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The question of the precise location of mycolic acids, the single most distinctive cell wall entity of members of the Mycobacterium genus, has now been addressed. The free hydroxyl functions of the arabinogalactan component of the mycobacterial cell wall were O-methylated under conditions in which the mycolyl esters were not cleaved. Subsequent replacement of the mycolyl functions with O-ethyl groups resulted in an acid- and base-stable differentially O-alkylated surrogate polysaccharide, more amenable to analysis. Complete hydrolysis, reduction, acetylation, and gas chromatography/mass spectrometry revealed the unexpected finding that the mycolyl substituents were selectively and equally distributed on the 5-hydroxyl functions of terminal- and 2-linked arabinofuranosyl (Araf) residues. Further analysis of the O-alkylated cell wall through partial acid hydrolysis, NaB[2H]4 reduction, pentadeuterioethylation, and gas chromatography/mass spectrometry demonstrated that the mycolyl units are clustered in groups of four on the previously recognized nonreducing terminal pentaarabinosyl unit [beta-Araf-(1----2)-alpha-Araf)2-3, 5-alpha-Araf. However, only about two-thirds of the available pentasaccharide units are so substituted. Thus, the antigenicity of the arabinan component of mycobacterial cell walls may be explained by the fact that about one-third of the pentaarabinosyl units are not mycolyated and are available for interaction with the immune system. On the other hand, the extreme hydrophobicity and impenetrability of the mycobacterial cell may be explained by the same motif also acting as the fulerum for massive esterified paraffin residues. New fundamental information on the structure of mycobacterial cell walls will aid in our comprehension of its impenetrability to antibiotics and role in immunopathogenesis and persistence of disease.

We found that Mycobacterium porcinum ATCC 33776T (T = type strain) contains a new kind of mycolic acid with a methoxy group at the omega-1 position. This mycolic acid was identified by comparing it with the previously described methoxymycolic acids. The patterns of mycolic acid methyl esters from 418 strains belonging to 44 species of mycobacteria were studied by using thin-layer chromatography. In addition to M. procinum ATCC 33776T, representative strains of M. porcinum, Mycobacterium fortuitum, "Mycobacterium peregrinum," Mycobacterium senegalense, and a recently isolated Mycobacterium sp. contained appreciable amounts of the newly described mycolic acid.

Cellular fatty acid (FA) of 28 species of mycobacteria were analysed by capillary gas chromatography (GC). There were more than sixty volatile FA before C12:0 have no any difference between species both in quality and quantity. The identification scheme of Mycobacterium based on cellular FA was presented here. According to this scheme, all the tested species could be divided into 8 groups by the FA peak height order of C18:1, C18:0 and TBSA which were quite stable; after that, 17 species (60%) might be identified to species level with help of some distinctive FA such as C19:0, C21:0. Most pathogenic bacteria could be separated by the scheme (M. tuberculosis, M. bovis and some complexes). This scheme was also demonstrated to be objective rationality by computer cluster analysis with cellular FA components.

Laser microprobe mass analysis of single bacterial organisms allows the determination of their intrabacterial ratio of sodium-to-potassium ions and the registration of fragment ions originating from their organic bacterial cell matrices as mass fingerprint spectra. It has been established previously that the intrabacterial cation ratio provides information on the physiological state of an individual bacterial cell. In the present experiments it is also shown, with different cultivable mycobacterial species and strains (drug sensitive and resistant) exposed to various drugs, that data derived from the evaluation of the mass fingerprint spectra reflect changes in the degree of impairment. The analysis of Mycobacterium leprae derived from a limited number of skin biopsies of lepromatous/borderline lepromatous leprosy patients under World Health Organization-recommended multiple-drug therapy (WHO/MDT) showed impairment of the organisms with both of the methods of measurement in proportion to the duration of treatment except in one case. In one M. leprae population from a patient who had been treated for 19 months, the fingerprint evaluation gave the first evidence for an insufficient response to treatment. This was further confirmed by the unusual frequency distribution of the Na+,K+ ratios which revealed the existence of two subpopulations, one impaired and one unimpaired.

