Branched fatty acids from Mycobacterium aurum.

New methyl-branched fatty acids were isolated from the lipids of Mycobacterium aurum, belonging to both saturated and non-saturated series. The most abundant component of the former series was identified as a C22-mycosanoic acid (2-L, 4-L-dimethyleicosanoic acid). The unsaturated fraction contained a mixture of 2-L, 4-L-dimethyl-11-eicosenoic acid and 2-L, 4-L-dimethyl-14-eicosenoic acid. The biosynthetic precursors of these, according to the hypothesis of elongation by propionate units, were found in the non-branched hexadecenoic fraction. The lipidic fraction containing mycosanoic acid was a partially acylated oligosaccharide devoid of sulfate or phosphate groups.

Structural elucidation by field desorption and electron-impact mass spectrometry of the C-mycosides isolated from Mycobacterium smegmatis.

C-mycosides are superficial type-specific glycopeptidolipids of mycobacterial origin. In the present work, we have shown that field desorption mass spectrometry using the cationization method is a useful method for the molecular weight determination of such compounds. Complementary structural information has been obtained by electron-impact mass spectrometry. Combination of both methods has permitted the elucidation of the structure of the C-mycosides of Mycobacterium smegmatis, ATCC 607. This structure is similar to those described elsewhere, but some minor differences are observed in the lipid portion, mainly in the double bond location.

Analysis of bacterial biotin-proteins.

The biotin-protein populations in several bacterial strains were analyzed by solubilization of [3H]biotin-labeled cells with sodium dodecylsulfate followed by electrophoresis on polyacrylamide gels containing the detergent. A variety of patterns of biotin-labeled polypeptide chains was seen, ranging from a single biotin-protein in Escherichia coli, corresponding to the biotin carboxyl carrier protein component of acetyl-CoA carboxylase, to multiple species in Enterobacter aerogenes, Pseudomonas citronellolis, Bacillus cereus, Propionibacterium shermanii, Lactobacillus plantarum, and Mycobacterium phlei, which probably represent subunits of multiple biotin-dependent enzymes present in these organisms. In the case of Pseudomonas citronellolis two major biotin-containing polypeptides with approximate molecular weights of 65 000 and 25 000 were shown to correspond to the biotin carboxyl carrier components of pyruvate carboxylase and acetyl-CoA carboxylase, respectively. Thus in the case of Pseudomonas citronellolis two different biotin-dependent enzymes in the same cell do not share common biotin carboxyl carrier subunits.

Primary structure of a 7Fe ferredoxin from Streptomyces griseus.

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12,291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the Fe-S clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two Fe-S clusters are conserved.

Purification and characterization of an extracellular lectin from Mycobacterium smegmatis.

Mycobacteria exist naturally in aggregated form and pathogenic strains colonize macrophages. A lectin has been isolated from the culture broth of M. smegmatis, which may, possibly, have an important role in either or both of these phenomena. The lectin of Mr 12,000-14,000 agglutinates erythrocytes from different species, and agglutination is reversed by arabinogalactan isolated from mycobacteria, as well as by yeast mannan. It has a pI of 5.5 and is rich in aspartic and glutamic acid residues.

A study on the receptor for a mycobacteriophage : phage phlei.

From Mycobacterium phlei, glycolipid fractions have been isolated which inactivate phage Phlei. On the basis of the characteristics of the inactivation (specificity, kinetics, requirement for Ca++) typical of the phage-host cell system, it was concluded that these fractions contain the receptor sites for phage Phlei ; this conclusion was supported by electron microscopic studies. All the active fractions contain four kinds of components : fatty acids, glycerol, sugars (D-lyxose, 6-0-methyl-D-glucose, and low amounts of glucose and mannose), and water-soluble acids. These acids are isolated by degradation of the receptor fractions as oxalic and pyruvic acids. Variations of the ratio oxalic acid/pyruvic acid according to the mode of degradation and the absence of the peak characteristic of the protons of a pyruvic acid residue in the NMR spectrum, suggest that these acids might arise from the splitting of oxaloacetic acid. A tentative structure of the receptor is proposed, in many monoglycerides are linked through keto-acid to a polysaccharide core.

Structure and stereochemistry of mycolic acids of Mycobacterium marinum and Mycobacterium ulcerans.

