Studies on monoacylglycerols from Gordona lentifragmenta,Gordona bronchialis and Gordona rubropertincta

Monoacylglycerols containing α-branched-β-hydroxylated fatty acids (mycolic acids) ranging from C₄₂ to C₅₀ and from C₆₀ to C₆₆, were isolated from Gordona lentifragmenta and from G. bronchialis, respectively. On the other hand, G. rubropertincta showed only a monoacylglycerol fraction which released non-hydroxylated fatty acids; they were identified as C_16:0-, C_16:1,- C_18:1- and branched C_19:0-fatty acids. This last component was identified as 10-methyl octadecanoic acid (tuberculostearic acid).     

Complex lipids of Rhodomicrobium vannielii

Eight components, seven of which contained phosphorus, were found in the phospholipid fraction of Rhodomicrobium vannielii. The major components were lipoamino acid (o-ornithine ester of phosphatidyl glycerol, 46.5%) and phosphatidyl choline (26.5%). The other six components were phosphatidyl glycerol (9.7%), bisphosphatidic acid (6.7%), phosphatidyl ethanolamine (4.5%), phosphatidic acid (1.8%), lysophosphatidyl glycerol-o-ornithine ester (3.2%), and N,N-ornithine amide of unidentified fatty acid (0.95%). Total phospholipid accounted for 4.2% of cell dry weight. The major fatty acid was vaccenic acid, C_18:1, which accounted for approximately 90% of the total fatty acids of the complex lipid fraction. The other four fatty acids were C_16:0 (6.25%), C_18:0 (3.8%), C_14:0 (0.7%), and C_16:1 (0.35%). The sulfolipid content was 0.01% of the cell dry weight or 0.14 μmoles per g of dried cells, assuming that its fatty acid component is vaccenic acid. No steroids were detected.     

Signature Lipids and Stable Carbon Isotope Analyses of Octopus Spring Hyperthermophilic Communities Compared with Those of Aquificales Representatives

The molecular and isotopic compositions of lipid biomarkers of cultured Aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis ruber, Thermocrinis sp. strain HI 11/12, Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus, and Aquifex aeolicus all contained glycerol-ether phospholipids as well as acyl glycerides. The n-C_20:1 and cy-C₂₁ fatty acids dominated all of the Aquificales, while the alkyl glycerol ethers were mainly C_18:0. These Aquificales biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C₁₈ and C₂₀ alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C_20:1 and cy-C₂₁, plus a series of iso-branched fatty acids (i-C_15:0 to i-C_21:0), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in ¹³C relative to source water CO₂ by 10.9 and 17.2‰, respectively. The C_20–21 fatty acids of the PSC were less depleted than the iso-branched fatty acids, 18.4 and 22.6‰, respectively. The biomass of T. ruber grown on CO₂ was depleted in ¹³C by only 3.3‰ relative to C source. In contrast, biomass was depleted by 19.7‰ when formate was the C source. Independent of carbon source, T. ruber lipids were heavier than biomass (+1.3‰). The depletion in the C_20–21 fatty acids from the PSC indicates that Thermocrinis biomass must be similarly depleted and too light to be explained by growth on CO₂. Accordingly, Thermocrinis in the PSC is likely to have utilized formate, presumably generated in the spring source region.     

Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids

An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6–10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C₆–C₁₀ carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C₆–C₁₀ dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C₆–C₁₀ ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.     

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.     

Temperature-Induced Changes in the Fatty Acid Composition of the Cyanobacterium, Synechocystis PCC6803

Changes in glycerolipid and fatty acid composition with a change in growth temperature were studied in the cyanobacterium, Synechocystis PCC6803. Under isothermal growth conditions, temperature did not significantly affect the composition of the various classes of lipids, but a decrease in temperature altered the degree of unsaturation of C₁₈ acids at the sn-1 position, but not that of C₁₆ acids at the sn-2 position of the glycerol moiety in each class of lipids. When the growth temperature was shifted from 38°C to 22°C, the desaturation of C₁₈ acids, but not that of C₁₆ acids, was stimulated. The desaturation of fatty acids occurred only in the light and was inhibited by chloramphenicol, rifampicin and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, but not by cerulenin, an inhibitor for fatty acid synthesis. These findings suggest that desaturase activities are induced after a shift from a higher to a lower temperature, and that the desaturation of fatty acids is connected with the reactions involved in photosynthetic electron transport.     

Structural characterization of teichoic acids from Lactobacillus brevis

Teichoic acids are a major constituent of the cell wall of Gram-positive bacteria. Structural characterization of lipoteichoic and teichoic acids isolated from Lactobacillus brevis was undertaken using 1D and 2D NMR experiments as well as chemical methodology. Compositional analysis indicated the presence of high amounts of glycerol, glucose, and alanine. In the case of LTA octadecenoic acid was also detected. The basic LTA/WTA structure was identified as 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at C-2 of the glycerol residues with d-Ala or α-d-Glc. In the case of LTA a higher amount of Ala could be detected and partial alanylation at position C-6 of the Glc could also be observed.     

