Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.     

Monomycoloylglycerol of Nocardia asteroides

Isolation of monoacylglycerol containing only hydroxy acids was achieved after fractionation of the diethyl ether-soluble lipids of Nocardia asteroides by column and thin layer chromatography. The isolated lipid, which was liquid, liberated only glycerol into the aqueous phase, and had (C₅₀C₅₆) mycolic acids as the sole constituent fatty acids.     

Thehalose mycolates from Nocardia asteroides,Nocardia farcinica,Gordona lentifragmenta and Gordona bronchialis

Isolation of glycolipids from Nocardia asteroides, N. farcinica, Gordona lentifragmenta and G. bronchialis, by column chromatography of lipid extracts on a 50% (w/w) mixture of silicic acid and silica gel H, is described. The isolated materials were partially characterized by infrared spectroscopy, optical rotation and refractive index measurements, and by identifying the products of alkaline hydrolysis. Analytical studies showed that the glycolipids released only trehalose in the aqueous phase while mycolic acids were the constituent fatty acids identified.The isolated lipids are trehalose esters in which the trehalose molecule is esterified with mycolic acids.     

Time course dependent changes in contents and physical properties of glycolipid species in Rhodococcus rhodochrous.

The yield of trehalose dimycolate (TDM), the major glycolipid species elaborated by Rhodococcus rhodochrous, a producer of approx. C40-mycolic acid, was not constant in cells cultured for different periods of time. From cells collected at 24, 36, 72, 144 and 172 h of cultivation the following percentages of TDM in diethyl ether soluble lipids (DESL) were found: 10.8%, 23.4%, 10.0%, 9.0% and 5.0%, respectively. In turn, the cellular content accounted for approx. 0.6%, 1.2%, 0.9%, 0.6% and 0.2%, respectively. On the other hand, the yield of galactose monomycolate (GalMM), a minor glycolipid species maintained at approx. 3.4% in DESL during the different periods of time examined; this value represented about 0.3% of the cellular content. The melting temperatures of TDMs fell between 37 degrees C to approximately 97 degrees C with the lowest value from cells grown for 36 h, whereas the melting temperatures of the GalMMs were in a narrow range between 56 degrees C and 64 degrees C. The methyl ester derivatives of the constituent fatty acid moieties of DTMs and GalMMs migrated on thin layer chromatography like methyl esters of C40-C46 mycolic acids, therefore faster than methyl esters of C28-C34 mycolic acids but slower than methyl esters of C50-C56 mycolic acids. Further analysis of the products of pyrolysis of the methyl ester derivatives of the fatty acid moiety released from TDM after alkaline hydrolysis was carried out using gas chromatography combined mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of mononocardomycoloylglycerol from Nocardia rhodochrous

A component of the acetone-soluble lipids of Nocordia rhodochrous grown on glycerol, was purified by column chromatography on silicic acid and characterized by infrared, nuclear magnetic resonance spectroscopy, optical rotation measurement and product identification after alkaline hydrolysis. Glycerol was the sole water-soluble component and nocardomycolic acids with chain lengths ranging from C₄₀ to C₄₄ were the constituent fatty acids identified. On the basis of the evidence obtained, the substance isolated from N. rhodochrous is identified as a mixture of mononocardomycoloylglycerols in which nocardomycolic acids are bound to one of the primary hydroxyl groups of the glycerol molecule.     

Composition and toxicity of lipids from Rhodococcus rhodochrous grown on medium containing galactose, glucose or mannose

Rhodococcus rhodochrous, a producer of mycolic acid of approx. C40, exhibited a higher cellular mass yield when grown on glucose than when grown on galactose or mannose. The cellular content of the diethyl ether-soluble lipids in microorganisms cultivated on glucose or mannose varied with the incubation time, while that of microorganisms grown on galactose remained constant. The lipids extracts from cells cultivated on different hexoses and collected at the exponential phase of growth were more toxigenic; this property was related in general to the content of glycolipid. On the other hand, cells cultivated on galactose or mannose had a higher quantity of glycolipid in the exponential phase, while the glycolipid content of those grown on glucose remained approximately constant. Amongst the components of the lipid extract, the glycolipid fraction was the sole fraction bearing toxic property. Neutral plus fatty acids and phospholipids displayed no similar characteristic.     

Effect of growth stage on mycolic acid structure in cell walls of Nocardia asteroides GUH-2

Mycolic acids were extracted from the cell walls of Nocardia asteroides GUH-2 during different phases of growth at 37 degrees C. These were subjected to structural analysis by combining thin-layer chromatography and gas-liquid chromatography with UV and infrared spectrophotometry and mass spectroscopy of both methyl esters and trimethyl silyl derivatives. By analyzing the fragmentation patterns of these derivatives by three different methods of mass spectroscopy combined with gas-liquid chromatographic separation, the different structural subclasses of mycolic acids were quantitated. Significant qualitative and quantitative modifications of specific mycolic acid subclasses occurred in the cell walls of N. asteroides GUH-2 that were growth stage dependent. The mycolic acids that were predominant in the log phase were polyunsaturated (greater than 2 double bonds per molecule), with long chain lengths and even carbon atom numbers (i.e., C54, C56). In contrast, those that were prominent in the stationary phase were more saturated (few or no double bonds) and of shorter overall carbon chain length (less than or equal to C52). Furthermore, stationary-phase cells had significantly increased amounts of mycolic acids with odd-numbered carbon chain lengths (i.e., C49, C51, C53).     

Molecular weight determination of methyl esters of mycolic acids using thermospray mass spectrometry.

Methyl esters of normal fatty acids, corynomycolate and corynomycolenate were used as model compounds for thermospray mass spectrometric procedures for molecular weight determination of the related nocardial mycolic acids. By using ammonium acetate at the positive ion generator, in both cases, a family of ions was produced. The following members were found and corresponded to the adducts: (1) M + H; M + NH4 and M + H + NH4 for methyl esters of normal fatty acids, whereas M + H, M + 2H and M + H + NH4 were the adducts most frequently observed with methyl corynomycolates. The methyl esters of C40-C48 mycolic acids from Rhodococcus rhodochrous exhibited prominent peaks corresponding to adducts M + H + NH4 whereas those corresponding to M + 2H showed slightly lower intensities. The structure M + H had no significant representatives with this subclass of mycolic acids. A similar pattern was observed with methyl esters of C50-C54 mycolic acids from Nocardia asteroides GUH-2. Ion peaks C50-C54 representing adducts M + 2H and M + H + NH4 prevailed in the mass spectrum. In this case, the intensities of peaks corresponding to M + 2H were slightly higher than those of the M + H + NH4. Essentially three main species of nocardomycolic acids were detected: (1) monounsaturated C50:1, C52:1 and C54:1; (2) diunsaturated C50:2, C52:2 and C54:2 and (3) triunsaturated C52:3 and C54:3 mycolic acids. The most abundant mycolic acid was C52:2 followed in decreasing abundance by C52:1, C54:2, C50:2, C52:3 and C54:3 mycolic acids.