Mycobacterium marinum and M. ulcerans were previously shown to synthesize lipid compounds which are stereochemically different from the corresponding molecules isolated from M. tuberculosis and other species. Stereochemical and biogenetic studies of mycolic acids isolated from M. marinum showed that the absolute configurations of the chiral centres occurring in the mycolates are identical to those of the other mycobacterial species examined so far. Furthermore, all the methyl branches were found to come from methionine whatever the configuration of the centre. The structures of the mycolates synthesized by M. marinum and M. ulcerans were found to be identical, consisting of dicyclopropyl and monocyclopropyl monoenoic mycolates, monoenoic keto- and methoxymycolates, thus reinforcing the taxonomical relationship between the two species.

Further stereochemical studies of phthiocerol and phenol phthiocerol in mycobacteria.

Mycobacterium tuberculosis, M. marinum and some other pathogenic species elaborate waxes A, based on a long-chain beta-diol (phthiocerol and companion compounds) and polymethyl-branched fatty acids. The stereochemical studies conducted on waxes A showed that those of M. tuberculosis, M. leprae and M. kansasii differ from waxes A isolated from M. marinum and M. ulcerans by the absolute configuration of the methyl-branched chiral centres occurring in both the long-chain beta-diols and the fatty acyls. Furthermore, the two mycobacterial groups also differ in the stereochemistry of the beta-diol chiral centres.

Gas chromatography of mycobacterial fatty acids and alcohols: diagnostic applications.

Capillary gas chromatography of cellular fatty acids and alcohols has been used as a routine method for a period of two years in the mycobacterial diagnostic laboratory of Statens institutt for folkehelse, Oslo, Norway. All mycobacteria (165 isolates) other than Mycobacterium tuberculosis (MOTT) and 24 randomly selected M. tuberculosis isolates were studied. Twelve characteristic lipid constituents allowed the construction of a diagnostic scheme. Without exceptions, all 36 examined isolates belonging to the M. tuberculosis-complex were characterized by a relatively high concentration level of hexacosanoic acid (mean: 4%, range: 1-13%), low level of tetracosanoic acid (mean: 1%, range: 0.1-3%), lack of methylbranched acids other than tuberculostearic acid, and lack of fatty alcohols. Members of the MAIS-complex (73 isolates) were all characterized by the general presence of the fatty alcohols 2-octadecanol (mean: 2%, range: 0.1-5%) and 2-eicosanol (mean: 7%, range: 2-21%), relatively high levels of tetracosanoic acid (mean: 5%, range: 1-15%) and lack (or trace) of hexacosanoic acid and methylbranched acids other than tuberculostearic acid. All 16 isolates of M. gordonae were easily recognized by their unique lack of tuberculostearic acid and their content of 2-methyl-tetradecanoic acid (mean: 5%, range: 2-12%), and the M. xenopi isolates were the only examined strains containing the fatty alcohol 2-docosanol (mean: 9%, range: 2-13%). The six M. malmoense strains contained the two unique constituents 2-methyl eicosanoic acid (mean: 3%, range: 1-4%) and 2,4,6-trimethyl tetracosanoic acid (mean: 3%, range: 2-4%). The ten strains of M. kansasii were characterized by 2,4-dimethyl tetradecanoic acid (mean: 5%, range: 1-11%), whereas the seven strains of M. marinum shared 2,4-dimethyl hexadecanoic acid (mean: 4%, range 0.2-12%) as a specific marker.

Characteristic new members of the phthiocerol and phenolphthiocerol families from Mycobacterium ulcerans.

Diacyl phthiodiolone A and phenolphthiodiolone A lipids were isolated from two strains of Mycobacterium ulcerans. The diol units of the phthiodiolone A and phenolphthiodiolone A components were shown to have erythro stereochemistry by infrared spectroscopy and proton nuclear magnetic resonance of an acetal derivative. This stereochemistry is shared only by related diols from M. marinum, the diols from M. bovis, M. kansasii, M. leprae and M. tuberculosis having threo stereochemistry.

Calmodulin-like protein and the phospholipids of Mycobacterium smegmatis.

When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed. Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above). Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium. Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM. Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium. Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.

Isolation and structural characterization of a new non-phosphorylated lipoamino acid from Mycobacterium phlei.