Hydrocarbon pseudosolubilizing and emulsifying proteins produced by Pseudomonas cepacia N1

Significant specific pseudosolubilizing activity of the n-hexadecane pseudosolubilizing factor (C₁₆PSF), n-octadecane pseudosolubilizing factor (C₁₈PSF) and pristane pseudosolubilizing factor (PrisPSF) was found. From the partial chemical analysis of the chromatographically purified samples the pseudosolubilizing factors (PSFs) and n-hexadecane emulsifying factor (HEF-C₁₆) were tentatively identified as glycoprotein and lipoprotein, respectively. Each of the purified PSFs showed molecular weight 14,000 in SDS-PAGE but they had different amino acid compositions. The carbohydrate component of the C₁₆PSF consists of 93% glucose residues. C₁₂-, C₁₃-, C₁₄- and C₁₆- are the major fatty acids constituting 64% of the total fatty acids of HEF-C₁₆.     

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.     

A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These compounds show not only excellent surface-active properties, but also versatile biochemical actions. During a survey of new MEL producers, we found that a basidiomycetous yeast, Pseudozyma crassa, extracellularly produces three glycolipids. When glucose and oleic acid were used as the carbon source, the total amount of glycolipids reached approximately 4.6 g/L in the culture medium. The structures of these glycolipids were similar to those of well-known MEL-A, -B, and -C, respectively. Very interestingly, in all the present glycolipids, the configuration of the erythritol moiety was entirely opposite to that of conventional MELs. The present glycolipids were identified to have the carbohydrate structure of 4-O-β-d-mannopyranosyl-(2R,3S)-erythritol, stereochemically different from 4-O-β-d-mannopyranosyl-(2S,3R)-erythritol of conventional MELs. Furthermore, these new glycolipids possessed both short-chain acids (C₂ or C₄) and long-chain acids (C₁₄, C₁₆, or C₁₈) on the mannose moiety. The major component of the present glycolipids clearly showed different interfacial and biological properties, compared to conventional MELs comprising two medium-chain acids on the mannose moiety. Accordingly, the novel MEL diastereomers produced by P. crassa should provide us with different glycolipid functions, and facilitate a broad range of applications of MELs.     

Determination of structure and composition of suberin from the roots of carrot, parsnip, rutabaga, turnip, red beet, and sweet potato by combined gas-liquid chromatography and mass spectrometry

Suberin from the roots of carrots (Daucus carota), parsnip (Pastinaca sativa), rutabaga (Brassica napobrassica), turnip (Brassica rapa), red beet (Beta vulgaris), and sweet potato (Ipomoea batatas) was isolated by a combination of chemical and enzymatic techniques. Finely powdered suberin was depolymerized with 14% BF₃ in methanol, and soluble monomers (20-50% of suberin) were fractionated into phenolic (<10%) and aliphatic (13-35%) fractions. The aliphatic fractions consisted mainly of ω-hydroxyacids (29-43%), dicarboxylic acids (16-27%), fatty acids (4-18%), and fatty alcohols (3-6%). Each fraction was subjected to combined gas-liquid chromatography and mass spectrometry. Among the fatty acids very long chain acids (>C₂₀) were the dominant components in all six plants. In the alcohol fraction C₁₈, C₂₀, C₂₂, and C₂₄ saturated primary alcohols were the major components. C₁₆ and C₁₈ dicarboxylic acids were the major dicarboxylic acids of the suberin of all six plants and in all cases octadec-9-ene-1, 18-dioic acid was the major component except in rutabaga where hexadecane-1, 16-dioic acid was the major dicarboxylic acid. The composition of the ω-hydroxyacid fraction was quite similar to that of the dicarboxylic acids; 18-hydroxy-octadec-9-enoic acid was the major component in all plants except rutabaga, where equal quantities of 16-hydroxyhexadecanoic acid and 18-hydroxyoctadec-9-enoic acid (42% each) were found. Compounds which would be derived from 18-hydroxyoctadec-9-enoic acid and octadec-9-ene-1, 18-dioic acid by epoxidation, and epoxidation followed by hydration of the epoxide, were also detected in most of the suberin samples. The monomer composition of the six plants showed general similarities but quite clear taxonomic differences.     

