Cellulose-inducible Ultrastructural Protuberances and Cellulose-affinity Proteins of Eubacterium cellulosolvens

Scanning electron microscopy detected ultrastructural protuberances on the cellulolytic anaerobeEubacterium cellulosolvens . Such cell surface structures were found only when cells were cultivated in cellulose containing medium, suggesting these structures play a role in cellulose degradation. Organisms cultivated in medium containing cellobiose, glucose, fructose, maltose, or carboxymethylcellulose (CMC) contained few, if any, of these protuberances. Also, when a soluble carbohydrate or CMC was added to cellulose-grown cells, the ultrastructural protuberances were no longer detected. In fact, a time course study revealed that the loss of these protuberant structures occurred within 5 min of the addition of glucose, cellobiose, fructose, or a glucose analog to the medium. On the other hand, formation of these protuberances required at least 2 h, and 4 h before large numbers were present on the cells. Cellulose-grown cells also bound the FITC-labeled lectin BSI-B₄, obtained from Bandeiraea (formerly Griffonia) simplicifolia. Less detectable levels of lectin were bound by cellobiose-grown cells, and glucose- and fructose-grown cells did not bind any detectable levels of the lectin. Moreover, the addition of glucose or 2-deoxyglucose to the medium of a cellulose-grown culture resulted in the loss of detectable lectin binding. A cellulose-affinity protein fraction, which contained cellulase activity, was also isolated from the cellular extracts of cellobiose- and cellulose-grown cultures of E. cellulosolvens. This affinity fraction could not be eluted from the cellulose column with either sodium dodecyl sulfate (SDS), urea, or a 2-M solution of NaCl, but was eluted by Tris buffer containing ethylenediaminetetraacetic acid (EDTA). The fraction possessed cellulase activity, and consisted of numerous polypeptides. However, this protein fraction could not be detected in the extract of glucose-grown cultures, or in the extract of cellulose-grown cultures within 5 min of the addition of glucose (or a glucose analog) to the medium. The immediate loss of the cellulose-affinity protein fraction and protuberant structures when a soluble carbohydrate was added to the medium indicated some, as yet unknown, regulatory mechanism.     

Symbiotic Relationship of Bacteroides cellulosolvens and Clostridium saccharolyticum in Cellulose Fermentation

In coculture, Bacteroides cellulosolvens and Clostridium saccharolyticum fermented 33% more cellulose than did B. cellulosolvens alone. Also, cellulose digestion continued at a maximum rate 48 h longer in coculture. B. cellulosolvens hydrolyzes cellulose and supplies C. saccharolyticum with sugars and a growth factor replaceable by yeast extract. Alone, B. cellulosolvens exhibited an early cessation of growth which was not due to nutrient depletion, low pH, or toxic accumulation of acetic acid, ethanol, lactic acid, H₂, CO₂, cellobiose, glucose, or xylose. However, a 1-h incubation of B. cellulosolvens spent-culture medium with C. saacharolyticum cells starved for growth factor allowed a resumption of B. cellulosolvens growth. The symbiotic relationship of this naturally occurring coculture is one of mutualism, in which the cellulolytic microbe supplies the saccharolytic microbe with nutrients, and in turn the saccharolytic microbe removes a secondary metabolite toxic to the primary microbe.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 °C) and low (20 °C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T_m (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 °C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T_m was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 °C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T_m by 10.5 °C. In mineral media at 20 °C the corresponding changes of T_m were almost negligible. After a temperature shift from 40 to 20 °C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

Studies on Tetrahymena membranes:: Temperature-induced alterations in fatty acid composition of various membrane fractions in Tetrahymena pyriformis and its effect on membrane fluidity as inferred by spin-label study

The fatty acid distribution pattern of lipids extracted from different subcellular components of Tetrahymena pyriformis was found to be significantly different from one type of membrane to another.The growth-temperature shift caused alterations in fatty acid composition. The ratio of palmitoleic to palmitic acid, especially, showed a sharp linear decline with increase of temperature in all of the membrane fractions.The spin labels were rapidly incorporated into Tetrahymena membranes. The order parameter of 5-nitroxide stearate spin label incorporated into various membrane fractions was found to be different for the different membrane fractions, suggesting the following order of the fluidity; microsomes > pellicles > cilia.The fluidity of the surface membranes, cilia and pellicles isolated from Tetrahymena cells grown at 15°C was noticeably higher than that of the membranes from cells grown at 34°C but was not so different with microsomal fractions.The motion of the spin label in the pellicular membrane was more restricted than in its extracted lipids, thus indicating the assumption that in Tetrahymena membranes the proteins influence the fluidity.It was also suggested that a sterol-like triterpenoid compound, tetrahymanol, which is principally localized in the surface membranes, would be involved in the membrane fluidity.     