An unknown immunogenic glycopeptidolipid, named GPL X-1, was isolated from Mycobacterium xenopi, which is a nontuberculous mycobacterium responsible for pulmonary and disseminated infectious diseases mainly occurring in immunocompromised patients. The glycopeptidolipid was purified until homogeneity, in the native form, by direct phase high performance liquid chromatography. A new route is proposed for the structural elucidation of its unusual lipopeptidic core. The presence of allothreonine (aThr), phenylalanine, and serine in the molecular ratio 1:1:2, respectively, was established by reverse phase high performance liquid chromatography analysis of the phenylthiocarbamyl amino acid derivatives. From the molecular mass (1828 Da) of the native glycopeptidolipid, determined by cesium ion liquid secondary ion mass spectrometry using the amphipathic triethylene glycol monobutyl ether matrix, it was deduced that the tetrapeptide was amidified by a dodecanoic acid. The complete structure, C12-Ser-Ser-Phe-aThr-OCH3, of the lipopeptidic core was established by pyrolysis electron impact-mass spectrometry of the native glycopeptidolipid. To date, this is the first example of a mycobacterial glycopeptidolipid with a C12-tetrapeptidic core containing serine. A novel approach, based on two dimensional 1H,1H correlated spectroscopy analysis of the native and peracetylated GPL X-1, was developed, allowing the structural determination of the monosaccharidic residues with their alkali-labile groups "in situ" on the whole complex molecule. 2-O-Acyl-alpha-L-Rhap, alpha-L-Rhap, 2,4-di-O-acyl-6-deoxy-alpha-L-Glcp, 2,3,4-tri-O-Me-alpha-L-Rhap, and 3-O-Me-6-deoxy-alpha-L-Talp were identified, where Me, Rhap, and Talp are methyl, rhamnopyranosyl, and talopyranosyl, respectively. The latter two were localized at the carbohydrate non-reducing ends, and the C-3's of the remaining monosaccharide residues were found involved in the interglycosidic linkage. The alpha anomeric configurations were inferred from the JC-1,H-1 heteronuclear coupling constants, and the L absolute configurations for all the monosaccharide residues were established by gas chromatography analysis of the trimethylsilyl (+/-)-2-butyl glycosides. Finally, by pyrolysis electron impact mass spectrometry of peracetylated GPL X-1, the following tetrasaccharide appendage structure was proposed: 2,3,4-tri-O-Me-L-Rhap(alpha 1----3)-2-O-lauryl-L-Rhap(alpha 1----3)-L-Rhap- (alpha 1----3)-2,4-di-O-(acetyl,lauryl)-6-deoxy-alpha-L-Glcp. Compared to the oligosaccharidic glycopeptidolipid structures, the particular features of the GPL X-1 tetrasaccharide structure arise from the presence of monosaccharide residues esterified by C12 fatty acids and from the absence of the basal disaccharide core, L-Rhap-(alpha 1----2)-6-deoxy-alpha-L-Talp.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.

Thin-layer chromatographic analysis of 72 Finnish clinical mycobacterial isolates presumptively identified as Mycobacterium malmoense revealed four major glycolipid profiles with two minor variations. An additional glycolipid profile was found in three British M. malmoense-like strains. No clear distinction between the strains could be made by means of gas chromatography of cellular fatty acids. The two M. malmoense-specific constituents, 2-methyleicosanoate and 2,4,6-trimethyltetracosanoate, were detected in all strains. The frequency of chemotypes other than that of the type strain was 8% among the Finnish isolates. This variation should be recognized when confirmative identification of mycobacteria is based on thin-layer chromatography of glycolipid extracts.

A fast-growing, non-photochromogenic mycobacterium isolated from the environment exhibited, on thin-layer chromatograms, a characteristic pattern of mycolates composed of unsaturated mycolates and also an unknown more polar component. Spectroscopic analysis and chemical degradation showed that this latter component was a novel mycolic acid containing a methoxy group at the omega-1 position (instead of omega-17 and omega-18 in known methoxymycolates), and two double bonds in the long mero aldehyde chain (instead of one as in known mycolates with additional oxygenated groups).

Rapidly growing mycobacteria of clinical significance were identified by mycolic acids detected with high-performance liquid chromatography. Mycolic acids from whole cells were extracted, derivatized, and detected by a modified high-performance liquid chromatography procedure in less than 3 h. Use of an internal standard allowed differentiation of Mycobacterium chelonae and Mycobacterium fortuitum by comparison of relative retention times. Peak height ratios were used for subidentification of M. chelonae strains; however, M. fortuitum and Mycobacterium smegmatis could not be separated by this system.

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.

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.

Mycobacterium avium strain P-55 and M. avium strain DENT differ from M. avium strain 16909-338 on the basis of their fatty acid spectra (C14:0, C18:0 and tuberculostearic [TBS] acids) studied by multivariate statistical analyses. Strains P-55 and DENT are closer to M. paratuberculosis strain 5889 than to M. avium strain 16909-338, a finding which is in harmony with earlier immunological observations. The recently isolated M. paratuberculosis strain 385 has proved different from M. paratuberculosis strain 5889.

