatty acids, C11 and C14. Membrane fluidity remained stable in response to growth medium salinity. The findings on membrane responses to salinity will facilitate a better understanding of this halophyte's tactics for salt tolerance.     

Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles.

1. Intact cells, cell fragments (membranes) and matrix vesicles were isolated from the proliferating and calcifying layers of epiphyseal cartilage by sequential hyaluronidase and collagenase digestion and differential centrifugation. Lipids were extracted and analyzed for various lipid classes and their fatty acid composition by column, thin-layer, paper and gas-liquid chromatography. 2. On a protein basis the isolated matrix vesicles had more total lipid than either the membrane or cell fractions, the vesicles and membranes being richer in non-polar lipids and containing smaller quantities of phospholipids than whole cells. Expressed as a percentage of the total lipid, the cells were richer in triacylglycerols and lower in free fatty acids than in the membrane or vesicle fractions. The proportion of free cholesterol and the cholesterol/phospholipid ratio were nearly twice as high in the matrix vesicles as in the other tissue fractions. Choline and ethanolamine phosphoglycerides progressively declined in the membrane and matrix vesicle fractions, whereas serine phosphoglycerides and sphinogomyelin increased. Non-phosphorus-containing polar lipids were present in all fractions, the vesicles being richer in polyhexosyl ceramides, cerebrosides, glycosyldiacylglycerols and certain uncharacterized acidic polar lipids. 3. Fatty acid patterns of the matrix vesicles were distinctive from those of isolated cells, being generally richer in 18 : 0 and 18 : 2, and lower in 16 : 1 and 18 : 1 fatty acids. Monoacyl forms were similarly increased in 16 : 0 and/or 18 : 0, and reduced in 16 : 1, 18 : 1 or 20 : 2 fatty acids, depending on the lipid class. The fatty acid composition of diphosphatidylglycerol from cells and matrix vesicles was markedly different, providing evidence that the cardiolipin in the vesicles was not from mitochondrial components. 4. Based on the fact that the matrix vesicles were significantly enriched in free cholesterol, sphingomyelin, glycolipids and serine-phosphoglycerides, it is concluded that they are derived from the plasma membrane of the cell, supporting earlier conclusions based upon morphological and enzymological evidence.     

Distribution of phospholipid molecular species containing arachidonic acid and cholesterol in V79-UF cells

V79-UF cells were isolated from Chinese hamster V79 cells as a cell line that requires exogenous unsaturated fatty acids for growth. V79-UF cells incorporated arachidonic acid into phospholipids. The molecular species of diacyl phosphatidylcholine and phosphatidylethanolamine containing arachidonic acid comprised 61.4 and 70.5% of the total phospholipid molecular species in total membranes and 58.1 and 64.7% in plasma membrane, respectively. Polyunsaturated molecular species were distributed in a higher amount in the intracellular membranes than in the plasma membrane. No significant difference was seen in the diffusion coefficient between the plasma membranes from cells supplemented with oleic and arachidonic acids in spite of a distinct difference in the degree of unsaturation between the molecular species of these plasma membranes. The amount of cholesterol in the plasma membrane was higher in the cells grown in the presence of arachidonic acid than in those grown in the presence of oleic acid.     

Regulation of fatty acid biosynthesis by hydrocarbon substrates in Mycobacterium convolutum.

When Mycobacterium convolutum R22 was grown on the n-alkanes C13 through C16, the predominant fatty acids were of the same chain length as the growth substrate. Cells grown on C13 through C16 n-alkanes incorporated between 15 and 85 pmol of acetate per microgram of lipid into the fatty acids, whereas acetate- or propane-grown cells incorporated 280 and 255 pmol of acetate per microgram of lipid, respectively. In vivo experiments demonstrated that hexadecane, hexadecanoic acid, and hexadecanoylcoenzyme A (CoA) all inhibited de novo fatty acid synthesis. Hexadecanoyl-CoA was the most potent inhibitor. Hexadecane and hexadecanoic acid inhibited acetyl-CoA carboxylase by up to 37 and 39%, respectively, at 1 mM. Hexadecanoyl-CoA inhibited the enzyme activity by 65% at 50 micrometer. Cells that were grown on C14 through C16 n-alkanes had about 25 times less acetyl-CoA carboxylase activity than did cells grown on acetate or propane, suggesting repressed levels of the enzyme. Hexadecane- or pentadecane-grown cells were found to have 5 to 10 times more intracellular free fatty acid than cells grown on acetate, propane, or ethane.     

