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.

[Lipid analysis in bacterial taxonomy: proposal for a standardized method]. / Analyse lipidique en taxonomie bactérienne: proposition d'une méthode standardisée.

To increase the application of lipid analysis for taxonomic purposes, a standardized method would be useful. This method has to be simple enough to be used routinely. Such a method, based on thin-layer and gas chromatography, is proposed. It can be applied to any kind of bacteria when a few milligrams of cells are available. Comparison of the results provided by this analysis with data found in the literature (given in a schematic form in six tables) allows the identification of the studied strain in the most favorable cases, or gives information for the choice of conclusive complementary tests in the other cases.

[Bacteriophage occurrence in "Mycobacterium phlei" (author's transl)]. / Présence d'une bactériophage chez Mycobacterium phlei.

Surface growth of synchronized bacteria was obtained by means of a suspension of Mycobacterium phlei cells in pentane, the dispersion of which resulted from passage through glass (Ballotini) column. By using standardized conditions, a series of identical cultures were obtained, suitable for studying their evolution as a function of time. By counting colonies every twenty minutes, during ten hours, two doublings were observed, with a generation time of five hours. At the end of a plateau, just before the next doubling, the curve exhibited a marked decrease. Bacteriophages were found in culture medium at the time corresponding to this decrease. In thin sections of the pellicles collected at this time, condensations resembling DNA from phage heads could be noticed within the bacterial cells, as well as free phages in th close neighbourhood of burst cells. The relations between phage and bacteria, and the possible relation between the presence of the phage and the synthesis of phleates has not been determined.

[Study of the biosynthesis of phleic acids, polyunsaturated fatty acids synthesised by Mycobacterium phlei (author's transl)]. / Etude du processus de biosynthèse des acides phléiques, acides polyinsaturés synthétisés par Mycobacterium phlei

Because of their structures, phleic acids (general formula: CH3-(CH2)m-(CH=CH-CH2-CH2)n-CO2H; main component: m = 14, n = 5) cannot be synthesized by the same kinds of enzymatic systems as other natural polyunsaturated fatty acids. By using specifically labelled 14C compounds, we have tested the ability of different molecules to be incorporated in the phleate skeletons by Mycobacterium phlei. The localisation of radioactive carbon atoms has been studied by chemical degradation of labelled phleates, isolation and purification of the degradation products, and determination of their specific radioactivity. When M. phlei cells are incubated with labelled acetate, the unsaturated and saturated parts of the molecules of phleic acids are unequally labelled. The radioactivity of succinate monoester on the one hand and fatty acids (mixture of myristic and palmitic acids) on the other hand, measured after oxidative degradation of phleate esters, shows a constant ratio under definite conditions. Whether [1-14C]acetate or [2-14C]acetate is used for incubation, the same ratio is observed. Therefore acetate is the precursor of the unsaturated part as well as of the saturated part of the phleate molecules. By using labelled fatty acid esters, it has been found that palmitic acid is the precursor of phleates with m = 14, while myristic acid is the precursor of phleates with m = 12. Stearic and eicosanoic acids are not incorporated without degradation. The hypothesis of a condensation of a saturated fatty acid with a preformed polyunsaturated molecule was examined. Search for such a molecule in the lipids of M. phlei gives negative results. Pentaunsaturated phleate arising from palmitate is more abundant than pentaunsaturated phleate arising from myristate, while the reverse is true for hexaunsaturated phleates. These observations make very unlikely such an hypothesis. An elongation process fits well with the observed facts provided that this process involves elongation by two acetate units simultaneously, making elongation by four carbon atoms at a time. Such a requirement would be easily satisfied if two molecules of acetate are condensed together before their utilization in the elongation process. In such a hypothetical process, crotonate would be the most probable substrate of the elongation reaction.

[Lipid composition of cultivable Mycobacteria isolated from livers of armadillos infected by Mycobacterium leprae]. / Etude de la composition lipidique de mycobactéries cultivables isolées de foies de tatous infectés par Mycobacterium leprae.

