Halorubrum salinum sp. nov., isolated from a marine solar saltern

The halophilic archaeal strain GX71^T was isolated from the Gangxi marine solar saltern near the Weihai city of Shandong Province, China. Cells of the strain were pleomorphic and lysed in distilled water, stained Gram-negative and formed red-pigmented colonies. Strain GX71^T was able to grow at 25–45 °C (optimum 30 °C), in the presence of 1.7–4.8 M NaCl (optimum 2.6 M NaCl), with 0.005–0.7 M MgCl₂ (optimum 0.05 M MgCl₂) and at pH 5.5–9.5 (optimum pH 7.0–7.5). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 10 % (w/v). The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-3) and an unidentified lipid was also detected. The 16S rRNA gene sequence of strain GX71^T showed 94.0–97.0 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB′ gene sequence of strain GX71^T was 87.3–93.4 % similarity to current members of the genus Halorubrum. The DNA G+C content of GX71^T was 67.1 mol%. Strain GX71^T showed low DNA–DNA relatedness with Halorubrum lipolyticum CGMCC 1.5332^T, Halorubrum saccharovorum CGMCC 1.2147^T, Halorubrum kocurii CGMCC 1.7018^T and Halorubrum arcis CGMCC 1.5343^T, the most closely related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX71^T represents a novel species of the genus Halorubrum, for which the name Halorubrum salinum sp. nov. is proposed. The type strain is GX71^T (= CGMCC 1.10458^T = JCM 17093^T).     

Haloplanus salinus sp. nov., an extremely halophilic archaeon from a Chinese marine solar saltern

Halophilic archaeal strain YGH66^T was isolated from the Yinggehai marine solar saltern near the Sanya city of Hainan Province, China. Cells were pleomorphic, flat, stained Gram-negative, and produced pink-pigmented colonies. Strain YGH66^T was able to grow at 20–50 °C (optimum 37 °C), at 0.9–4.8 M NaCl (optimum 3.1 M NaCl), at 0.005–1.0 M MgCl₂ (optimum 0.05 M MgCl₂), and at pH 6.0–8.0 (optimum pH 7.0). The cells of strain YGH66^T were lysed in distilled water, and the minimum NaCl concentration that prevented cell lysis was 5 % (w/v). The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid (GL1) chromatographically identical to sulfated mannosyl glucosyl diether and a minor unidentified lipid (GL2), respectively. On the basis of 16S rRNA gene sequence analysis, strain YGH66^T was closely related to Haloplanus natans JCM 14081^T, Haloplanus aerogenes TBN37^T, and Haloplanus vescus RO5-8^T with the similarities of 98.0, 97.6, and 96.9 %, respectively. The rpoB′ gene similarity between strain YGH66^T and the current three members of Haloplanus were 90.4–92.8 %. The DNA G+C content of strain YGH66^T was 67.2 mol %. The DNA–DNA hybridization values between strain YGH66^T and three members of Haloplanus, H. natans JCM 14081^T, H. aerogenes TBN37^T, H. vescus RO5-8^T were 50, 46 and 39 %, respectively. It was concluded that strain YGH66^T represents a novel species of the genus Haloplanus, for which the name Haloplanus salinus sp. nov. is proposed. The type strain is YGH66^T (=CGMCC 1.12127^T = JCM 18368^T).     

Halorubrum rubrum sp. nov., an extremely halophilic archaeon from a Chinese salt lake

Two halophilic archaeal strains, YC87^T and YCA11, were isolated from Yuncheng salt lake in Shanxi, China. Cells of the two strains were observed to be pleomorphic rod-shaped, stained Gram-negative and produced red-pigmented colonies. Strain YC87^T was able to grow at 20–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.1 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.3 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.0) while strain YCA11 was able to grow at 20–50 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0.01–0.7 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.5). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 8 % (w/v) for strain YC87^T and 12 % (w/v) for strain YCA11. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether; another major glycolipid and trace amounts of several unidentified lipids were also detected. The 16S rRNA gene sequences of the two strains were 99.8 % identical, showing 93.2–98.2 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB′ gene similarity between strains YC87^T and YCA11 was 99.3 % and showed 87.5–95.2 % similarity to the closest relative members of the genus Halorubrum. The DNA G+C content of strains YC87^T and YCA11 were determined to be 64.9 and 64.5 mol%, respectively. The DNA–DNA hybridization value between strain YC20^T and strain YC77 was 87 % and the two strains showed low DNA–DNA relatedness with Halorubrum cibi JCM 15757^T and Halorubrum aquaticum CGMCC 1.6377^T, the most related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains YC87^T and YCA11 represent a novel species of the genus Halorubrum, for which the name Halorubrum rubrum sp. nov. is proposed. The type strain is YC87^T (=CGMCC 1.12124^T = JCM 18365^T).     

