Structure of membrane lipids and physico-biochemical properties of the plasma membrane from Thermoplasma acidophilum, adapted to growth at 37 degrees C.

Thermoplasma acidophilum, a mycoplasma-like organism, grows optimally at 56 degrees C and pH2. The low temperature extreme of growth is 37 degrees C. The plasma membrane of cells grown at 37 degrees C was isolated and characterized physicobiochemically. Membrane lipids which comprise 25% of the membrane dry weight consist mainly of two repetitively methyl-branched C40 side chains that were ether-linked to two glycerol molecules. The lipid structures were elucidated by combined gas chromatography-mass spectroscopy, direct probe mass spectroscopy and 13C NMR. 37 degrees C-grown cells contained lipids with 42% more pentane cyclization than the 56 degrees C-grown cells. In 37 degrees C-grown cells, phospholipid and serine content decreased by about 10% each, carbohydrate content increased by 5%. EPR studies demonstrated an increase in membrane lipid fluidity of 37 degrees C-grown cells with an upper transition temperature at 35 degrees C which was shifted down by 10 degrees C compared with cells grown at 56 degrees C. Membrane-bound ATPase activities also indicated similar changes upon adaptation. There is a close correlation between membrane fluidity and physiological functioning of this membrane-bound enzyme.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385-392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15 degrees C after the shift down of growth temperature from 39 degrees C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Candida dubliniensis and Candida albicans display surface variations consistent with observed intergeneric coaggregation

Adherence of yeasts to other microorganisms and epithelial cell surfaces is important in their colonization. Comparative studies based on the coaggregation of Candida dubliniensis versus Candida albicans with Fusobacterium nucleatum and other oral bacteria suggested differences in the surfaces of these yeasts. Transmission electron microscopy was used to test the hypothesis that there are morphologic variations in the cell surface of these two species. C. dubliniensis type strain CD36 and C. albicans ATCC 18804 were grown on Sabouraud's dextrose agar at various growth temperatures. In some experiments suspensions of yeast cells were treated with dithiothreitol. Fixation for transmission electron microscopy was accomplished using dimethylsulfoxide and alcian blue added to 3% paraformaldehyde and 1% glutaraldahyde in cacodylate buffer. The cell wall of both species was predominantly electron lucent and was visibly differentiated into several layers. A thin electron dense outer layer was seen with clearly visible fibrillar structures, closely associated to the cytoplasmic membrane. The length of the fibrils of the C. albicans cells grown at 37 degrees C was approximately two times greater than those of the cells grown at 25 degrees C. The fibrils of the 37 degrees C-grown cells were thin, distinct and tightly packed whereas those of the 25 degrees C-grown cells appeared blunt, loosely spaced and aggregated. C. dubliniensis demonstrated short, blunt fibrils appearing similar to those of the 25 degrees C-grown C. albicans cells. C. dubliniensis showed no difference in the density, length and arrangement of fibrils between the 25 degrees C and 37 degrees C growth temperatures. The shortest and most aggregated fibrils seen were of the 45 degrees C-grown C. albicans cells. Dithiothreitoltreated 37 degrees C-grown C. albicans cells revealed a distorted and partially destroyed fibrillar layer. In this investigation C. dubliniensis, unlike C. albicans, displayed an outer fibrillar layer that did not vary with variations in growth temperature. In addition, the fibrils on the C. dubliniensis cells were similar to those of the 25 degrees C-grown C. albicans in that they were considerably shorter and less dense than those of the 37 degrees C-grown C. albicans cells. It can be postulated, that C. dubliniensis exhibits constant cell surface characteristics consistent with hydrophobicity and that this property may give this species an ecological advantage. Therefore, C. dubliniensis may compete well in oral environments via enhanced attachment to oral microbes and other surfaces, perhaps even more efficiently than C. albicans.     

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28 degrees C and 37 degrees C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Several proteins with molecular weights ranging from 34 kDa to 71 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could also be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discussed.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43°C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20°C, but in a liquid crystalline state when cells were grown at 37 and 43°C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15 degrees C to 34 degrees C or decreased from 39 degrees C to 15 degrees C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34 degrees C swam at 570 microns/s. On incubation at 15 degrees C these cells swam at 100 microns/s. When the temperature was increased to 34 degrees C after a 90-min incubation at 15 degrees C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34 degrees C-grown cells decreased to 210 microns/s, and the cells ceased to move when the temperature was decreased to 15 degrees C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15 degrees C increased gradually during incubation at 15 degrees C. On the other hand, the fluidity of the heated cell decreased during incubation at 34 degrees C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 microns/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Characterization and environmental regulation of outer membrane proteins in Xenorhabdus nematophilus.