ESR studies on the membrane properties of a moderately halophilic bacterium. II. Effect of extreme growth conditions on liposome properties

Lipid preparations from the cells of a moderately halophilic bacterium, Pseudomonas halosaccharolytica grown under the two extreme conditions of high temperature-high NaCl concentration and low temperature-low NaCl concentration showed distinctively different profiles in phospholipid and fatty acid composition. Cells grown at 40 degrees C in medium containing 3.5 M NaCl had high concentrations of saturated and C19 cyclopropanoic fatty acids (about 50 per cent of the total), whereas cells grown at 20 degrees C in medium containing 0.5 M NaCl had decreased concentrations of these fatty acids with increased concentrations of the corresponding unsaturated fatty acids. The phospholipid composition was also affected ty the culture conditions; cells grown at 40 degrees C in 3.5 M NaCl had large amounts of acidic phospholipids, whereas those grown at 20 degrees C in 0.5 M NaCl had small amounts. ESR studies on liposomes prepared from lipids of cells grown under the two conditions showed characteristic profiles for correlation times and order parameters of three spin labels of stearic acid derivatives similar to those of membranes of whole cells of this bacterium. ESR studies showed that the physical properties of the liposomes from the total extractable lipids and isolated phosphatidylglycerol from the cells were completely different from those of synthetic dioleoylphosphatidylglycerol. Liposomes of the lipids extracted from cells grown at 40 degrees C in 3.5 M NaCl showed change in rotational viscosity on altering the NaCl concentration to 0.5M, whereas liposomes of lipids extracted from cells grown at 20 degrees C in 0.5 M NaCl did not show change in rotational viscosity on increasing the NaCl concentration to 3.5 M.     

Lipid Composition of Purified Vesicular Stomatitis Viruses

Methods are described for the production of vesicular stomatitis (VS) virus of sufficient purity for reliable chemical analysis. VS virions released from infected cells were concentrated and purified at least 150-fold by sequential steps of precipitation with polyethylene glycol, column chromatography, rate zonal centrifugation, and equilibrium centrifugation. The Indiana serotype (VS(Ind) virus) propagated in L-cells was found to contain 3% ribonucleic acid, 64% protein, 13% carbohydrate, and 20% lipid; the molar ratio of cholesterol to phospholipid was 0.6 or greater. Thin-layer chromatography revealed no unusual neutral lipids or phospholipids and gas-liquid chromatography revealed no unusual fatty acids incorporated into VS virions. The antigenically distinct New Jersey serotype (VS(NJ) virus) grown in L-cells showed a similar lipid profile except that the proportion of neutral lipids was larger than in VS(Ind) virus also grown in L-cells. This differences was less pronounced when the lipid composition of VS(Ind) and VS(NJ) viruses grown in chick embryo cells was compared, but VS(NJ) virus grown in either cell type always contained larger amounts of neutral lipids other than cholesterol than did VS(Ind) virus. The lipid composition of both VS(Ind) and VS(NJ) viruses grown in L-cells or chick embryo cells more closely resembled that of plasma membrane than of whole cells. A consistent finding was the relatively large amounts of phosphatidylethanolamine and sphingomyelin and the relatively small amounts of phosphatidylcholine in both VS viruses compared with uninfected whole L-cells and chick embryo cells or their plasma membranes. The methods available for isolation of plasma membranes were inadequate for conclusive comparison of the lipids of VS virions with the lipids of the plasma membranes of their host cells. Nevertheless, the data obtained are consistent with two hypotheses: (i) the lipid composition of VS viruses primarily reflects their membrane site of maturation, and (ii) the newly synthesized viral proteins inserted into cell membranes influence the proportions of phospholipids and neutral lipids selected for incorporation into the viral membrane.     

Lipid and fatty acid composition of Gluconobacter oxydans before and after intracytoplasmic membrane formation