Structural analysis by mass spectrometry and NMR spectroscopy of the glycolipid sulfate from Halobacterium salinarium and a note on its possible function

The major glycolipid sulfate of the extreme halophile Halobacterium salinarium was isolated and characterised mainly by mass spectrometry and NMR spectroscopy. The mass spectrum of the permethylated, desulfated and trimethylsilylated derivative showed the molecule to be a trihexosyl glycerol C₂₀-diether with the sulfate group on the terminal hexose. A 3-position of the sulfate was indicated by the mass spectrum obtained after acetylation and trimethylsilylation (solvolysis of sulfate and replacement by a trimethylsilyl group). The NMR spectrum of the desulfated permethylated glycolipid gave conclusive evidence for the presence of one β and two α anomeric protons. With the knowledge of degradation data it was possible to assign the β signal to galactose (terminal hexone), and the α signals to glucose and mannose. These data together make it likely that the glycolipid sulfate is identical in structure with the glycolipid from Halobacterium cutirubrum characterised previously (M. Kates and P.W. Deroo, J. Lipid Res., 14 (1973) 438).On the basis of a suggested function of cerebroside sulfate of animal origin (identical polar end with the bacterial glycolipid: β-galactopyranose-3-sulfate) and the present knowledge of ion transport in Halobacteria, it is proposed that the bacterial glycolipid may function as a selective K⁺ receptor for the K⁺ transport from a high-Na⁺ and low-K⁺ outside medium.     

Electron Spin Resonance Studies of Ionic Permeability Properties of Thylakoid Membranes of Beta vulgaris and Avicennia germinans

Measurement of intrathylakoid aqueous volumes by electron spin resonance spectroscopy was used to study ionic permeability properties of thylakoid membranes isolated from Beta vulgaris L. and Avicennia germinans L. The thylakoids behaved as perfect osmometers in the presence of sorbitol and betaine. Thylakoids exposed to hypertonic solutions of NaCl and KCl shrank and subsequently swelled, requiring 10 minutes to regain their original volume. The initial influx rate calculated from the kinetics of changes in intrathylakoid volume in response to 450 millimolar gradients of NaCl and KCl was 2.3 × 10⁻¹³ moles per square centimeter per second. These data show that the passive permeability to NaCl and KCl was low.     

Bleaching herbicide effects on plastids of dark-grown plants: lipid composition of etioplasts in amitrole and norflurazon-treated barley leaves

The effects of the bleaching herbicides amitrole (125 micro M) and norflurazon (100 micro M) on etioplast lipids were studied in barley plants (Hordeum vulgare L. cv. Express) grown for 7 d either at 20 degrees C or 30 degrees C in darkness. Total lipid, glycolipid and phospholipid contents of control etioplasts were increased at 30 degrees C in comparison with those at 20 degrees C. The two herbicides caused a decrease in the total lipid, glycolipid and phospholipid amounts compared to the untreated etioplasts and lowered the lipid to protein ratio. In the controls, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) accounted for about 66 mol% of the etioplast polar lipids, while the remainder was represented by sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), in approximately equal proportions. Both amitrole and norflurazon increased MGDG at both temperatures, but decreased DGDG except with norflurazon at 30 degrees C. As a consequence, the MGDG to DGDG molar ratio was higher in the herbicide-treated etioplasts compared to the controls at both the growth temperatures. The amount of the negatively charged polar lipids SQDG and PG were decreased by treatments with amitrole at 20 degrees C and norflurazon at 30 degrees C. The two herbicides determined different responses in the fatty acid unsaturation of the individual polar lipids. Changes in the lipid composition of etioplasts and the interaction between the pigment-protein complex, protochlorophyllide-NADPH-protochlorophyllide oxidoreductase, and polar lipids are discussed.     

The effect of the polar moiety of lipids on the ion permeability of bilayer membranes

Summary Bilayer membranes were prepared with the negatively charged lipids phosphatidylglycerol and diphosphatidylglycerol, the positively charged lipid lysyl phosphatidylglycerol, the zwitterionic lipid phosphatidylethanolamine, and an uncharged glycolipid, diglucosyldiglyceride, all isolated from gram-positive bacteria. Bilayer membranes of all these lipids manifested specific resistances of 10⁷ to 10⁹ cm² and capacitances of 0.3 to 0.4 F cm^–2. The membrane potentials of these bilayers were measured as a function of the sodium chloride, potassium chloride, and hydrogen chloride transmembrane concentration gradients (0.01 to 0.10m) and were found to be linear with the logarithm of the salt activity gradients. Membranes made from lysyl phosphatidylglycerol (one net positive charge) were almost completely chloride selective, whereas membranes from phosphatidylglycerol and diphosphatidylglycerol (one and two net negative charges, respectively) were highly cation selective. Membranes prepared with either diglucosyldiglyceride or phosphatidylethanolamine showed only slight cation selectivity. These findings indicate that the charge on the polar head group of membrane lipids plays an important role in controlling the ion-selective permeability of the bilayer.     