We have examined the production of the outer membrane proteins of the primary and secondary forms of Xenorhabdus nematophilus during exponential- and stationary-phase growth at different temperatures. The most highly expressed outer membrane protein of X. nematophilus was OpnP. The amino acid composition of OpnP was very similar to those of the porin proteins OmpF and OmpC of Escherichia coli. N-terminal amino acid sequence analysis revealed that residues 1 to 27 of the mature OpnP shared 70 and 60% sequence identities with OmpC and OmpF, respectively. These results suggest that OpnP is a major porin protein in X. nematophilus. Three additional proteins, OpnA, OpnB, and OpnS, were induced during stationary-phase growth. OpnB was present at a high level in stationary-phase cells grown at 19 to 30 degrees C and was repressed in cells grown at 34 degrees C. OpnA was optimally produced at 30 degrees C and was not present in cells grown at lower and higher temperatures. The production of OpnS was not dependent on growth temperature. In contrast, another outer membrane protein, OpnT, was strongly induced as the growth temperature was elevated from 19 to 34 degrees C. In addition, we show that the stationary-phase proteins OpnA and OpnB were not produced in secondary-form cells.     

Outer membrane proteins and cell surface structure of Selenomonas ruminantium.

The protein compositions of the membrane preparations from Selenomonas ruminantium grown in glucose or lactate medium were determined by sodium dodecyl sulfate- and two-dimensional (first, isoelectric focusing; second, sodium dodecyl sulfate) polyacrylamide slab gel electrophoresis. The outer membrane from both glucose- and lactate-grown cells contained two major proteins with apparent molecular weights of 42,000 and 40,000. These proteins existed as peptidoglycan-associated proteins in the outer membrane. The critical temperature at which they were dissociated completely into the monomeric subunits of 42,000 and 40,000 daltons was found to be 85 degrees C. The amount of each protein varied considerably depending upon the cultural conditions. The absence of the lipoprotein of Braun in S. ruminantium was suggested in our preceding paper (Y. Kamio, and H. Takahashi, J. Bacteriol. 141:888--898, 1980), and the possible absence of the protein components corresponding to the Braun lipoprotein in this strain was confirmed by electrophoretic analysis of the outer membrane and the lysozyme-treated peptidoglycan fractions. Examination of the cell surface of S. ruminantium by electron microscopy showed that the outer membrane formed a wrinkled surface with irregular blebs, some of which pinched off forming vesicles of various sizes. Rapid cell lysis occurred with the addition of a low level of lysozyme to the cell suspension. These findings led us to conclude that the physiological and morphological properties of this strain were similar to those of "deep rough" and mlp or lpo mutants of Escherichia coli K-12, respectively.     

The mechanism of membrane response to chilling. Effect of temperature on phospholipid deacylation and reacylation reactions in the cell surface membrane

The ciliary membrane of Tetrahymena pyriformis is physically and metabolically remote from the main centers of lipid metabolism. Nevertheless, it possesses an independent capacity to modify its phospholipid molecular species composition rapidly under stress. The role of ciliary phospholipid deacylating and reacylating enzymes in this phenomenon has been evaluated. Isolated cilia showed substantial phospholipase A (combined A1 and A2), acyl-CoA synthetase and acyltransferase activities. Activities of all the three enzymes of cilia from 39 degrees C-grown cells were greatly reduced when the cilia were incubated at 15 degrees C. In contrast, the phospholipase A and acyltransferase activities in cilia from 15 degrees C-grown cells were surprisingly high at 15 degrees C and twice as high at 37 degrees C as were the equivalent activities in preparations from 39 degrees C-grown cells. While the in vivo substrate specificity of phospholipase A could not be meaningfully assessed, the acyltransferases exhibited a temperature-dependent substrate specificity in vivo. Growth temperature also affected the positional distribution of fatty acids incorporated into ciliary phospholipids in vivo. The ability of acyltransferases to utilize added [14C] acyl-CoA could be markedly stimulated, and their lipid class specificity could be significantly altered in vitro by supplementing the incubation mixture with exogenous lysophospholipid acceptors. These findings suggest that the rate-limiting factor in acyl chain turnover is not the activity of acyltransferases per se but rather the availability of suitable substrates and acceptors. Therefore, we postulate that temperature alters the rate and specificity of ciliary membrane phospholipid retailoring primarily by controlling the in situ phospholipase A activity.     