Lipid and Fatty Acid composition of chloroplast envelope membranes from species with differing net photosynthesis

Lipid and fatty acid compositions were determined for chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) leaves. The lipid composition was similar in sunflower, spinach, and undifferentiated maize chloroplast envelope membranes and different in maize mesophyll chloroplast envelope membranes. The predominant lipid constituents in all envelope membranes were monogalactosyldiglyceride (27 to 46%), digalactosyldiglyceride (18 to 33%), and phosphatidylcholine (7 to 30%). The fatty acid composition was also similar in sunflower and spinach chloroplast envelope membranes in comparison to those from maize. The major acyl fatty acids of the chloroplast envelope membrane were palmitic (C_16:0, 41 and 36%) and linolenic (C_18:3, 29 and 40%) acids for spinach and sunflower; palmitic (77%) and stearic (C_18:0, 12%) acids for young maize; and palmitic (61%), stearic (14%), and linolenic (13%) acids for mature maize. The differences in lipid and acyl fatty acid compositions among these plants which vary in their rates of net photosynthesis were largely quantitative rather than qualitative.     

Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius

Cytochrome P450 (CYP) 147F1 from Streptomyces peucetius is a new CYP subfamily of that has been identified as ω-fatty acid hydroxylase. We describe the identification of CYP147F1 as a fatty acid hydroxylase by screening for the substrate using a substrate binding assay. Screening of substrates resulted in the identification of fatty acid groups of compounds as potential hits for CYP147F1 substrates. Fatty acids from C_10:0 to C_18:0 all showed type I shift spectra indicating their potential as substrates. Among several fatty acids tested, lauric acid, myrsitic acid, and palmitic acid were used to characterize CYP147F1. CYP147F1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida as surrogate electron transfer partners. Kinetic parameters, including the dissociation constant, K_m, NADH consumption assay, production formation rate, and coupling efficiency for CYP147F1 were also determined.     

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”.     

Fatty acid composition of lipids from differentiated tissues and cell cultures of Euonymus europaeus

The fatty acid patterns of Euonymus europaeus callus cultures and cell suspension cultures were analysed at the beginning of stationary growth phase and compared with those from the respective differentiated tissues. The lipid and fatty acid patterns in cell cultures differed remarkably from those in the tissues of the mother plant. No glycerol triacetate was detected in the callus cultures derived from differentiated tissues whereas in seeds this lipid compound amounts to 29%. In addition to fatty acids normally occurring in differentiated tissues, lipids in cultured cells also contained short-chain (C₁₂–C₁₄) as well as very long-chain fatty acids (C₂₀–C₂₄). In tissue culture cells the major fatty acids were found to be saturated, whereas in the mother cells unsaturated fatty acids were predominant. Palmitic acid is the most abundant fatty acid in most of the cultures. Lauric, myristic and palmitic acid amount to 50% in lipids of cell suspension cultures.     

Adaptation of Saccharomyces cerevisiae cells to high ethanol concentration and changes in fatty acid composition of membrane and cell size

Background

Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium.

Methodology/Principal Findings

First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the cell membrane showed that the contents in oleic acid (C_18:1) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C_16:0) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strain.

Conclusions

Our results suggest that activity of cell growth defined by specific growth rate of the yeast cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of cell membrane and the size of ethanol-adapted yeast cells were changed during adaptation process. Those might be the typical phenotypes of yeast cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother cell between the non-adapted and ethanol strains suggests that the cell size, cell cycle and adaptation to ethanol are thought to be closely correlated.     

Phenotypic changes in the fluidity of the tonoplast membrane of crassulacean-acid-metabolism plants in response to temperature and salinity stress

Electron paramagnetic resonance-spectroscopic studies on spin-labeled purified tonoplast membranes showed that in the obligate crassulacean-acid-metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. the fluidity of the tonoplast decreased during acclimation to higher temperatures. This phenotypic change in tonoplast fluidity was paralleled by a decrease in the mobilization of malic acid from the vacuoles during CAM in the light. The shift from the C₃ to the CAM mode of photosynthesis in the facultative CAM plant Mesembryanthemum crystallinum L. also led to a decrease in the fluidity of the tonoplast membrane. The results are consistent with the hypothesis that the ability to store malic acid during CAM in the vacuoles depends largely on the actual fluidity of the tonoplast membrane.     

Changes in Cellular Fatty Acid Composition of Cephalosporium acremonium during Cephalosporin C Production

