Mycolic acid analysis in Nocardia species. The mycolic acid compositions of Nocardia asteroides, N. farcinica, and N. nova.

Mycolic acids from twelve Nocardia species were analyzed for structure using capillary gas chromatography and mass spectrometry. This high-resolution procedure permitted good separation of the trimethylsilyl (TMS) ether derivatives of mycolic acid methyl ester according to the total number of carbon and double bonds. The profiles of the mycolic acid molecular species were used as models to illustrate the difference in the structures of each species, even in the case of N. asteroides complex; N. asteroides, N. farcinica and N. nova. Although N. asteroides and N. farcinica had similar lengths of carbon skeleton, i.e., 51.9-53.7 was the average carbon number (Av.Nc.), they had different compositions of unsaturated acids. Mycolic acids from N. asteroides were composed of abundant saturated acids and less than 1% tetraenoic acids; mycolic acids from N. farcinica were composed of unsaturated acids, which were composed of abundant dienoic acids, 2-12% of tetraenoic acids and a trace of pentaenoic acids. In contrast, Av.Nc. of mycolic acids from N. nova were 55.7-56.3, which were relatively longer than those from N. asteroides or N. farcinica. Regarding the characteristics of the structure of alpha-branch, major components were C16:0 and C18:0 for N. asteroides 23206T, and C16:0 and C14:0 for N. farcinica 23157T, respectively. The presence of monounsaturated alpha-branch (C18:1 and C16:1) was characteristic of N. nova.     

Free mycolic acids as criteria in the classification of Gordona and the 'rhodochrous' complex.

The methyl esters of free mycolic acids from representative strains of Gordona bronchialis, G. rubra, G. terrae and Nocardia kirovani each gave, on mass spectroscopy, homologous series of anhydromycolic esters containing from one to four double bonds with the main components of the parent mycolic acids centered on 56, 58, 62 or 64 carbon atoms (total range from C52 to C66). The mycolic acids from the Gordona strains, with chain lengths centered around C60, form a group intermediate in size between nocardomycolic acids (centered around C50) and mycolie different from those of the 'rhodochrous' complex which have anhydromycolates ranging from C34 to C50. Gordonae are thus more closely related in their mycolic acid composition to Nocardia than to Mycobacterium but can be distinguished from each of these genera.     

Free mycolic acids as criteria in the classification of Nocardia and the 'rhodochrous' complex.

The methyl esters of free mycolic acids from representative strains of Nocardia asteroides, N. brasiliensis, N. caviae and the 'rhodochrous' complex were subjected to detailed mass spectral analysis. The anhydromycolic esters of the Nocardia strains consisted of homologous series containing from zero to three double bonds, with the main components of the parent mycolic acids centred on C52 to C54 (range C46 to C58). The anhydromycolates from one rhodochrous strain, Nocardia opaca, had a molecular weight range similar to the nocardiae (C46 to C57) but the remaining rhodochrous strains gave an homologous series of anhydromycolates containing from zero to two double bonds, with the main components of the parent mycolic acids centred on C38, C42, C44 or C46 (total range from C34 to C50). The mycolic acids from the rhodochrous strains with chain lengths centred around C40 form a group intermediate in size between corynomycolic acids (centred around C32) and nocardomycolic acids (centred around C50). These data weaken the case for retaining the 'rhodochrous' complex in the genus Mycobacterium, and also show that many rhodochrous strains can be distinguished from true nocardiae and corynebacteria. These results confirm the value of lipid characters in the classification of these organisms.     

Changes in molecular species composition of nocardomycolic acids in nocardia rubra by the growth temperature