A new non-phosphorylated lipoamino acid was extracted from Mycobacterium phlei, strain IST. It is particularly sensitive to alkaline hydrolysis, and contains a lysine residue joined to a 1,2-diglyceride via an ester linkage. The FAB-positive mass spectrum shows the presence of various molecular species of which the most abundant contains a palmitic and a tuberculostearic acid residue. An analogue of this lipid was synthesized, 1,2-dipalmitoyl 3-lysyl glycerol. Both its chromatographic behavior (TLC), and the decomposition pathways of the MH+ ions, studied by FAB MS and MIKE spectroscopy, were identical to the natural product.

Molecular species analysis of lipids by metastable ion mass spectrometry.

A convenient universal and fast mass spectrometrical method designed for the molecular species analysis of natural lipids is described. In contrast to the commonly employed procedures the method does not require chemical or enzymatic treatment and does not include chromatographic steps. The method relies on the recognition of ions characteristic of individual molecular species in the mass spectrum of a particular lipid fraction, that is accomplished on the basis of metastable ion spectra. The efficiency of this approach is demonstrated with a variety of natural lipids: triglycerides, glycerophospholipids, sphingomyelin and ornithinolipids. The advantages and limitations of the method as well as possible further developments are discussed.

Rapid identification of mycobacteria using gas liquid chromatography.

Methyl esters of fatty acids derived from 110 strains of previously identified mycobacteria representing 17 species and a group of unidentified rapid growers, were examined by gas-liquid-chromatography (GLC). Ten species had specific GLC profiles, which enabled accurate identification; but in 2 groups of species strains shared common profiles. M. bovis, and M. xenopi usually had specific profiles but one strain of each could not be distinguished from M. tuberculosis. The group comprising M. terrae, M. fortuitum, M. chelonei, M. flavescens and rapid growers were generally not well separated by GLC; however, 6 of 12 M. terrae strains, 2 of 3 M. flavescens, and all 5 M. fortuitum strains had specific profiles. Other strains of this group had only common peaks and by GLC were indistinguishable from each other. Using a table of specific and characteristic peaks, 34 of 54 (63%) recent isolates were correctly identified, and 18 (33%) were correctly allocated to groups sharing similar GLC profiles: only 2 isolates were wrongly identified. At present, GLC analysis provides easy and rapid identification of a majority of mycobacteria but cannot replace fully biochemical tests in the identification of medical mycobacteria.

The role of cell-wall associated lipids in the pathogenesis of lesions in experimental murine nocardiosis vs lesions produced by Mycobacterium fortuitum.

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.

Structural and immunochemical characterization of the acidic arabinomannan of Mycobacterium smegmatis.

A serologically active, acidic arabinomannan has been isolated from Mycobacterium smegmatis. The polysaccharide contains approximately 56 arabinosyl and 11 mannosyl residues, and 2 phosphate, 6 monoesterified succinate, and 4 ether-linked lactate groups. After saponification to remove succinyl groups, the polysaccharide can be separated into phosphorylated (55%) and nonphosphorylated (45%) forms, the former containing a little more arabinose and a little less mannose than the latter. The structures of these polysaccharides were investigated by 1H- and 13C-n.m.r. spectroscopy and methylation analysis, before and after selective cleavage of furanosyl linkages. The phosphorylated and nonphosphorylated forms of the polysaccharide were found to have similar, if not identical, structures. The main structural feature of the polysaccharides is the presence of chains of contiguous arabinofuranosyl residues linked alpha-(1 leads to 5). These chains are attached at 0-4 of arabinopyranosyl residues that are present in a core region of the polysaccharide that also contains mannopyranosyl residues. Immunochemical studies demonstrated that the polysaccharide is an effective, precipitating antigen with antisera from rabbits immunized with cell walls or heat-killed cells of M. smegmatis. The polysaccharide is, however, more effective as a precipitating antigen after removal of the succinate groups, and completely ineffective after removal of arabinofuranosyl residues. The polysaccharide therefore contains an important antigen in common with the arabinogalactan lipopolysaccharide of the cell wall of the bacterium, i.e., chains of contiguous alpha-(1 leads to 5)-linked arabinofuranosyl residues.

Isolation and analysis by the reductive-cleavage method of linkage positions and ring forms in the Mycobacterium smegmatis cell-wall arabinogalactan.