Phenolic glycolipids were first discovered as cell-wall constituents of M. bovis, M. bovis BCG, M. marinum, and M. kansasii. Recently, such compounds were also isolated from M. leprae and have been shown to be specific-species serological markers. Moreover, they seem to be involved, in the case of lepromatous leprosy, in the stimulation of the suppressor T-cells. The functional activities of these phenolic glycolipids over the immune cells stimulation emphasized the role played by these molecules in the mycobacteria pathogenicity. Phenolic glycolipids have also been found in M. gastri and M. tuberculosis strain Canetti. From a structural point of view, these glycolipids contain the same aglycon moiety mainly assigned to phenolphthiocerol diester while the sugar part structure confers to some of these glycolipids their antigenic specificity. The search of immunoreactive glycolipids and their function analysis remain a challenge for chemists and immunologists for the understanding of the mycobacteria pathogenicity.

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.

CHEMICAL ANALYSES OF THE CELL WALLS OF ORGANISMS ISOLATED IN VARIOUS PARTS OF THE WORLD FROM CASES OF LEPROMATOUS AND TUBERCULOID LEPROSY MAKE POSSIBLE THEIR ASSIGNMENT TO ONE OF THE THREE GENERA: Corynebacterium, Mycobacterium, or Propionibacterium. One, bacterium 22M, remains unassigned. The combined chemical and enzymatic properties attributed to leprosy bacilli freshly harvested from lepromata are found collectively, but not individually, in these three genera.

Field desorption mass spectrometry has been used to analyze carbohydrate polymers with 5 to 14 hexose units without prior derivatization. In all examples, the molecular weight of the oligosaccharide could be determined by means of the abundant quasimolecular ions of the type MNa(+), MH(+), MNa(2) (2+), and MNa(3) (3+). Fragmentation at glycosidic linkages was observed in varying extents. The reduced oligosaccharide Man(8)GlcNAcH(2), obtained from IgM [Cohen, R. E. & Ballou, C. E. (1980) Biochemistry 19, 4345-4358], gave quasimolecular ion signals MNa(+) at m/z 1544, MH(+) at m/z 1522, MNa(2) (2+) at m/z 784, and MNa(3) (3+) at m/z 530, all corresponding to its assumed molecular weight of 1519.5. Mycobacterial methylmannose polysaccharides with the general structure Man(x)MeMan(y)-OCH(3) [Yamada, H., Cohen, R. E. & Ballou, C. E. (1979) J. Biol. Chem. 254, 1972-1979] were also successfully analyzed. Man(1)MeMan(13)-OCH(3), the largest homolog, gave the expected signal of the quasimolecular ion MNa(+) at m/z 2506. The larger polysaccharides were analyzed by using a KRATOS MS-50 mass spectrometer with a high-field magnet enabling full sensitivity to be maintained up to 3000 atomic mass units. Polysaccharides up to m/z 1978 were analyzed by using a KRATOS MS-9 mass spectrometer operated at 4 Kv. The signal-to-noise ratio, which becomes a serious problem in field desorption mass spectrometry at low accelerating voltages, and the low instrument sensitivity were improved considerably by our use of a method of adding scans with low total ion currents obtained over a longer desorption time. In this way, we obtained complete sequence information on methylmannose polysaccharides up to Man(1)MeMan(9)-OCH(3)(MNa(+) at m/z 1802). Analysis of a presumed Man(1)MeMan(7)-OCH(3), gave a spectrum consistent only with the structure Man(2)MeMan(6)-OCH(3), revealing the existence of a methylmannose homolog with 2 unmethylated mannoses at the nonreducing end of the chain.

The object of this work was to study the effect of vacuum on Endomyces magnusii, Serratia marcescens, Escherichia coli and Mycobacterium luteum. The zone of tolerance to the water activity was determined for the intact cells of E. magnusii and for the cells subjected to vacuum. Suspensions of the above cells were studied by UV spectroscopy with the aim of detecting changes in the permeability of cell membrane after the action of vacuum.

Deoxyribonucleic acid modification in six strains of mycobacteria was investigated. The presence of 5-methylcytosine in the virulent strain Mycobacterium tuberculosis H37Rv and its absence in the avirulent strain M. tuberculosis H37Ra and other saprophytic, fast-growing mycobacteria appear to be the salient features. However, deoxyribonucleic acid from M. smegmatis SN2 lysogenized with the temperature phage I3 showed the presence of 5-methylcytosine. All of the strains had N6-methyladenine.

