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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.

The models "population--Nocardia-like spontaneous mutants" for 14 cultures of Streptomycetes and Streptoverticillium were used to assess the fatty acid composition of lipids as a criterion for generic differentiation of Streptomyces and Nocardia cultures. The composition of fatty acids in the Nocardia-like mutant Str. kanamyceticus RIA-771 was found to be identical with that of Nocardia asteroides RIA-43 and Nocardia brasiliensis RIA-440, i. e. generic chemotaxonomic traits "overlapped" in the process of spontaneous intraspecial variability. In different strains of one and the same species as well as in five different species, the quantitative composition of fatty acids either hardly changed in the process of intraspecial variability or the ratio between fatty acids changed, so that Nocardia-like mutants resembled typical cultures of the Nocardia genus in this characteristics. The results suggest that the quantitative composition of fatty acids of lipids cannot be regarded as a sufficiently reliable criterion for generic differentiation of Steptomyces and Nocardia cultures. This trait should be used only for additional characterization of cultures.

Nocobactin NA, a lipid-soluble iron-chelating product with an unusual and characteristic u.v.-absorption spectrum, was isolated from Nocardia asteroides grown under conditions of iron deficiency. Its structure was determined by physical methods and by synthesis of one of its degradation products. Nocobactin NA was obtained as a homologous mixture of compounds with side chains of differing length, and resembles mycobactin M in structure except that it has an oxazole ring in place of an oxazoline ring, and the side chains in the cobactin fragment are considerably shorter.

The nocardomycolic acid compositions of extractable and the cell wall-bound lipids from five strains of Nocardia asteroides (A-23007, A-23094, B-23006, B-23095, and IFO 3384) were compared by using gas chromatography-mass spectrometry. The molecular species composition of mycolic acid differed significantly among the strains of N. asteroides. The A-23007 strain possessed the shortest species, centering at C(44(46)), and the A-23094 and IFO-3384 strains followed, each centering at C(52). The B-23006 and B-23095 strains possessed the longest species, centering at C(56) or C(54), thus indicating that N. asteroides strains accommodate a heterogeneous group in respect to carbon numbers of mycolic acids. The doublebond isomers of mycolic acids from the representative strain IFO 3384 were fully separated and analyzed by argentation thin-layer chromatography, followed by gas chromatography-mass spectrometry. The reference strain (IFO 3384) possessed up to four double bonds on the straight chain of mycolic acids ranging from C(46) to C(60). All of the species possessed a C(14) alkyl branch at C-2. The more highly unsaturated subclasses consisted of the longer-chain mycolic acids. Marked changes in mycolic acid composition were induced by altering the growth temperature of strain IFO 3384. The cells grown at the higher temperature (50 degrees C) contained more saturated mycolic acids, whereas those grown at the lower temperature (17 degrees C) had more polyunsaturated (up to tetraenoic) mycolic acids, although a significant difference in carbon chain length was not detected. These changes in the degree of unsaturation of mycolic acids occurred shortly after shifting the growth temperature from 17 to 50 degrees C at logarithmic stages of the bacterial growth, thus indicating that N. asteroides can adapt to changes in the environmental temperature by altering the structure of mycolic acids of the cell walls.

As with other bacteria belonging to the corynebacteria, mycobacteria, and nocardia group, Nocardia possess in their cell walls a neutral polysaccharide. Structural analysis of the cell wall polysaccharide of Nocardia asteroides R 399 was undertaken. The carbohydrate polymer contained D-arabinose and D-galactose as in mycobacteria. Besides these two carbohydrates we pointed out the occurrence of two additional components: D-glucose and a polyol. This polyol, because of its small amount and its uneasy detection, had been for a long time ignored. It has been proven to be the 6-deoxy-D-altritol or 1-deoxy-D-talitol. The polymer consists of a main strand composed of----5 Araf 1----and----4Galp1----or----5Galf1----; oligoarabinosyl side chains were localized on C3 of an arabinosyl residue. Other shorter ramifications also occur on some galactosyl units. A characterization of the linkage between polysaccharide and peptidoglycan inside the cell wall has also been carried out. The two polymers are joined by a phosphodiester bond which involves 6-deoxyaltritol. As some corynebacteria previously analyzed were also shown to contain mannose (and sometimes glucose), we can conclude that the main skeleton of cell wall polysaccharides of the corynebacteria, mycobacteria, and nocardia group of bacteria is an arabinogalactan; however, individual structural features of the polysaccharide are varying according to the bacterial species. These results might be connected with variations that were observed in immunological analysis.

The adjuvant activity of cell wall skeletons (mycolic acid-arabino-galactan-mucopeptide, CWS) prepared from the cells of mycobacteria, nocardia and corynebacteria was examined in vivo in mice and guinea pigs. The cell wall skeletons of Mycobacterium bovis BCG (BCG-CWS), Nocardia asteroides 131 and Corynebacterium diphtheriae PWC suspended in Freund's incomplete adjuvant (FIA) as water-in-oil emulsions showed potent adjuvant activity on the formation of circulating antibody and cell-mediated immunity to bovine serum albumin (BSA), sheep erythrocytes (SRBC) and sulfanylazo-bovine serum albumin (SA-BSA) in mice and guinea pigs. After acetylation or acid treatment, BCG-CWS retained its adjuvant activity, but the activity of BCG-CWS was destroyed completely by alkaline treatment. The cell wall constituents, arabinose-mycolate and arabino-galactan, prepared from BCG-CWS showed no adjuvant activity. It was also shown that BCG-CWS suspended in phosphate buffered saline or associated with oil droplets augmented cell-mediated cytotoxicity in which thymus-derived lymphocytes (T-cells) are effector cells.