The Mycobacterium smegmatis arabinogalactan polysaccharide has been isolated from the cell wall by saponification and extraction to remove lipids and subsequent solubilization by treatment with lysozyme. Analysis for neutral sugars demonstrated the presence of D-arabinose and D-galactose in a ratio of 3:1, respectively. Reductive cleavage of the fully methylated polysaccharide in the presence of triethylsilane and trimethylsilyl trifluoromethanesulfonate and subsequent acetylation in situ gave six partially methylated 1,4-anhydroalditol acetates as the major products and three partially methylated 1,5-anhydroalditol acetates as minor products. Partially methylated 1,5-anhydroalditol acetates were not formed when reductive cleavage was accomplished with triethylsilane and a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate as the catalyst, demonstrating that the polysaccharide is exclusively comprised of furanosyl residues. The partially methylated anhydroalditols so produced were identified by comparison to authentic standards. Their identifies are consistent with the presence in the M. smegmatis arabinogalactan of an octasaccharide repeating unit comprised of a nonreducing terminal D-arabinofuranosyl group, a 2-O-linked D-arabinofuranosyl residue, three 5-O-linked D-arabinofuranosyl residues, a 3,5-di-O-linked D-arabinofuranosyl residue, a 5-O-linked D-galactofuranosyl residue, and a 6-O-linked D-galactofuranosyl residue.

Molecular species of mycolic acid subclasses in eight strains of Mycobacterium smegmatis.

Thin layer chromatographic and gas chromatographic separation and mass spectrometric identification of mycolic acid subclasses and molecular species from eight strains of Mycobacterium smegmatis were established. Two major adjacent spots and a lower minor one were detected on silica gel thin layer chromatograms of methyl esters. The most abundant subclass showing the highest Rf value on TLC was that of alpha-mycolic acids (M1), the second was that of alpha-mycolic acids (M1), a shorter homologue than alpha-mycolates, and the third was the hydroxy mycolic acids (M4) derived from epoxy mycolic acids. They were identified by gas chromatography-mass spectrometry as their trimethylsilylether derivatives. alpha'-Mycolic acids were monoenoic acids ranging from C60 to C66 and possessing an alpha-unit of C24:0. Such profiles of alpha'-mycolic acids were common in eight strains. alpha-Mycolates were dienoic acids ranging from C75 to C79 and possessing an alpha-unit of C24:0. In most strains, the major molecular species of alpha-mycolates were odd-carbon-numbered, centering at C77 and C79, possessing a methyl branch in the even-carbon-numbered straight chain. The average carbon number of alpha-mycolates, from seven strains examined, was about 78, but that of the Takeo strain was 76.3. The profiles of epoxy mycolic acid molecular species composition from eight strains ranging from C75 to C81 were very similar to their M1 subclass profiles.

Fatty acid composition of mycobacterial lipids as an index of pathogenicity.

Five species of Mycobacterium were analyzed for the fatty acid composition of total and neutral lipids and of individual classes of phosphatides. The methyl esters of fatty acids were analyzed using the technique of gas liquid chromatography. It was found that myristic, palmitic, palmitoleic, and tuberculostearic acids were the major fatty acids present in all of the lipid components of these strains of tubercle bacilli. The ratio of unsaturated to saturated fatty acids is lowest in the most pathogenic strain, indicating a progressive increase in this ratio, and the highest ratio is represented by a saprophytic strain. An avian pathogenic organism falls in the center of this classification among the examined strains of mycobacteria. These results indicate that the ratio of unsaturated to saturated fatty acids of lipids can be used to determine and establish the pathogenic species of mycobacteria.

Comparative study of the lipid composition of particular pathogenic and nonpathogenic species of Mycobacterium.

A comparative study was undertaken of lipid composition of Mycobacterium tuberculosis H(37)Rv and H(37)Ra, M avium, M phlei, and M 607. Neutral lipids and phosphatides constituted about 55 and 25 percent of the total lipids, respectively. Seven different phosphatides were isolated and identified in varying proportions in all of the above species of mycobacteria. These were polyglycerophosphatide, phosphatidic acid, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol dimannoside, and phosphatidylinositol pentamannoside. Choline-containing phosphatides and cholesterol (steroids) were not detected in lipids of any of the five species under these defined experimental culture medium and growth conditions. Interestingly enough, neutral lipids of M tuberculosis (H(37)Rv and H(37)Ra) contained a higher percentage of diglycerides than monoglycerides, whereas in the other species (M avium, M phlei, and M 607) the monoglyceride content exceeded that of the diglycerides. It appears that the lipid composition of mycobacteria can be an additional useful parameter in distinguishing pathogenic from nonpathogenic species of mycobacteria.