Temperature-sensitive mutants in rfaI and rfaJ, genes for galactosyltransferase I and glucosyltransferase II, for synthesis of lipopolysaccharide in Salmonella typhimurium

Certain rough mutants of Salmonella typhimurium LT2 were shown to be temperature sensitive for the production of lipopolysaccharide (LPS). When grown at the restrictive temperature (42 or 45 degrees C), the cells contained LPS deficient in O (somatic) side chains, based on phage-sensitivity data and gel electrophoresis of the LPS. Cells grown at the permissive temperature, 30 degrees C, made LPS resembling that of smooth cells. The mobility of the LPS in gels, the phage sensitivity patterns, and gas chromatographic analysis indicate that LPS of 45 degrees C-grown cells of SA126 (rfaJ3012) is of chemotype Rb2, with one glucose and two galactose units (and thus inferred to be due to a mutation in rfaJ), and LPS of 45 degrees C-grown cells of SA134 (rfa13020) is of chemotype Rb3, with one glucose and one galactose unit (inferred to be rfaI). These inferences were confirmed, for pKZ26 (pBR322-rfaGBIJ) and pKZ27 (pBR322-rfaGBI) both complement rfaI3020, but only pKZ26 complemented rfaJ3012. In addition, pKZ26 carrying a Tn5 insertion resulting in loss of complementation of a known rfaJ mutation, but not of rfaG, B, or I, also resulted in loss of rfaJ3012 complementation. Based on gel analysis, there is a small amount of the LPS containing smooth side chains in cells of SA126 grown at 45 degrees C; following a switch to 30 degrees C, the amount of LPS with O side chains gradually increased, and the amount of core LPS was reduced, though even after 3 h the LPS does not fully resemble that of smooth strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Translocation of nascent non-signal sequence protein in heated Escherichia coli

Exposure of Escherichia coli to heat resulted in 1) selective inhibition of protein synthesis, 2) synthesis of heat shock proteins, and 3) altered subcellular distribution of newly synthesized proteins. Either 5 min or 1 h at 48 degrees C increases outer membrane proteins of Coomassie Blue-stained gels. After 1 h, there was a loss of stained proteins from the soluble fraction. Much greater changes in the distribution of radiolabeled (newly synthesized) proteins were observed, with marked increases in the number of outer membrane protein species and a corresponding loss of soluble fraction proteins. Three major species of radiolabeled proteins from heat-treated cells remain in the soluble fraction; these proteins have apparent Mr 56,000, 69,200, and 79,400. Cells were labeled with L-[35S] methionine at either 37 or 48 degrees C and chased with non-radiolabeled methionine before a temperature shift to either 48 or 37 degrees C, respectively. Only proteins synthesized at elevated temperature participated in translocation. It is suggested that heat disordering of membrane lipids promotes interlipidic connections between the inner and outer membrane providing pathways for protein movement to the outer membrane and may be the mechanism whereby a cell quickly responds to environmental temperature stress. The response does not require but may trigger synthesis of mRNA.     

Effect of Temperature on the Growth and Cell Wall Chemistry of a Facultative Thermophilic Bacillus

The morphology and cell wall composition of Bacillus coagulans, a facultative thermophile, were examined as a function of growth temperature. The morphology of the organism varied when it was grown at different temperatures; at 37 C the organism grew as individual cells which increased in length with increasing growth temperature. At 55 C it grew in long chains of cells. Cell wall prepared from cells grown at 37 C contained 44% teichoic acid by weight, whereas cells grown at 55 C contained 29% teichoic acid. Teichoic acid from these cells was a polymer of glycerol phosphate containing galactose and ester alanine. The ratio of ester alanine to phosphate was significantly higher in cell walls and teichoic acid from 37 C-grown cells compared with those from 55 C-grown cells. Other differences observed were that cells grown at 55 C contained a lower level of autolytic ability, produced cell walls which bound more Mg(2+), and contained less peptide cross-bridging in its peptidoglycan layer than cells grown at 37 C.     

Growth at Low Temperature Mimics High-Light Acclimation in Chlorella vulgaris

Structural and functional alterations to the photosynthetic apparatus after growth at low temperature (5[deg]C) were investigated in the green alga Chlorella vulgaris Beijer. Cells grown at 5[deg]C had a 2-fold higher ratio of chlorophyll a/b, 5-fold lower chlorophyll content, and an increased xanthophyll content compared to cells grown at 27[deg]C even though growth irradiance was kept constant at 150 [mu]mol m-2 s-1. Concomitant with the increase in the chlorophyll a/b ratio was a lower abundance of light-harvesting polypeptides in 5[deg]C-grown cells as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by western blotting.The differences in pigment composition were found to be alleviated within 12 h of transferring 5[deg]C-grown cells to 27[deg]C. Furthermore, exposure of 5[deg]C-grown cells to a 30-fold lower growth irradiance (5 [mu]mol m-2 s-1) resulted in pigment content and composition similar to that in cells grown at 27[deg]C and 150 [mu]mol m-2 s-1. Although both cell types exhibited similar measuring-temperature effects on CO2-saturated O2 evolution, 5[deg]C-grown cells exhibited light-saturated rates of O2 evolution that were 2.8-and 3.9-fold higher than 27[deg]C-grown cells measured at 27[deg]C and 5[deg]C, respectively. Steady-state chlorophyll a fluorescence indicated that the yield of photosystem II electron transport of 5[deg]C-grown cells was less temperature sensitive than that of 27[deg]C-grown cells. This appears to be due to an increased capacity to keep the primary, stable quinone electron acceptor of photosystem II (QA) oxidized at low temperature in 5[deg]C- compared with 27[deg]C-grown cells regardless of irradiance. We conclude that Chlorella acclimated to low temperature adjusts its photosynthetic apparatus in response to the excitation pressure on photosystem II and not to the absolute external irradiance. We suggest that the redox state of QA may act as a signal for this photosynthetic acclimation to low temperature in Chlorella.     