Different stratum corneum lipid liposomes as models to evaluate the effect of the sodium dodecyl sulfate

The stability of stratum corneum (SC) liposomes against the action of surfactants has been revised. To this end, two types of vesicles were used; vesicles formed with the lipid and protein material extracted from SC, and lipid mixtures approximating the SC composition. In this case, the proportion of ceramides (Cer) and cholesteryl sulfate (Chol-sulf) was varied and the relative proportion of the other lipids remained constant. The increasing presence of these two lipids increased the resistance of liposomes against the action of the anionic surfactant sodium dodecyl sulfate (SDS). The rise in the cell-to-cell cohesion that occurred in recessive X-linked ichthyosis due to the accumulation of Chol-sulf could be associated in part to the enhanced stability of (Chol-sulf)-enriched bilayers. It is noteworthy that the surfactant partitioning between bilayers and the aqueous phase increased and decreased, respectively, as the proportion of Cer and Chol-sulf increased. This effect may be attributed to the variations in both the electrostatic interactions lipid-surfactant (electrostatic repulsion between the sulfate groups of both Chol-sulf and SDS), and the hydrophilic lipophilic balance of the lipid mixtures, in which Cer is replaced by the major polar lipid of the mixture (Chol-sulf). The fact that the free surfactant concentration was always smaller than its critical micelle concentration indicates that the permeability alterations were mainly ruled by the action of surfactant monomers, in agreement with the results reported for sublytic interactions of this surfactant with PC liposomes.     

Uniform preparations of large unilamellar vesicles containing anionic lipids

A general procedure for the preparation of large unilamellar vesicles of selected sizes has been developed. The procedure consists of dissolving the lipid in organic solvent, washing with mild acid, removing the solvent, adding salt (0.15 M KCl) solution, and adjusting the pH (raising it to about pH 10 and lowering it immediately to pH 7.55). The procedure takes less than 30 min. The resulting unilamellar vesicles are of a single size with a rather low standard deviation. The sizes of these preparations range between 150 and 1000 nm in diameter. Sizes and polydispersities were measured to within 1-2% by photon correlation spectroscopy. Vesicle size varies with the phospholipid structure, the composition of the phospholipid mixture, the ionic strength of the medium, the alkyl chain composition, the cholesterol content of the phospholipid mixture, and the timing of the pH adjustment procedure. Uniform preparations of vesicles have been obtained from the dioleoyl esters of phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine, from diphytanyl ethers of glycolipid sulfate, phosphatidylglycerol, phosphatidylglycerol phosphate, and phosphatidylglycerol sulfate, from bovine liver phosphatidylinositol, from Escherichia coli phosphatidylethanolamine, from membrane lipid extracts from E. coli and Holabacterium cutirubrum, and from dodecanesulfonate-alkanol mixtures and free oleic acid. The preparation of unilamellar vesicles from oleic acid is novel, and the size range is 600-3000 nm; the preparations are relatively uniform. Vesicles of phospholipids in which sucrose and trehalose replace salt as the impermeant do not differ significantly from those prepared in pentaerythritol.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

Lipid mixing during freeze-thawing of liposomal membranes as monitored by fluorescence energy transfer

A new pair of fluorescence-energy-transferring probes, dansylphosphatidylethanolamine and dioctadecylindocarbocyanine, were incorporated separately into phospholipid vesicles to monitor intervesicle lipid mixing under various conditions. The transfer efficiencies of mixtures of sonicated vesicles labeled with 2 wt% donor dansylphosphatidylethanolamine (DnsPE) or with 1 wt% acceptor dioctadecylindocarbocyanine (DiI-C18) were negligible, but increased to about 25% after the vesicles had been frozen in a solid CO2/ethanol bath, thawed and diluted. The freeze-thaw-induced mixing of lipids between vesicles, signified by energy transfer, was dependent on lipid concentration and was promoted by 0.5-1.5 M KCl, 0.5 M potassium trichloroacetate and 5 mM sodium acetate (pH 4) and inhibited by 0.5 M LiCl, 0.5 M glycerol, 0.5 M sucrose, 0.15 M KCl and 0.15-1.5 M NaCl. These results support and complement previously reported measurements of the trapped volumes, turbidities and population size distributions of similarly treated liposomes. Comparison of the responses of paucilamellar vesicles with those of multilamellar vesicles suggests that lipid mixing during freeze-thawing can occur either during interaction of the outermost bilayers of vesicles or during interaction of all bilayers, possibly as a result of breakdown and reformation of bilayer structure.     