Mycobacterium microti walls contained three types of mycolic acids, very similar to those found in Mycobacterium tuberculosis. An alpha-mycolate with two cyclopropane rings, a methoxymycolate with one cyclopropane ring and a methoxyl group, and a ketomycolate with one cyclopropane ring and a keto group were partially characterized. The mycolates made up 34% (by weight) of the peptidoglycan-arabinogalactan-mycolate wall skeleton. Young exponential phase cultures and organisms harvested from mouse lungs contained high proportions of ketomycolates; older cultures had roughly equal proportions of keto- and methoxymycolates. The proportion of alpha-mycolates increased slightly with age of culture, but was always less than one-third of the total.

Data from the literature indicate the presence, in the Mycobacterium kansasii wall, of a phenolic glycolipid called mycoside A. A tentative trisaccharide structure was proposed for the oligosaccharide part, whereas the aglycone was found to correspond to a phenol phthiocerol residue esterified by two mycocerosic acids. In the present work, structural information mainly arising from fast atom bombardment-mass spectrometry, tandem mass spectrometry, and 1H NMR of native and chemically degraded phenolic glycolipid indicates a tetrasaccharide structure for the inherent oligosaccharide. This structure is now determined as: 2,6-dideoxy-4-O-methyl-arabino-hexopyranosyl(1 alpha----3)2-O-methyl-4-O- acetylfucopyranosyl(1 alpha----3)2-O-methyl-rhamnopyranosyl(1 alpha----3)2-4-di- O-methylrhamnopyranosyl-1 alpha----phenolglycol. Evidence for the structure of the dideoxyhexose, unique in mycobacteria, is presented in the following paper (Fournié, J.-J., Rivière, M., Papa, F., and Puzo, G. (1987) J. Biol. Chem. 262, 3180-3184).

Phenolic glycolipids play a key role as an antigenic probe for serodiagnosis of some human pathogen mycobacterial infections. The lipidic part which corresponds to a phenolphthiocerol dimycocerosate molecule, and the presence of partial O-methylated sugars, confer a high hydrophobicity to this kind of molecule. Fast atom bombardment (FAB) mass spectrometric analysis with standard matrices such as glycerol or thioglycerol was unsuccessful. Using a new matrix--monobutyltriethylene glycol--FAB analysis allows molecular weight determination and partial structural elucidation of the saccharidic and the lipidic part of those compounds.

The analysis of a complex mixture of monounsaturated fatty acids from mycobacterium phlei is achieved by capillary gas chromatographic separation of their pentafluorobenzyl esters, formation of gas-phase carboxylate anions by electron capture ionization and decomposition of these anions by collision activation. This method allows the structural determination of fatty acid isomers by examination of their collisionally activated dissociation mass-analysed ion kinetic energy spectra. A good correlation is observed between the structure of the unsaturated fatty acids ranging from C24 to C27 and the methyl terminal part of the main chain of diunsaturated mycolic acids.

The biochemical tests, namely, niacin, catalase, nitrate reduction, tween hydrolysis, tellurite reduction, arylsulfatase and urease tests were carried out for all the mycobacteria which are immunogenically closely related to M. leprae. Among them only M. vaccae shows closest relationship with M. leprae when compared with its communicated data. Except for the tellurite reduction test which was variable in case of M. leprae, all the other tests were found similar to that of M. leprae. In the next experiment, the thin-layer chromatographic pattern of mycolates from M. leprae has been compared with that of M. leprae. The presence of Keto-mycolate in the cell wall structure of both M. vaccae and M. leprae also reflects their biochemical relationship at their ultrastructural level.

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.

The presence of lipids has been demonstrated in mycobacteriophage I3. The total lipid was composed of 69% phospholipids and 31% neutral lipids. More than two-thirds of phospholipids present in the phage were synthesized in the host prior to infection. The fatty acid composition of the phage differed markedly from that of its host, both in chain length and the degree of saturation. The phage lipid was mostly composed of saturated fatty acids of which more than 50% were short chain fatty acids. Changes in growth temperatures reflected variations in fatty acid composition, characteristic of the phage, and which were distinctly different from those of the host. Electron microscopic observations revealed that the phage has a membranous bilayer structure. The presence of lipids may facilitate the phage-host interaction especially in lipid-rich organisms like mycobacteria.

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.

We examined the mycolic acid composition of 133 strains belonging to MAIS complex (Mycobacterium avium-M. intracellulare-M. scrofulaceum) and MAIS intermediate strains and the related species M. asiaticum, M. malmoense, M. shimoidei, and M. simiae. The analysis revealed that about 10% of the strains identified as M. avium-M. intracellulare complex by conventional cultural and biochemical tests were in fact M. simiae strains according to their mycolate composition. Of 25 strains previously studied by the International Working Group on Mycobacterial Taxonomy, 2 (MAIS intermediate and M. asiaticum) presented patterns incompatible with the clusters to which they had been assigned. M. malmoense and both M. simiae serovars shared the same pattern with alpha-, alpha'-, and ketomycolates. We describe here the method used to identify the mycolic acid profiles in detail. We found it to be highly reproducible and convenient for use in mycobacterial reference laboratories.

