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.     

Lipid Composition in the Classification of Nocardiae and Mycobacteria

Ninety-six strains of aerobic actinomycetes with a type IV cell wall (major amounts of meso-diaminopimelic acid, arabinose, and galactose) were analyzed for the presence of mycolic acids and nocardomycolic acids. The method used was comparatively simple and permits the separation of these organisms into two groups: the mycobacteria and the nocardiae. In general, strains received as mycobacteria contained mycolic acids, confirming the generic assignment made by other methods. On the basis of nocardomycolic acid content, Mycobacterium brevicale, M. rhodochrous, and M. thamnopheos should be placed in the genus Nocardia, and on the basis of mycolic acid content, strains recently isolated from bovine farcy should be placed in the genus Mycobacterium. Nocardia farcinica should be considered a nomen dubium and N. asteroides should be considered the type species of the genus.     

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.     

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).     

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.     

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.     

A comparative study of the 'rhodochrous' complex and related taxa by delayed-type skin reactions on guinea pigs and by polyacrylamide gel electrophoresis.

Cell extracts prepared by ultrasonic disruption of 17 strains of the 'rhodochrous' complex and related taxa were compared by polyacrylamide gel electrophoresis and for immunologic relatedness, by skin test reactions. Two organisms, Jensenia canicruria and Nocardia calcarea, gave similar gel patterns and skin test reactions, and are considered to be identical. Extracts of nocardia rubra showed a strong antigenic relationship with those of three Nocardia pellegrino organisms (N325, N324 and N420) previously assigned to the 'rhodochrous' complex. Two Gordona organisms appeared to be less antigenically related to the 'rhodochrous' complex. Extracts of three of four organisms designated Lspi (Rhodococcus coprophilus Rowbotham & Cross 1976) elicited skin test reactions similar to those of the 'rhodochrous' strains. One Lspi strain, N650, showed striking similarities to the 'rhodochrous' complex strain N420 (Nocardia pellegrino).     

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.     

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.     

Tetraenoic and pentaenoic mycolic acids from Mycobacterium thamnopheos. Structure, taxonomic and biosynthetic implications

On the basis of the analysis of mycolates, the type strain of Mycobacterium thamnopheos has been considered as a member of the genus Nocardia. In a comparative study conducted on mycobacterial species we found that M. thamnopheos synthesized two types of mycolate having the same mobilities on thin-layer chromatography as those of mycobacteria, but different from nocardomycolates. Mass spectrometry analyzes showed that the major series of both types consisted of polyunsaturated mycolic acids, ranging from C72 to C78 with four or five double bonds. On pyrolytic mass spectrometry or gas chromatography, the least polar mycolates released mainly monounsaturated C22 esters whereas the other type yielded saturated C20 and C22 esters. These results suggested that M. thamnopheos might be more related to the Aurantiaca taxon than to mycobacteria and Nocardia. The permanganate-periodate oxidation products of esters obtained by pyrolysis of the least polar mycolates showed that they contained docosen-4-oic and docosen-6-oic acids. Both types of mycolate esters yielded the same set of long-chain meroaldehydes on pyrolysis. These meroaldehydes were significantly distinct from those of mycobacterial mycolates in the location of the double bonds. After hydrogenation of the double bond located in the alkyl-branched chain, the two types of mycolates had the same mobility on thin-layer chromatography, indicating that the difference of migration was due to the additional double bond found in the least polar mycolates. Based on stereochemical data, the relative configuration of both mycolates was found to be threo, like that established for all mycolates studied so far.     

A case of lung infection caused by an unusual strain of Nocardia farcinica

A case of lung infection caused by an unusual strain of Nocardia farcinica is reported. This is the third case of the N. farcinica infection in this country. The strain failed to utilize rhamnose as sole carbon source, but could be identified by a numerical identification method. The mycolic acids contained 1-3 double bonds and the numbers of the carbon atoms of the mycolic acids were 50 to 60, average 56.     

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.     

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)     

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.     

The Cell Wall Composition and Distribution of Free Mycolic Acids in Named Strains of Coryneform Bacteria and in Isolates from Various Natural Sources

The major cell wall amino acids and sugars in 177 strains of coryneform bacteria were determined using a 'rapid'method. Representatives were examined for free mycolic acids and the oxygen requirements of all strains were determined. Included were named strains, most of which were labelled Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium or Microbacterium , and a similar number of unnamed isolates from various natural sources. Strains which contained meso -diaminopimelic acid (DAP) were divided into four groups according to their oxygen requirements, the wall sugars and the occurrence and nature of free mycolic acids. Group 1 strains were mainly facultatively anaerobic and contained arabinose and mycolic acids of the Corynebacterium type. They were considered to be members of Corynebacterium sensu stricto and included Cor. diphtheriae and related animal parasites, Microbact. flavum , and Cor. glutamicum and similar species. Group 2 strains were aerobic, contained arabinose and mycolic acids of the 'rhodochrous'type and were considered members of the ' rhodochrous 'complex. Group 3 strains were aerobic, contained ribose and no mycolic acids. Most were Br. linens strains from cheese but a few, possibly related strains, were from other habitats. Group 4 strains were aerobic and contained neither a pentose sugar nor mycolic acids and were of unknown taxonomic status. Most remaining strains contained lysine or ornithine in the wall and smaller numbers contained L-DAP or diaminobutyric acid; none contained mycolic acids. The chemotaxonomic data are discussed in relation to recent numerical taxonomic studies of coryneform bacteria.     

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.     

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.     

Cell wall modification resulting from in vitro induction of L-phase variants of Nocardia asteroides.

The chemical composition of the cell walls of several L-form revertants derived from Nocardia asteroides 10905 was determined at different stages of growth. It was observed that each L-form revertant had a cell well that differed from that of the parental strain when grown under identical conditions. In some strains the peptidolipid and mycolic acid components were affected the most, whereas in other strains the fatty acid, sugar, and mycolic acid moieties were altered. Shifts in mycolic acid size were prominent, whereas the basic peptidoglycan structure appeared to be affected the least. Both the method used to induce the L-form of N. asteroides 10905 and the length of time these organisms were maintained in the wall-less state affected the degree of cell wall modification during the reversion process. Thus, removal of the cell wall appeared to potentiate and select for mutational alterations within the cell envelope of N. asteroides, and these changes resulted in altered cellular and colonial morphology.     

Purification and characterization of C28-55 fatty acids from Mycobacterium smegmatis

The nonmycolic C16 to C55 fatty acids obtained from Mycobacterium smegmatis ATCC 356 by saponification were enriched with respect to the C28 to C55 acids by successive chromatography on silicic acid and Sephadex LH-20 columns. These partially purified fatty acids were then derivatized to the p-bromophenacyl ester and further fractionated by argentation thin-layer chromatography and reverse-phase high-performance liquid chromatography into their individual components. The esters were characterized by electron impact mass spectrometry. Two structural series of C28:1 to C42:1 and C45:2 to C55:2 fatty acids were identified as possible precursors of the monoenyl and dienyl mycolic acids, respectively. These acids were structurally related to the alpha-alkylhydroxyl group of the corresponding mycolic acid. The results suggest that these C28 to C55 fatty acids (meromycolic acids) of M. smegmatis might be precursors of mycolic acids.