Cephalosporium acremonium was cultivated in fermentation medium containing sucrose or methyl oleate as a carbon source for cephalosporin C production. The level of antibiotic production was 48 g of cephalosporin C per liter under optimum conditions when methyl oleate was used. The C_18:1 (oleic acid) methyl ester appeared to be utilized faster than the C_18:2 (linoleic acid) methyl ester in fermentation broth. Physiological characteristics of C. acremonium were investigated by determining the fatty acid composition of the total cellular free lipid. Significant changes in cellular fatty acid composition occurred during inoculum cultivation and fermentation. The percentage of C_18:1 increased from 19.1 to 38.5%, but the percentage of C_18:2 decreased from 56.7 to 36.1%, and there was an increase in pH during inoculum cultivation. The cellular fatty acid composition of C. acremonium grown in fermentation medium containing methyl oleate (methyl oleate medium) was significantly different from that in fermentation medium containing sucrose (sucrose medium). The major fatty acids detected were C_16:0 (palmitic acid), C_18:1, and C_18:2. In methyl oleate medium, the ratio of C_18:1 to C_18:2 increased from 0.34 to 1.37, while the cell morphology changed from hyphae to arthrospores and conidia. In contrast, in sucrose medium, the ratio of C_18:1 to C_18:2 decreased from 0.70 to 0.43, and most of the cells remained hyphal at the end of fermentation. We observed that hyphae contained a higher proportion of C_18:2 but arthrospores and conidia contained a higher proportion of C_18:1.     

Ethanol Tolerance in the Yeast Saccharomyces cerevisiae Is Dependent on Cellular Oleic Acid Content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ⁹Z-C_16:1) and oleic acid (Δ⁹Z-C_18:1), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C_16:0) and stearic acid (C_18:0), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ⁹ and Δ¹¹) and substrate chain-length preferences (i.e., C_16:0 and C_18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Δ¹¹Z-C_18:1), whereas neither Δ¹¹Z-C_16:1 nor palmitoleic acid (Δ⁹Z-C_16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

Adaptation of Escherichia coli O157:H7 to pH Alters Membrane Lipid Composition, Verotoxin Secretion, and Resistance to Simulated Gastric Fluid Acid

The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1ω7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10⁸ CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.     

Cell Envelope Phospholipid Composition of Burkholderia multivorans

Burkholderia multivorans causes opportunistic pulmonary infections in cystic fibrosis and immunocompromised patients. The purpose of the present study was to determine the nature of the phospholipids and their fatty acid constituents comprising the cell envelope membranes of strains isolated from three disparate sources. A conventional method for obtaining the readily extractable lipids fraction from bacteria was employed to obtain membrane lipids for thin-layer chromatographic and gas chromatography-mass spectrophotometric analyses. Major fatty acid components of the B. multivorans readily extractable lipid fractions included C_16:0 (palmitic acid), C_16:1 (palmitoleic acid), and C_18:1 (oleic acid), while C_14:0 (myristic acid), ΔC_17:0 (methylene hexadecanoic acid), C_18:0 (stearic acid), and ΔC_19:0 (methylene octadecanoic acid) were present in lesser amounts. Fatty acid composition differed quantitatively among strains with regard to C_16:0, C_16:1, ΔC_17:0, C_18:1, and ΔC_19:0 with the unsaturated:saturated fatty acid ratios being significantly less in a cystic fibrosis type strain than either environmental or chronic granulomatous disease strains. Phospholipids identified in all B. multivorans strains included lyso-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol in similar ratios. These data support the conclusion that the cell envelope phospholipid profiles of disparate B. multivorans strains are similar, while their respective fatty acyl substituent profiles differ quantitatively under identical cultivation conditions.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Membrane lipid composition, fluidity, and surface charge changes in response to growth of the fresh water cyanobacterium Synechococcus 6311 under high salinity

The effect of adaptation to saline growth of a fresh water cyanobacterium Synechococcus 6311 on components of the cytoplasmic membranes and thylakoids was investigated. Significant changes in membrane surface charge, lipid, fatty acid, and carotenoid composition were observed upon transfer of the cells from a low salt (0.015 M NaCl) to a high salt (0.50 M NaCl) growth medium. Very similar changes in the polar lipid classes and fatty acid composition were observed in both membranes, but changes in fluidity and surface charge and a significant shift in the protein to lipid ratio were only apparent in the cytoplasmic membranes. The fluidity and surface charge data correlate well with functional studies and we can attribute the cytoplasmic membrane as the major site of interaction and adaptation to the saline environment.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.     

A small molecule screen for paqr-2 suppressors identifies Tyloxapol as a membrane fluidizer for C. elegans and mammalian cells

Defects in cell membrane homeostasis are implicated in numerous disorders, including cancer, neurodegeneration and diabetes. There is therefore a need for a powerful model to study membrane homeostasis and to identify eventual therapeutic routes. The C. elegans gene paqr-2 encodes a homolog of the mammalian AdipoR1 and AdipoR2 proteins that, when mutated, causes a membrane homeostasis defect accompanied by multiple phenotypes such as intolerance to dietary saturated fatty acids, intolerance to cold and a characteristic tail tip morphology defect. We screened a compound library to identify molecules that can suppress the paqr-2 phenotypes. A single positive hit, Tyloxapol, was found that very effectively suppresses multiple paqr-2 phenotypes. Tyloxapol is a non-ionic detergent currently in use clinically as an expectorant. Importantly, we examined the potential of Tyloxapol as a fluidizer in human cells and found that it improves the viability and membrane fluidity of AdipoR2-deficient human cells challenged with palmitic acid, a membrane-rigidifying saturated fatty acid.