Fatty Acids of Myxococcus xanthus

Fatty acids were extracted from saponified vegetative cells and myxospores of Myxococcus xanthus and examined as the methyl esters by gas-liquid chromatography. The acids consisted mainly of C₁₄ to C₁₇ species. Branched acids predominated, and iso-pentadecanoic acid constituted half or more of the mixture. The other leading component (11–28%) was found to be 11-n-hexadecenoic acid. Among the unsaturated acids were two diunsaturated ones, an n-hexadecadienoic acid and an iso-heptadecadienoic acid. No significant differences between the fatty acid compositions of the vegetative cells and myxospores could be detected. The fatty acid composition of M. xanthus was found to be markedly similar to that of Stigmatella aurantiaca. It is suggested that a fatty acid pattern consisting of a large proportion of iso-branched C₁₅ and C₁₇ acids and a substantial amount of an n-16:1 acid is characteristic of myxobacteria.     

Die Lipide von Endomycopsis vernalis bei verschiedener Stickstoff-Ernährung

1. Endomycopsis vernalis was cultivated on media with different N supply: series A 1%, series B 0,125% asparagine. Sonified cells were extracted and yielded 14.3% (A) and 65.3 (B) total lipids/non lipid dry matter respectively.
2. Neutral and complex lipids were separated by rubber membrane dialysis. There is no difference in the percentage of complex lipids of both series. The increase of lipids in cells grown on low N level is due to a higher content of neutral lipids.
3. Components of the neutral lipids, analysed by DC, were diglycerides, triglycerides, free and esterified ergosterol. Their percentage is influenced by the nutritional conditions. There is a significant increase of triglycerides and of sterol esters in the high lipid cells of series B.
4. Methyl esters of component fatty acids of glycerides and sterol esters were analyzed by GLC. Saturated acids C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, monoenic acids C₁₆ and C₁₈, linoleic and linolenic acids were found to be present. Major acids were in all cases 18:1 (17–57%), 18:2 (18–50%) and 16:0 (10–18%). Linolenic acid is higher in di-and triglycerides of low lipid cells of series A than in high lipid cells of series B. Both qualitative and quantitative differences of fatty acids were found in sterol esters of series A and B respectively.
5. The major components of complex lipids, identified by DC and isolated by CC, in both series, were phosphatidyl choline (A:36.5, B:41.0%) and phosphatidyl ethanolamine (A:24.9, B:20.5%) in addition to small amounts of lysophosphatidyl choline, lysophosphatidyl ethanolamine, phosphatidyl serine, monophosphoinositide, diphosphatidyl glycerol and, possibly cerebroside like substances.
6. Methyl esters of the fatty acids of phosphatidyl choline and ethanolamine from both series were determined by GLC. In all samples 16:0, 18:0, 18:1, 18:2 and 18:3 acids were present besides of traces of 16:1 and 17:0. In contrast to neutral lipids the major acid of phospholipids is linoleic (53–58%), followed by oleic (8–24%) and linolenic acid (1–18%). The percentages of palmitic (4–8%) and stearic acids (tr.-1%) are small. Low lipid cells of series A differ from high lipid cells of series B by an increase of linolenic, and a decrease of linoleic acids, both in phosphatidyl choline and phosphatidyl ethanolamine.

     

Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa

The mycolic acid compositions of Nocardia rubra and related bacteria grown in media containing different concentrations of antituberculous isonicotinic acid hydrazide (INH) were determined in detail by gas chromatography-mass spectrometry. On the basis of molecular species composition, average carbon numbers of mycolic acids were calculated. In Nocardia rubra, N. lutea and Rhodococcus rhodochrous IFO-13161, the ratio of mycolic to non-mycolic fatty acids and the average carbon numbers of mycolic acids were decreased at the INH concentrations of higher than 1 g/ml, paralleling with the significant inhibition of growth. In above three species the synthesis of longer chain mycolic acids (longer than C₄₄ or C₄₆) was inhibited more significantly than shorter homologues such as C₃₈ or C₄₀. In contrast, neither growth inhibition nor change in corynomycolic acid composition was observed in Corynebacteria xerosis and Rhodococcus rhodochrous IFO-13165 at the concentration region of INH up to 100 g/ml. The direct mass fragmentographic analysis of the trimethylsilylated (TMS) derivatives of mycolic acid methyl esters, monitoring [M-15] ions of individual molecular species, revealed that the chain shortening of total mycolic acid molecule by INH occurred more greatly in more highly unsaturated subclasses than in less unsaturated subclasses. Furthermore, mass fragmentographic analysis, monitoring fragment ions (A) and (B), due to straight chain and branched chain alkyl units, respectively, demonstrated the inhibition of mycolic acids was not attributed to the shortening of -alkyl chain, but to the inhibition of chain elongation of C₂₈ to C₃₂ straight chain meromycolic acids. It was also indicated the amounts of trehalose mono- and di-mycolate (cord factor) decreased significantly with the addition of INH (1 to 20 g/ml) in the above strains. From the results obtained above, INH appeared to inhibit the synthesis of mycolic acids longer than C₄₄ or C₄₆ specifically by inhibiting chain elongation or desaturation of precursor long chain fatty acids longer than C₂₈ or C₃₀.     