Gluconobacter oxydans differentiates by forming quantities of intracytoplasmic membranes at the end of exponential growth, and this formation occurs concurrently with a 60% increase in cellular lipid. The present study was initiated to determine whether this newly synthesized lipid differed from that extracted before intracytoplasmic membrane synthesis. Undifferentiated exponential-phase cells were found to contain 30% phosphatidylcholine, 27.1% caridolipin, 25% phosphatidylethanolamine, 12.5% phosphatidylglycerol, 0.4% phosphatidic acid, 0.2% phosphatidylserine, and four additional unidentified lipids totaling less than 5%. The only change detected after formation of intracytoplasmic membranes was a slight decrease in phosphatidylethanolamine and a corresponding increase in phosphatidylcholine. An examination of lipid hydrolysates revealed 11 different fatty acids in the lipids from each cell type. Hexadecanoic acid and monounsaturated octadecenoic accounted for more than 75% of the total fatty acids for both cell types. Proportional changes were noted in all fatty acids except octadecenoate. Anteiso-pentadecanoate comprised less than 1% of the fatty acids from undifferentiated cells but more than 13% of the total fatty acids from cells containing intracytoplasmic membranes. These results suggest that anteiso-pentadecanoate formation closely parallels the formation of intracytoplasmic membranes. Increased concentrations of this fatty acid may contribute to the fluidity necessary for plasma membrane convolution during intracytoplasmic membrane development.     

Component from the cell surface of the hydrocarbon-utilizing yeast Candida tropicalis with possible relation to hydrocarbon transport.

A polysaccharide-fatty acid complex was isolated from the cell surface of Candida tropicalis growing on alkanes. This complex was solubilized by Pronase treatment of whole cells. A decrease in alkane-binding affinity was observed after Pronase treatment, resulting in 10 to 12% of the yeast dry cell weight being released as polysaccharide. The isolated polysaccharide contained 2.5% fatty acids. C. tropicalis and Saccharomyces cerevisiae grown with glucose contained only traces of fatty acids in the corresponding polysaccharide fraction. The fatty acids were not removed from the polysaccharide moiety by gel filtration. Extraction of the polysaccharide with chloroform-methanol showed that fatty acids were covalently bound to the polysaccharide. The amphipathic nature of the isolated polysaccharide and the hydrocarbon-induced formation suggest a possible role in alkane metabolism.     

Effects of growth temperature on protoplast membrane properties in Bacillus megaterium.

Changes in the protoplast membrane of the KM strain of Bacillus megaterium were assessed after growth at 20, 30, or 37 degrees, C. Although the overall membrane concentrations of lipids and proteins were virtually unchanged, increased culture temperature resulted in cells with membranes that contained relatively more unbranched and long-chain fatty acids and more acidic phospholipids, as well as different proportions and numbers of individual proteins. Electrophoretic analysis revealed 23, 31, or 29 protein bands, respectively, in membranes from cells grown at the three temperatures. Protoplasts from cells grown at higher temperatures were considerably less susceptible to lysis by shearing forces. As judged by passive leakage at 30 degrees C, intact cells from cultures grown at 37 degrees C were the least permeable to erythritol. Relatively low ambient concentrations of Ca2+ or Mg2+ protected protoplasts from osmotic lysis but even much higher concentrations left erythritol leakage virtually unaffected. Thus, growth temperature affected not only membrane lipis but also membrane proteins and these changes resulted in membranes with altered mechanical properties and permeabilities.     

Composition and Biophysical Properties of Lipids in Xenorhabdus nematophilus and Photorhabdus luminescens, Symbiotic Bacteria Associated with Entomopathogenic Nematodes

Primary and secondary forms of Photorhabdus luminescens Hm and Xenorhabdus nematophilus N2-4 were grown at 18 and 28(deg)C for 24 to 96 h, and we made determinations of the fatty-acid compositions of total lipids and of the fluidity measured by 5-doxyl-stearic acid embedded in liposomes made from total lipids. The levels of the unsaturated fatty acids 16:1 and 18:1 (those with chain lengths of 16 or 18 and one double bond) generally were higher in primary-phase variants of P. luminescens grown at 18(deg)C than in those grown at 28(deg)C. Prolonged culture at 18(deg)C caused the level of 18:1 to fall and reach that observed at 28(deg)C. The ratio of saturated to unsaturated fatty acids rose with prolonged culture times in variants of each species at both phases. When grown at 18(deg)C, the proportion of 16:1 in X. nematophilus was lower than in P. luminescens; the patterns of temperature-induced changes were similar in these species. X. nematophilus contained a greater percentage of short-chain fatty acids (i.e., with chain lengths of <14.0) than P. luminescens. Lipid liposomes from primary and secondary cultures of both bacterial species grown at 18(deg)C were more ordered (i.e., less fluid) than those grown at 28(deg)C. This result suggests the surprising absence of homeoviscous adaptation of membranes to temperature. Also, liposomes from primary cultures were more ordered than those from secondary cultures and membranes from primary cultures of P. luminescens were more ordered at both culture temperatures than membranes from X. nematophilus. The biological significance of the effect of growth conditions on membrane biophysical properties in these bacteria is discussed.