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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.

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.

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.

Ribosomal subunits from Mycobacterium smegmatis were analyzed by using sedimentation velocity, sedimentation equilibrium, and acrylamide gel electrophoresis experiments. These s(20.w) values for the subunits are 48.7S and 28.1S. The molecular weight of the 49S subunit is about 1.65 x 10(6), and that of the 28S subunit is 7.8 x 10(5). Both subunits contain about 37% protein and 63% ribonucleic acid. A protein-deficient particle having an s(20.w) value of 15.7S contains about 11% protein and 89% ribonucleic acid. Skin tests showed all subunits and proteins to be active as agents in provoking delayed hypersensitivity, but the 16S protein-deficient particle, as well as the proteins derived from it, was more specific than the subunits themselves.

Protein spectra of 19 strains of typical and atypical mycobacteria were studied. The advantage of protein extracts obtained from the living cells destroyed by press was shown in experiments of fractionation in polyacrylamide gel. On the basis of similarity indices of the protein spectra a conditioned scheme reflecting the extent of similarity of the cultures under study was drawn. there were revealed protein fractions common for the majority of the cultures studied; It was impossible to isolate the fractions strictly specific of individual groups.

A study of proteinograms of some strains of mycobacteria with the use of the method of electrophoresis in polyacrylamide gel showed that individual strains had characteristic protein spectra which could serve as one of the possible taxonomic characteristics. Analysis of proteinograms of three groups of mycobacteria (tuberculosis, atypical and saprophytes) by electrophoretic mobility of the fractions, with consideration to their homologous character and by the intensity of protein fractions in the mobility zones pointed to heterogeneity of the group of atypical strains and their greater similarity to tuberculosis mycobacteria than to acid-fast saprophytes. Proteinograms of strains H37 Rv and Bov-8 proved to be identical; a marked similarity between the protein spectra of M. avium and M. battey was revealed.

Mycobacterium lacticolum 121 grows and synthesizes exopolysaccharide at the initial values of pH of the medium from 5.0 to 11.5. The accumulation of biomass is maximal (10 to 12 gram/litre) at pH 6.0 to 11.5; the rate of growth is highest at pH 8.0 to 11.5. The largest amount of exopolysaccharide (2.0 to 2.4 gram/litre) is produced in the medium at pH 8.0 to 11.0; the rate of its production is highest at pH 9.0 to 11.0. The growth of Mycobacterium lacticolum 121 and the biosynthesis of polysaccharide are optimal at pH 8.0 to 8.2. Changes in acidity of the medium have no effect on the qualitative composition, structure, and molecular weight of the polysaccharide.

Partitioned between aqueous methanol and petroleum ether, the unsaponifiable pigments of Mycobacterium kansasii were all epiphasic. Thin-layer chromatography of these carotenoids showed that M. kansasii formed at least nine pigments. These pigments were identified by their chromatographic properties and spectral characteristics as phytoene, zeta-carotene, neurosporene, lycopene, leprotene, gamma-carotene, delta-carotene, alpha-carotene, and beta-carotene. Three additional pigmented spots on thin-layer chromatography found in trace amounts were possibly degradation products of the major carotenoids.

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 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.

Culture filtrates prepared from 12 species of mycobacteria were examined for the presence of 3 mycobacterial polysaccharides previously purified by concanavalin A affinity chromatography. Four commercial Old Tuberculins, tuberculin tine test units, and purified protein derivative were also studied. All 3 polysaccharides were demonstrated in all of the filtrates and each of the Old Tuberculins and purified protein derivative. The amount of concanavalin A-nonreactive polysaccharide was measured in clinical skin testing antigens and found to be in the range of potential significance as a cause of false positive reactions to Old Tuberculin.

A rapid staining method using auramine O and acridine orange (AOAO) is described for staining mycobacteria and fungi in paraffin sections of bovine tissues. One hundred seventy-seven tissue sections from specimens divided into two general groups on the basis of previous histopathology results were examined with a fluorscent microscope. Group I, a total of 77 sections, were from 47 mycobacterial and 30 fungal granulomas. Mycobacteria were found by the AOAO procedure in 44 of the 47 tissues previously diagnosed as positive for mycobacteria. All 30 fungal granulomas previously diagnosed using convential fungal stains were positive with the AOAO procedure. Group II consisted of sections prepared from 100 granulomas in which typical mycobacterial lesions were observed by histopathologic examination but in which no mycobacteria had been detected. Using the AOAO procedure, two of these 100 granulomas were found to contain mycobacteria and two were found to contain bacterial colonies. In the remaining 96 no etiologic agent could be demonstrated.