Terpenoid and other extractives of western white pine bark

A detailed chemical analysis of the benzene extract of western white pine bark was conducted. The extract consisted of 13% phlobaphenes, 18% strong acids, 21% polar weak acids, 6.5% fatty acids, 9.5% resin acids, and 32% neutrals. The fatty acids consisted mainly of C_20:0, C_22:0, and C_24:0 acids. The resin acids were identified as: isopimaric, anticopalic, dehydroabietic, sandaracopimaric, abietic, 6,8,11,13-abietatetraen-18-oic and pimaric acids. The neutrals on saponification gave fatty acids, sterols, wax alcohols, nonsaponifiables, and other components. The esterified fatty acids consisted primarily of the C_16:0, C_18:0, C_20:0 and C_24:0 acids. The sterols included major amounts of sitosterol, campesterol, and stigmasterol, and traces of cholesterol. Over 70 individual compounds were isolated and identified from the nonsaponifiables. These included borneol, sesquiterpenes, diterpenes, steroidal ketones, as well as lanostane and serratane triterpenes. The characterization of12 new natural products or natural products isolated for the first time from Pinus species is reported.     

Epicuticular wax of Agropyron smithii leaves

Wax on the leaves of Agropyron smithii contains hydrocarbons (3%, C₂₇–C₃₃), esters (4%, C₂₆–C₅₆), free acids (5%, C₁₄–C₃₆), hentriacontane-14,16-dione (17%), 25-oxohentriacontane-14,16-dione (3%), 25-hydroxyhen-triacontane-14,16-dione (30%), 26-hydroxyhentriacontane-14,16-dione (18%) and unidentified material (20%). Free and combined acids and combined alcohols did not contain a characteristic major component and free alcohols were absent. A method of estimating proportions of isomeric hydroxy-β-diketones by ¹³C NMR spectroscopy of the hydroxy acids obtained from hydrolysis is described.     

Action of Lipases of Staphylococcus aureus on Milk Fat

The activity of the lipase(s) of two strains of coagulase-positive Staphylococcus aureus was determined in milk fat incubated at 15, 22, and 30 C for 8 days. Total fat hydrolysis was measured by acid degree values (ADV). Neutral lipids were separated into component groups on a Florisil column. Free fatty acids were determined by temperature-programmed gas-liquid chromatography. The ADV were 25 to 50% greater at 22 than at 15 C and 4 to 7 times greater at 30 than at 22 C. The lipases liberated as much as 0.48 g of fatty acids per gram of fat during 8 days at 30 C. The enzyme showed a predilection for the palmitic acid-glycerol bond. Addition of fatty acids C₁₄ to C₁₈ inclusive to inoculated sterile skim milk caused inhibition of S. aureus as follows: (i) complete at 0.05 and 0.10% concentration of C₁₀ and (ii) partial at 0.05 and complete at 0.10% concentration of C₈. The samples showing inhibition were negative for peptonization, coagulase, and change in pH. Addition of oleic and stearic acid to sterile skim milk inoculated with S. aureus resulted in an increase in nonprotein nitrogen, and the C₄ to C₁₂ acids caused a decrease in protease activity.     

Isolation and partial characterization of esters of trehalose from Corynebacterium ovis (C. pseudotuberculosis)

A glycolipid fraction was isolated from Corynebacterium ovis (C. pseudotuberculosis). It had [α]_D²⁵ = + 63.2° (C = 0.5, CHCl₃) and m.p. 43–46°C; the sugar content was 26%, determined as trehalose. Alkaline hydrolysis of the isolated fraction found trehalose as the sole water-soluble component, while glucose was found only after acid hydrolysis of the aqueous phase. Saturated and unsaturated short-chain mycolic acids with carbon atoms ranging from C₃₀ to C₃₆ were the constituents of the fatty acid moiety. The glycolipid fraction of C. ovis is therefore assumed to be a mixture of trehalose esters in which the trehalose molecule is esterified by saturated and unsaturated short-chain (C₃₀–C₃₆) mycolic acids.     

A range of ether core lipids from the methanogenic archaebacterium Methanosarcina barkeri

The core ether lipids of the methanogenic archaebacterium Methanosarcina barkeri were found to consist of C₂₀,C₂₀ glycerol diether and trace amounts of C₂₀,C₂₅ glycerol diether, C₂₀ glycerol monoether, C₄₀,C₄₀ diglycerol tetraethers with 1–3 cyclopentane rings, and a novel C₂₀,C₂₀ tetritol diether.     

Plasmid pRTL1 Controlling 1-Chloroalkane Degradation by Rhodococcus rhodochrous NCIMB13064

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C₃ to C₉). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C₁₂ to C₁₈) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064.