Nocardomycolic acids from Nocardia rubra were fully separated and characterized by a combination of argentation thin-layer chromatography and gas chromatography — mass spectrometry (GCMS). The occurrence of 20 or more different molecular species of mycolic acids was demonstrated. GCMS analysis of each subclass of mycolic acids after separation on AgNO₃ thin-layer chromatography revealed that in general the major species consisted of the even-carbon mycolic acids ranging from C₃₈ to C₅₂. However, the most abundant species differed by the subclasses; C₄₄ being in saturated, C₄₆ in monoenoic and C₄₆ in dienoic mycolic acids, respectively. All these acids were shown to possess C₁₂ or C₁₄ alkyl branch at 2 position, while double bonds were located in longer straight chain alkyl unit.By using this method, distinctive changes in mycolic acid composition by growth temperature were observed. The ratios of saturated, monoenoic to dienoic mycolic acids in a mixture of certain carbon numbered mycolic acids varied greatly, according to the shift of growth temperature. The mass fragmentographic analysis, monitoring M-15 ions derived from the loss of methyl group from the molecular ions showed the lower temperature (15°C) grown cells contained more unsaturated (especially dienoic) mycolic acids, while the higher temperature (40°C) grown cells contained more saturated mycolic acids in both extractable and cell-wall bound lipids. These changes in mycolic acid composition occurred shortly after shifting up the growth temperature from 20°C to 43°C at a logarithmic stage of the bacterial growth.     

Purification and structure analysis of mycolic acids in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is widely used for producing amino acids. Mycolic acids, the major components in the cell wall of C. glutamicum might be closely related to the secretion of amino acids. In this study, mycolic acids were extracted from 5 strains of C. glutamicum, including ATCC 13032, ATCC 13869, ATCC 14067, L-isoleucine producing strain IWJ-1, and L-valine producing strain VWJ-1. Structures of these mycolic acids were analyzed using thin layer chromatography and electrospray ionization mass spectrometry. More than twenty molecular species of mycolic acid were observed in all 5 strains. They differ in the length (20–40 carbons) and saturation (0–3 double bonds) of their constituent fatty acids. The dominant species of mycolic acid in every strain was different, but their two hydrocarbon chains were similar in length (14–18 carbons), and the meromycolate chain usually contained double bonds. As the growth temperature of cells increased from 30°C to 34°C, the proportion of mycolic acid species containing unsaturated and shorter hydrocarbon chains increased. These results provide new information on mycolic acids in C. glutamicum, and could be useful for modifying the cell wall to increase the production of amino acids.     

Structures of the homologous series of monoalkene mycolic acids from Mycobacterium smegmatis.

The positions of localization of the single carbon-carbon double bond in homologs of C series mycolic acids from Mycobacterium smegmatis have been established by 1) combined ammonia chemical ionization mass spectrometry/gas-liquid chromatography of aldehyde ozonolysis products, and 2) high resolution electron impact mass spectral analysis of vicinal glycol derivatives as their trimethylsilyl ethers. These studies have revealed that each homolog is a mixture of two isomers differing in double bond location. In each of the three homologs examined, approximately equal amounts of two isomers were present as follows: (formula: see text).     

Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization

We describe a simple tandem mass spectrometric approach toward structural characterization of mycolic acids, the long-chain α-alkyl-β-hydroxy fatty acids unique to mycobacteria and related taxa. On collisionally activated dissociation in a linear ion trap or tandem quadrupole mass spectrometer, the [M−H]⁻ ions of mycolic acid generated by electrospray ionization undergo dissociation to eliminate the meroaldehyde residue, leading to formation of carboxylate anions containing α-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths of the meromycolate chain and the α-branch. This study revealed that the mycolic acids isolated from pathogenic Rhodococcus equi 103 contained a series of homologous ions having C₃₀ to C₅₀ chain with 0–2 double bonds. The α-branch ranged from C₁₀ to C₁₈ with 0 to 1 double bond, in which 16:0 and 14:0 are the most prominent, whereas the meromycolate chain ranged from C₁₄ to C₃₄ with 0 to 2 double bonds. The major molecular species consisted of more than 3 isomers that differ by the lengths of the α-branch or meromycolate chain, and up to 10 isobaric isomers were identified for some minor ions. We also employed tandem quadrupole mass spectrometry with precursor ion and neutral loss scans for profiling mycolic acid with specific structure in mixtures. The tandem spectra obtained from precursor ion scans of m/z 255 (16:0-carboxylate anion) and m/z 227 (14:0-carboxylate anion) may provide a simple specific means for classification of Rhodococci species, whereas tandem spectra from neutral loss of meroaldehyde residue scans provided a simple approach to reveal the mycolic acid molecules with specific meromycolate chain in mixtures.     

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.     

Ultralong C100 mycolic acids support the assignment of Segniliparus as a new bacterial genus

Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus, Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated α-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane.     