After growth of Mycobacterium vaccae strain JOB5 on acetate or propane, the cellular fatty acids were isolated and identified by a combination of gas-chromatographic, mass-spectral, and chemical means. The fatty acids ranged from C12 to C19 and were a mixture of saturated, monounsaturated, and methyl-branched components. The double bond was in the delta9 position in the C15 to C18 unsaturated acids. The single methyl branch was located on the C10 position of Br-C17, Br-C18, and Br-C19 fatty acids. Branched-chain synthesis occurs at the expense of an unsaturated precursor fatty acid; the double bond serves as the site of methylation. Results suggest that S-adenosylmethionine is the methyl donor involved.

Experiences with the analysis of mycobacterial lipids by means of thin-layer chromatography are described. The influence of different media used for the cultivation of the strains, the age of cultures and heating of the bacteria on the lipid patterns was investigated. 25 strains of rapid growers belonging to various species were examined by a definite procedure. As the results indicate the lipid analysis allows a differentiation of more species than with the usual culture and enzymatic methods. The conclusions especially for taxonomic problems are discussed.

Immobilized deoxyribonucleic acid (DNA) preparations from eight species of mycobacteria were reacted with labeled reference DNA from Mycobacterium tuberculosis and M. kansasii. All mycobacteria showed some degree of homology. Immobilized DNA from Pseudomonas multivorans, which has a guanine plus cytosine content within the range established for the mycobacteria, showed no significant binding with either reference system. Relative per cent binding and thermal stability of bound DNA were used as parameters for determining degrees of relatedness among members of the genus. When these relationships were compared with those established by numberical taxonomic methods, a high correlation was found.

Guanine plus cytosine values of deoxyribonucleic acid derived from 30 cultures representing 14 mycobacterial species or varieties are presented. These data provide impressive reasons for maintaining the separation between the genera Corynebacterium and Mycobacterium; no conclusions can be arrived at from these data with respect to the Nocardia-Mycobacterium relationship. A bimodal clustering, in terms of guanine plus cytosine composition, is apparent within the genus Mycobacterium. In general, all members of any single phenetic species appear to fit into one or another of these clusters. The phenetic separation of species is, in some cases, confirmed by separation in terms of guanine plus cytosine values. The bimodal separation of guanine plus cytosine values within the genus Mycobacterium does not correspond to a division of the species into slow and rapid growers; it thus provides no justification for splitting Mycobacterium into two genera, composed of slow and rapid growers. This is not to say that such a split would not be useful, only that these data do not contribute to such a decision. Any further attempts to correlate phenetic classification with properties of mycobacterial deoxyribonucleic acid will require more specific techniques, such as molecular hybridization.

Fifty-eight strains of nonphotochromogenic mycobacteria representing nine different serotypes were studied, including 38 strains of Mycobacterium avium and 20 strains of Battey bacilli. The lipids were extracted from whole cells, saponified with potassium hydroxide, and esterified with diazomethane. The fatty-acid profiles determined by gas-liquid chromatography included saturated fatty acids ranging from C(8) to C(24), plus some unsaturated analogues and a branched-chain acid. No consistent differences in the fatty-acid profiles were observed between strains of M. avium and Battey bacilli. Quantitative differences in the means (significant at P < 0.01) were observed in the relative amounts of 14:0, 16:1, 18:0, 18:1, and a branched-chain acid among strains of the same serotype. We were unable to differentiate the fatty-acid profiles of recently isolated strains or those maintained in culture for more than 2 years.

Mycobactin S has been isolated from Mycobacterium smegmatis and from Mycobacterium sp. Olitzky & Gershon, strain 2, and mycobactin H from M. thermoresistible; all three organisms were grown on synthetic media of low iron content. These two mycobactins are mixtures of compounds having the same nucleus but differing in their fatty side chains. The nucleus of mycobactin S has a chemical structure identical with that of mycobactin T but differs in the optical configuration at the beta-carbon atom of the hydroxy acid fragment; the configuration in mycobactin S is S whereas that in mycobactin T is R (the previous assignment of this configuration was incorrect). The cobactin fragment of mycobactin H is identical with that of mycobactin S, but the mycobactic acid moiety differs in having methyl groups at position 6 in the benzene ring and at position 5 in the oxazoline ring. The configurations of all the asymmetric centres have been established for both mycobactins. Improved and simplified methods for the extraction and purification of mycobactins are described.