Structural studies on lamellated osmiophilic bodies isolated from pig lung. 31P NMR results and water content

1. Lamellated osmiophilic bodies are intracellular organelles in which pulmonary surfactant is stored prior to secretion. They contain about 85% phospholipid (per dry weight) and dipalmitoyl phosphatidylcholine is a major constituent, and although their ultrastructure is uncertain it is generally supposed that they resemble liposomes. However, liposomes are stable because layers of water are interposed between the lipid bilayers whereas an essential aspect of the function of lamellated bodies is that, subsequent to their secretion, they are rapidly disrupted to form a surface-active film which covers the respiratory epithelium of the lung. 2. A new method for isolating lamellated bodies from pig lung is described which has the advantage of speed and simplicity and which results in increased yields. The homogeneity of the preparation is similar to that obtained by other methods. 3. 31P NMR spectra of lamellated bodies showed that at 40 degrees C about 95% of the phospholipid was present as extended bilayers and that about 5% was present in a phase exhibiting isotropic head group mobility (tau R less than 10(-5) s). It is suggested that this phase may be due to apolar proteins which are present both in lamellated bodies and in liposomes prepared from lipids extracted from them. 4. The internal water content of lamellated bodies has been measured gravimetrically and the hydration of the phospholipid head groups has been examined by 31P NMR. The two methods gave results in good agreement and show that there are about seven molecules of water/molecule of phospholipid. It is concluded that although the phospholipid head groups in lamellated bodies are fully hydrated, there is no zone of free water. 5. Lamellated bodies are osmotically insensitive to NaCl whereas liposomes prepared from lipids extracted from them behave like perfect osmometers. It is suggested that the osmotic insensitivity and restricted water content of lamellated bodies are important to their function and dependent upon polar proteins in the outer limiting membrane.     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

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.     

Relation of the birefringence of a bilayer lipid membrane to the ionic strength of the medium]

A direct method of measuring the optical phase difference arising in a bilayer lipid membrane at the expence of its birefringent properties is offered. A dependence of the value and sign of lipid bilayer birefringence on ionic concentration in the medium has been observed. For example, the birefringence value of the bilayer from egg lecithin with eta-decan changes from -0.005 to 0.01 with a change of NaCl concentration from 0 to 5 M, the sign changing at about 1 M. It is suggested that the polar region of the bilayer has negative birefringence, and that it is its infrastructure that changes with the ionic strength of the solution.     

Clustering of α-Synuclein on Supported Lipid Bilayers: Role of Anionic Lipid, Protein, and Divalent Ion Concentration

α-Synuclein is the major component of Lewy body inclusions found in the brains of patients with Parkinson's disease. Several studies indicate that α-synuclein binds to negatively charged phospholipid bilayers. We examined the binding of α-synuclein to membranes containing different amounts of negatively charged lipids using supported lipid bilayers, epifluorescence microscopy, fluorescence recovery after photobleaching, and bulk fluorescence techniques. The membranes contained phosphatidylcholine and phosphatidylglycerol. In the absence of protein, these lipids mix uniformly. Our results show that the propensity of α-synuclein to cluster on the membrane increases as the concentration of anionic lipid and/or protein increases. Regions on the lipid bilayer where α-synuclein is clustered are enriched in phosphatidylglycerol. We also observe divalent metal ions stimulate protein cluster formation, primarily by promoting lipid demixing. The importance of protein structure, lipid demixing, and divalent ions, as well as the physiological implications, will be discussed. Because membrane-bound α-synuclein assemblies may play a role in neurotoxicity, it is of interest to determine how membranes can be used to tune the propensity of α-synuclein to aggregate.     

Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov., from a marine solar saltern and an aquaculture farm,respectively

Two halophilic archaea, strains GX21^T and R35^T, were isolated from a marine solar saltern and an aquaculture farm in China, respectively. Cells of the two strains were observed to be pleomorphic, flat, to contain gas vesicles, stain Gram-negative and produce red-pigmented colonies. Strain GX21^T was found to be able to grow at 25–50 °C (optimum 37 °C), at 2.6–4.8 M NaCl (optimum 3.4 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–8.5 (optimum pH 6.5) while strain R35^T was found to be able to grow at 25–45 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0–0.7 M MgCl₂ (optimum 0.03 M MgCl₂) and at pH 5.5–9.5 (optimum pH 6.5–7.0). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 15 % (w/v) for strain GX21^T and 12 % (w/v) for strain R35^T. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid and a minor lipid chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. 16S rRNA gene sequence analysis revealed that strains GX21^T and R35^T show 97.1 % sequence similarity to each other and are closely related to Haloplanus aerogenes TBN37^T (96.8 and 95.8 %), Haloplanus vescus RO5-8^T (96.7 and 96.1 %), Haloplanus salinus YGH66^T (96.4 and 95.8 %) and Haloplanus natans JCM 14081^T (96.3 and 95.4 %). The rpoB′ gene similarity between strains GX21^T and R35^T is 90.5 % and show 88.5–90.8 % similarity to the Haloplanus species with validly published names. The DNA G+C content of strain GX21^T and R35^T were determined to be 65.8 and 66.0 mol%, respectively. The DNA–DNA hybridization values between strain GX21^T and strain R35^T, and the two strains with the Haloplanus species with validly published names, showed less than 50 % DNA–DNA relatedness. It was concluded that strain GX21^T (=CGMCC 1.10456^T = JCM 17092^T) and strain R35^T (=CGMCC 1.10594 ^T = JCM 17271^T) represent two new species of Haloplanus, for which the names Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov. are proposed.     

Phase structure of liposome in lipid mixtures

Gas microbubbles present in ultrasound imaging contrast agents are stabilized by lipid aggregates that typically contain a mixture of lipids. In this study, the phase structure of the lipid mixtures that contained two or three lipids was investigated using three different methods: dynamic light scattering, ¹H NMR, and microfluidity measurements with fluorescence probes. Three lipids that are commonly present in imaging agents (DPPC, DPPE-PEG, and DPPA) were used. Two types of systems, two-lipid model systems and simulated imaging systems were investigated. The results show that liposomes were the dominant aggregates in all the samples studied. The polar PEG side chains from the PEGylated lipid lead to the formation of micelles and micellar aggregates in small sizes. In the ternary lipid systems, almost all the lipids were present in bilayers with micelles absent and free lipids at very low concentration. These results suggest that liposomes, not micelles, contribute to the stabilization of microbubbles in an ultrasound imaging contrast agent.     

Growth inhibition of Halobacterium cutirubrum by cerulenin,a potent inhibitor of fatty acid synthesis

The polar lipids of $\text{Halobacterium cutirubrum}$ are known to consist exclusively of diether derivatives of glycerol, and do not contain fatty acids. However, cerulenin, a specific and potent inhibitor of fatty acid synthesis, was shown to inhibit the growth of this organism. Protection from growth inhibition was demonstrated when fatty acids of 18 carbons were added to the growth medium, but not when palmitic or palmitoleic acids were used. Cerulenin appears to affect synthesis of all polar lipids in this organism while relative levels of protein and nucleic acids were not significantly affected. Growth inhibition by cerulenin supports the conclusion that the fatty acid synthetase system present in $\text{H. cutirubrum}$ is necessary for lipid biosynthesis, despite the fact that fatty acids are not structural components of the lipids of this bacterium. A pathway is proposed to account for these observations.     

Lipid metabolism in cultured lichen photobionts with different phosphorus status

Lipid metabolism was studied in different photobiont species from lichens by following incorporation of radiolabel from [1-14C]acetate. In four algal photobionts, Coccomyxa mucigena, C. peltigera variolosae, Trebouxia aggregata, T. erici, polar lipids were mainly (73-90%) labelled while triacylglycerols were the most highly labelled non-polar lipid class. A rhamnose-containing lipid was found in two Coccomyxa species, representing about 11% of the polar lipids of C. mucigena. All the major algal glycosyl- and phospho-glycerides were labelled with monogalactosyldiacylglycerol and phosphatidylglycerol, respectively, being the main labelled lipids in the polar lipid classes. The photobionts were grown in media differing in their phosphate content by one hundred-fold. Low phosphate levels caused only a small decrease in the proportion of phosphoglyceride labelling--mainly in phosphatidylglycerol. However, total lipid labelling was reduced (by 83.3 and 76.6% in two Coccomyxa spp. and 62.1 and 27% in two Trebouxia spp.) for the green algae. By comparison, variations in phosphate availability had no significant effect on a Nostoc sp. Examination of the algal species by electron microscopy revealed phosphorus-containing granules. This reserve of phosphorus explains why the algal photobionts were able to maintain the proportion of phosphoglyceride labelling well and may be an important adaptive mechanism for lichens.