Mycolic acids from Rhodococcus, Gordonia, and Dietzia

The mycolic acids from 11 species of Rhodococcus, seven species of Gordonia, and one species of Dietzia were analyzed using capillary gas chromatography and mass spectrometry (GLC/MS). All strains tested in this study were divided into three groups according to the degree of double bonds and the average carbon number (Av.Nc.) of their mycolic acids. The genus Gordonia belongs to the first group possessing an Av.Nc. in the upper 50s and 60s with 0 to 5 double bonds. Some Rhodococcus species possessed Av.Nc. in the 40s with a variety of distributions of polyunsaturated fatty acids from 0 to 4. The rest of the Rhodococcus species and the genus Dietzia possessed Av.Nc. in the 30s with saturated fatty acids. We previously reported on Nocardia strains whose Av.Nc. were in the 50s. Considering the identification of mycolic acid-containing Actinomycetales at the generic level, the Av.Nc. proved to be useful as a means of differentiating the genera Rhodococcus, Gordonia and Nocardia. The genus Dietzia was found to have its own characteristic constitution of mycolic acid molecular species. The mycolic acids from D. maris 58001T were characterized by an almost equal amount of constituents of even- and odd-numbered carbon chains, whereas the major components of mycolic acids in all other strains had even-numbered carbon chains. Another characteristic of Dietzia was some even-numbered mycolic acids which contained odd-numbered straight chains with odd-numbered alpha-branches. These characteristics indicated that Dietzia might possess a novel fatty acid biosynthesis system.     

Structural analysis of mycolic acids from phenol-degrading strain of Rhodococcus erythropolis by liquid chromatography–tandem mass spectrometry

We used reversed phase liquid chromatography?Celectrospray ionization tandem mass spectrometry for direct analysis of mycolic acids (MAs) from four different cultivations of Rhodococcus erythropolis. This technique enabled us to identify and quantify the specific molecular species of MAs directly from lipid extracts of the bacterium, including the determination of their basic characteristics such as retention time and mass spectra. We identified a total of 60 molecular species of MAs by means of LC/MS. In collision-induced dissociation tandem mass spectrometry, the [M-H]^? ions eliminated two residues, i.e., meroaldehyde and carboxylate anions containing ??-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths and number of double bond(s). Two strains, i.e., R. erythropolis CCM 2595 and genetically modified strain CCM 2595 pSRK 21 phe were cultivated on two different substrates (phenol and phenol with addition of humic acids as a sole carbon source). The addition of humic acids showed that there is a marked increase of unsaturated mycolic acids, mostly in the range of 20?C100?%. This effect is more pronounced in the R. erythropolis CCM 2595 strain.     

Defective mycolic acid biosynthesis in a mutant of Mycobacterium smegmatis.

A mutant of Mycobacterium smegmatis defective in mycolic acid biosynthesis was isolated following chemical mutagenesis. Fatty acids were extracted from the mutant and subjected to structural analysis by thin-layer chromatography and high-performance liquid chromatography (HPLC) of both methyl and p-bromophenacyl ester derivatives. Thin-layer chromatography did not show the presence of any fatty acid of RF comparable to that of standard methyl mycolate. The HPLC profile revealed a broad peak in the standard mycolic acid ester region. No characteristic peaks of mycolic acid esters comparable to the wild-type could be resolved. Mass spectral analysis of the HPLC-purified peak demonstrated the presence of shorter-chain fatty acids in the mutant. These data support the idea that the mutant accumulates precursors of mycolic acids and is incapable of carrying out the final conversion to mycolic acids of 60-90 carbon atoms.     

Characterization of 2,3-dihydroxybenzoic acid from Nocardia asteroides GUH-2.

Culture filtrates of virulent Nocardia asteroides GUH-2 after growth in acetate minimal medium displayed an absorbance maximum at 320 nm. After isolation by polyamide extraction and anion chromatography, a UV-active compound with this absorbance was shown to be 2,3-dihydroxybenzoic acid (DHB) by nuclear magnetic resonance, gas chromatographic, and mass spectrometric techniques. DHB production under several culture conditions was quantified by a standard high-pressure liquid chromatography assay. Under iron deficiency conditions, N. asteroides GUH-2 excreted up to 11 mg of DHB per liter into the culture medium. No DHB was detected when N. asteroides GUH-2 was grown in an iron-rich medium. With the less virulent strain N. asteroides 10905, DHB was not found under any condition tested.     