High temperature induced antibiotic sensitivity changes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which was resistant to a wide variety of antibiotics, became sensitive to several of these antibiotics when grown and tested at 46 degrees C. Cell wall antibiotics such as penicillin G and ampicillin were only effective when added to cells growing at 46 degrees C prior to a temperature shift to 37 degrees C. Antibiotics which penetrate the cytoplasmic membrane to express their inhibiting action present a pattern different from those which are active against the outer cell wall. In order that these compounds be effective, the permeability of the cytoplasmic membrane must be further altered with agents such as EDTA which allow the penetration of actinomycin D. Inhibitors of protein synthesis, such as streptomycin and chloramphenicol, have increased access to their sites of action in cells grown at 46 degrees C. Cells grown at 46 degrees C have 40% less lipopolysaccharide (LPS) than cells grown at 37 degrees C and the LPS aggregates were of large molecular size in cells grown at 46 degrees C. Growth at 46 degrees C affects the permeability properties of the outer cell wall more than the permeability properties of the cytoplasmic membrane and this was due, in part, to the selective release of LPS of LPS-protein complexes at elevated growth temperatures.     

Biosynthesis and assembly of the polysialic acid capsule in Escherichia coli K1. Role of a low-density vesicle fraction in activation of the endogenous synthesis of sialyl polymers

Escherichia coli K1 synthesizes a polysialic acid capsule when grown at 37 but not 15 degrees C. The derangement in sialyl polymer synthesis appears to result from the inability of 15 degrees C membranes to synthesize or assemble a functional endogenous acceptor (Troy, F.A., and McCloskey, M.A. (1979) J. Biol. Chem. 254, 7377-7387). Membranes from cells grown at 15 degrees C spontaneously gained the ability to synthesize sialyl polymer after incubation at 33 degrees C for 2-4 h. The incubation-dependent activation of the endogenous synthesis of sialyl polymer in 15 degrees C membranes possessed two unusual features. First, the sialyltransferase was localized in a low density vesicle fraction (LDV; rho = 1.11 g/cm3). Second, this fraction catalyzed protein synthesis, and protein synthesis was required for activation. A study of the LDV fraction showed: 1) their light density resulted from a 5- to 8-fold enrichment in lipid phosphate to protein ratio and their sialyltransferase activity was enriched 40-fold compared with unfractionated total membranes; 2) they contained proteins characteristic of inner and outer membranes including leader peptidase and lipoprotein; 3) they constituted 8% of the mass of unfractionated total membranes yet contained all of the endogenous sialyltransferase activity in 15 degrees C membranes. In contrast, LDV from 37 degrees C grown cells accounted for 4.8% of the membrane mass and only 12.5% of the endogenous sialyltransferase activity; 4) they were multilamellar and averaged 0.7 mu in diameter. Based on these results, we believe the LDV fraction is of physiological importance in sialyl polymer synthesis. Growth at 15 degrees C allowed identification and study of the LDV fraction possibly because of the altered thermotropic properties of the membrane phospholipids that occur when E. coli is grown at low temperature.     

Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO.

Growth of Pseudomonas aeruginosa PAO1 at 15 to 45 degrees C in tryptic soy broth resulted in changes in the lipids, lipopolysaccharides (LPSs), and outer membrane proteins of the cells. Cells grown at 15 degrees C contained, relative to those cultivated at 45 degrees C, increased levels of the phospholipid fatty acids hexadecenoate and octadecenoate and reduced levels of the corresponding saturated fatty acids. Furthermore, the lipid A fatty acids also showed thermoadaptation with decreases in dodecanoic and hexadecanoic acids and increases in the level of 3-hydroxydecanoate and 2-hydroxdodecanoate as the growth temperature decreased. In addition, LPS extracted from cells cultivated at the lower temperatures contained a higher content of long-chain S-form molecules than that isolated from cells grown at higher temperatures. On the other hand, the percentage of LPS cores substituted with side-chain material decreased from 37.6 mol% at 45 degrees C to 19.3 mol% at 15 degrees C. The outer membrane protein profiles indicated that at low growth temperatures there was an increase in a polypeptide with an apparent molecular weight of 43,000 and decreases in the content of 21,000 (protein H1)- and 27,500-molecular-weight proteins.