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.

An intracellular beta-hemolysin capable of lysing human, sheep and cow erythrocytes but not cells from some other animals was isolated from the cell walls of the three developmental cell-forms of Nocardia asteroides and characterised. The spherical cell-forms contained the highest amounts of the hemolysin (100 h.u./mg protein) and the least LD50 for mice suggesting that this may be the cell-form most pathogenic to susceptible animals. The hemolysin has the properties of a protein, was pH stable and sensitive to both catabolite repression and temperature. The activity of the hemolysin was enhanced by Ca++ and Na+ ions. The hemolysin was immunogenic in rabbits.

To reveal the taxonomical situation of Nocardia asteroides "sensu stricto", we compared the mycolic acid and mycolic acid-containing glycolipid composition and their granulomagenic activities in mice. The major glycolipids were glucose mono- and dimycolate, trehalose mono- and dimycolate and several unknown glycolipids, commonly, although the relative amount differed from strain to strains. On the other hand, molecular species composition of mycolic acids differed distinctively among the three closely related species: N. asteroides "sensu strico", N. farcinica and N. nova. GC/MS analysis showed the most abundant species of mycolic acids were C50(52) in N. asteroides, C54(52) in N. farcinica and C58(56) in N. nova, respectively with a different alpha-alkyl branch. Glucose mycolate and trehalose dimycolate possessing C50 mycolic acid showed a strong activity for granuloma formation in mice.

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.

Studies were conducted on the oxidation and assimilation of n-alkyl-substituted cycloalkane substrates by several hydrocarbon-utilizing microorganisms. These microorganisms utilized heptadecylcyclohexane and dodecylcyclohexane as the sole source of carbon and energy. Neither methylcyclohexane nor ethylcyclohexane was utilized as a growth substrate by any organisms tested. Gas-liquid chromatographic analyses of fatty acids present in cells after growth on dodecylcyclohexane confirm direct incorporation of both alpha- and beta-oxidation products. Growth patterns of these organisms on n-alkyl-substituted cyclohexane fatty acids of varying chain lengths suggest a greater probability of ring cleavage when the side chain contains an odd number of carbons.

The pulmonary pathogens, Nocardia asteroides and Mycobacterium fortuitum classically produce a markedly different tissue response ranging from the acute suppurative lesion of nocardiosis to the granulomatous disease produced by the Mycobacterium. Both organisms have similar cell-wall associated lipids which have been chemically characterized as types of saturated and unsaturated fatty acids. Earlier studies of virulence factors from M. tuberculosis and other Mycobacteria have shown that much of the host response is due to lipid constituency of the organism cell wall. In order to determine that contribution which the cell-wall associated lipids make in the pathogenesis of nocardiosis produced by N. asteroides and mycobacteriosis due to M. fortuitum, separate lipid fractions were obtained using the Anderson extraction technique as modified by Asselineau (Asselineau, J. 1966. The Bacterial Lipids. Hermann, Paris). These lipid fractions were injected into mice and the lesion development observed. Waxes A and D from the two organisms exhibited distinct differences in tissue response. Wax A from Nocardia produced a pronounced tissue response composed of multiple abscesses, macrophages, and reactive fibrous tissue. Wax A from Mycobacterium showed transient aggregations of polymorphonuclear leukocytes. Mycobacteria-derived wax D elicited a marked granulomatous response which persisted throughout the duration of the study, contrasting with a minimally acute inflammatory response to Nocardia-derived wax D. The phosphatide and soluble-fat fractions also showed aggressive lesions; however, these were similar for both organisms. These results indicate that the differences in tissue response elicited by lipids from N. asteroides and M. fortuitum may reside in wax fractions A and D.

The phospholipid of N. asteroides has been investigated. It was found to contain phosphatidyl ethanolamine, cardiolipin, phosphatidyl inositol and a family of mannophosphoinositides. Dimannophosphoinositides with 3 and 4 moles of fatty acid per phosphate residue represented the major glycophospholipids besides small amounts of other more glycosylated mannophosphoinositides.

The melting points of DNA were used to determine the nucleotide composition of DNA from the following microorganisms with a high growth rate: Mycobacterium phlei, M. smegmatis, M. fortuitum, Nocardia asteroides, the organism of the "rhodochrous"--N. opaca group, and organisms related to strain "Mycobacterium" rhodochrous ATCC 13808. M. fortuitum had a higher mol% G+C (67.5--69.5) than M. phlei (66.9--67.5) and M. smegnatis (64.4--68.1); the strains of M. smegmatis were characterized by the most heterogeneous nucleotide composition of DNA. The organisms of the "rhodochrous" complex (mol% G+C=69.5--72.2) were closer to N. asteroides (mol% G+C=72.5--73.5) than to the mycobacteria with a high growth rate.