Effects of culture conditions on the mycolic acid composition of isolates of Rhodococcus spp. from activated sludgefoams

The influence of two different carbon sources and three incubation temperatures on the mycolic acid compositions of three Rhodococcus isolates from activated sludge was examined using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry (GC-MS). Considerable qualitative and quantitative differences were detected in the mycolic acid compositions of the three very closely related isolates grown under the same conditions. Culture age also affected both the chain lengths and proportions of saturated mycolic acids detected in cell extracts, but not in the same manner for each isolate. Mycolic acids generally were of shorter chain lengths in cells grown with Tween 80 compared to glucose grown cells in strain 11R but the opposite situation occurred with strains A7 and D5. In all three, the proportion of unsaturated mycolic acids decreased with increasing growth temperatures. The taxonomic relevance of these observations and possible explanations for the observed changes in mycolic acid composition under various culture conditions are discussed.     

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.     

Quantitative comparison of the mycolic and fatty acid compositions of Mycobacterium leprae and Mycobacterium gordonae

The mycolic and fatty acids of three samples each of Mycobacterium leprae and Mycobacterium gordonae were compared. Acids released by whole-organism alkaline hydrolysis were converted to 4-nitrobenzyl esters and mycolic acids were further derivatized to t-butyldimethylsilyl ethers. Thin-layer chromatography of the derivatized long-chain extracts showed that all three M. leprae preparations contained so-called alpha-mycolates and ketomycolates but that the M. gordonae samples had a methoxymycolate in addition to the above types. Silica gel normal-phase high-performance liquid chromatography of the total mycolic acid derivatives confirmed the lack of detectable amounts of methoxymycolates in M. leprae and reverse-phase chromatography of the individual mycolate types demonstrated the homogeneity of the chain lengths of the mycolic acids in each species. Non-hydroxylated fatty acid 4-nitrobenzyl esters were transformed to methyl esters and examined by gas chromatography. Tuberculostearic (10-methyloctadecanoic) acid was a major component of the lipids of all three M. leprae preparations but it was absent in one M. gordonae strain and a very minor component in the other representatives of this latter species. On the basis of fatty and mycolic acid compositions, therefore, a previously suggested close relationship between M. leprae and M. gordonae was not supported.     

Nocardia in soils of southeastern Spain: abundance, distribution, and chemical characterisation

Actinomycetes belonging to the genus Nocardia were isolated from the surface horizon of 15 out of 46 soil samples examined. All the nocardiae strains isolated contained mycolic acids and saturated and unsaturated straight chain fatty acids (from 12 to 18 carbon atoms) and tuberculostearic acid and were biochemically identified as members of the Nocardia asteroides complex. Nocardiae were detected in alluvial, brown, and serosem great soil groups, but not in calcic brown, solontchack, and regosol great soil groups. Numbers of nocardiae isolated varied from 5.12 X 10(2) to 1.21 X 10(4) colony-forming units per gram of dry weight, and they were statistically correlated with the carbon content (percent C) of the soil. Soil samples were, in general, very dry.     

Structure determination of mycolic acids by using charge remote fragmentation

The collision-induced remote site fragmentation process of closed-shell ions, such as carboxylate anions, is a very potent analytical tool for the structural determination of fatty acids. This leads to an easy location of branch points, double bonds, cyclopropane rings and other functional groups. Although corynomycolic acid mixtures from Corynebacterium diphtheriae can be directly analyzed by negative-ion fast atom bombardment combined with collisionally activated decomposition spectra, mycolic acid mixtures from mycobacteria need a preliminary chemical cleavage. They are oxidized to beta-keto esters and then submitted to a retro-Claisen reaction. The resulting fatty acids were then converted into pentafluorobenzyl derivatives and introduced directly into a high pressure ion source working in the negative ion mode. The resulting gas phase carboxylate anions are activated to decompose by collision with helium atoms. When applied to M3-mycolic acids from Mycobacterium fallax, this method allows for the characterization of a new tri-unsaturated mycolic acid, which has the middle and the remote double bonds separated by two methylene groups.     

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.     

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.