Mycobactin, the lipid-soluble iron-binding compound of the mycobacteria, has been located using electron microscopy of whole cells stained with vanadate. It forms a discrete, discontinuous region close to, or even included in, the cytoplasmic membrane of Mycobacterium smegmatis. It does not occur throughout the whole thickness of the wall but is some distance within the envelope. Possible models for the accommodation of mycobactin are discussed. It is concluded from these observations that mycobactin may be acting either as a store of iron, or as an ionophore, or possibly fulfilling both roles.

Comparative immunodiffusion studies and thin-layer chromatographic analyses of whole-organism acid methanolysates were performed on 37 strains of Mycobacterium farcinogenes, Mycobacterium senegalense and Nocardia farcinica. The latter were clearly distinguished from the mycobacteria in containing a single mycolic acid methyl ester and showing more precipitinogens with nocardial than with mycobacterial and rhodococcal reference systems. The distribution of precipitinogens showed that M. farcinogenes and M. senegalense were very closely related and that both showed a greater affinity to Mycobacterium fortuitum than to any of the other established species of Mycobacterium tested. The complex pattern of alpha-mycolates and characteristic polar mycolates found in both M. farcinogenes and M. senegalense has only previously been found in M. fortuitum and Mycobacterium smegmatis.

The distribution of hydrocarbon and wax was studied in the cells of Mycobacterium ceroformans. The cells were disintegrated in French press and the content of hydrocarbon and wax was determined in the cell-free extracts. Hydrocarbon and wax are accumulated in the cell walls of Mycobacterium ceroformans.

A crude glycolipid fraction was isolated from Mycobacterium smegmatis ATCC 356 by ethanolic extraction and silica gel chromatography. After deacylation of the glycolipid fraction, a dipyruvylated pentasaccharide (acidic oligosaccharide A) and a monopyruvylated tetrasaccharide (acidic oligosaccharide B1) were purified by ion exchange and gel filtration chromatography. Methylation analysis, proton nuclear magnetic resonance, mass spectrometry, and periodate oxidation suggested that oligosaccharide A was 4,6-(1-carboxyethylidene)-3-O-methyl-beta-D Glcp-(1-3)-4,6-(1-carboxyethylidene)-beta-D-Glcp-(1-4)-beta-D-Glcp-(1-6)-alpha- D Glcp-(1-1)-alpha-D-Glcp and that oligosaccharide B1 was 4,6-(1-carboxyethylidene)-beta-D-Glcp-(1-4)-beta-D-Glcp-(1-6)-alpha-D-Glcp-(1-1 ) -alpha-D-Glcp. Both compounds contain a trehalose unit as a part of the oligosaccharide structure, and B1 appears to be a biosynthetic precursor of A because the two differ only in a pyruvylated 3-O-methylglucose unit. A third component, acidic oligosaccharide B2, differs from oligosaccharide A only in lacking 1 of the 2 pyruvate residues.

Mycobacterium carotenum is more tolerant to the action of hydrogen peroxide than its white, carotenoidless mutant. This elevated tolerance to H2O2 correlates with a higher content of DNA, RNA, and polysaccharides in the cells; it is not related to the content of proteins and lipids, or to the level of the catalase and peroxidase activity.

Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis was conducted of Total DNA using 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.

Using high-performance liquid chromatography, a new kind of phenolic glycolipid quantitatively minor, called phenolic glycolipid-II, was isolated from a lipidic fraction of Mycobacterium kansasii. The structure was determined by fast atom bombardment-mass spectrometry and proton nuclear magnetic resonance spectroscopy, as: 2,4-di-O-Me-alpha-D-Manp(1----3) 4-O-Ac-2-O-Me-alpha-L-Fucp(1----3)2-O-Me- alpha-L-Rhap(1----3) 2,4-di-O-Me-alpha-L-Rhap 1----phenolphthiocerol dimycocerosate. Phenolic glycolipids I and II differ only by their distal monosaccharide hapten which is 2,6-dideoxy-4-O-Me-alpha-D-arabinohexopyranosyl and the 2,4-di-O-Me-alpha-D-mannopyranosyl, respectively. This sugar appears to be characteristic and apparently unique in the Mycobacterium genus. Moreover, phenolic glycolipids I and II constitute with the lipooligosaccharides two classes of antigens of M. kansasii.