Four bacterial strains isolated from two livers of armadillos experimentally infected with Mycobacterium leprae were studied. Lipids obtained after saponification and methylation and complex lipids obtained by solvent extraction were examined. The presence of mycolates showed that the four strains belonged to the genus Mycobacterium, but the mycolate patterns, identical for the four strains, were different from those of all strains studied so far. Three of these strains contained phthioceranic acids, which were not found in the fourth one. Only the last strain contained mycosides of the C type, while the three others contained a new type of glycolipid. Their content in mycolates and in glycolipids demonstrated a clear-cut difference between these strains, on the one hand, and M. leprae on the other.

[Studies on the lipids of "Mycobacterium gordonae" in comparison with those of "M. leprae" and of some other scotochromogenic mycobacteria (author's transl)]. / Etude des lipides de Mycobacterium gordonae comparativement à ceux de M. leprae et de quelques mycobactéries scotochromogènes.

The mycolic acids were isolated from Mycobacterium gordonae (strain ATCC 14470), and purified by thin layer chromatography. Three species were studied by mass spectrometry. The analogy between M. gordonae and M. leprae, based on the lack of tuberculostearic acid, was supported by the comparison of the structures of their mycolic acids. Succinct analyses of the lipids of three other scotochromogenic strains of mycobacteria, using thin layer chromatography and gas-liquid chromatography, were performed. These strains were more remote from M. gordonae than is M. leprae, as far as the lipid content is concerned.

[Specific lipids from Mycobacterium ulcerans]. / Lipides spécifiques de Mycobacterium ulcerans.

The main lipids synthesized by Mycobacterium ulcerans are specific for the species. Three products were isolated by chromatography. Their structures were determined by means of spectrographic methods performed on the natural substances or on their split products. The most abundant products were phthiodiolone diphthioceranate and phenolphthiodiolone diphthioceranate . These structures have some analogies with those of phthiocerol dimycocerosate synthesized by M. tuberculosis and M. bovis, and with those of phenolphthiocerol mycocerosate synthesized by M. bovis. The reverse configuration of the polymethyl-branched-chain fatty acids isolated from the substances, according to their origin, remains to be pointed out. Little attention has generally been paid to the stereochemistry of such molecules. We verified that the branched-chain fatty acids found in diacyl phthiocerol and in the mycoside of M. leprae have the same configuration as in the analogous molecules isolated from M. tuberculosis or M. bovis, contrary to M. ulcerans. Another peculiarity of phenolphthiodiolone isolated from M. ulcerans is the occurrence of the phenol group in free form.

Comparative studies of the strains PA and PN of Mycobacterium phlei leading to their reclassification: examination of lipids and DNA, biochemical tests and phage typing.

Study of lipid and DNA, biochemical tests and phage typing performed on the strain PA previously labelled Mycobacterium phlei, lead to the conclusion that this strain belongs to the species M. smegmatis. Parallel studies performed on strain PN, isolated from a culture of strain PA, as well as DNA homology percentage of the two strains, do not support the assumption that strain PN could have resulted from a mutation of strain PA Strain PN produces mycolic acids similar to those found in Rhodococcus bronchialis; the few biological tests applied quite agree with such a classification.

[Structural and metabolic study of the mycolic acids of Mycobacterium fortuitum]. / Etude structurale et métabolique des acides mycoliques de Mycobacterium fortuitum.

The biosynthesis of mycolic acids was studied in whole cells of Mycobacterium fortuitum. At first the structures of the main mycolates produced by the used strain were established as diunsaturated and epoxymycolates. By using [1-14C]acetate as a radiotracer of the lipid synthesis, it was observed that the turnover of the mycolates during the exponential phase of growth of M. fortuitum is fast enough to make very difficult the identification of their precursors. If the growth of the bacterial cells is stopped or highly diminished, by the removal of a large part of their nutritional medium, mycolate synthesis, in contrast to the synthesis of other fatty acids, is stopped as shown by incubation of the concentrated bacterial culture with [1-14C]acetate. After removal of aliquots of the sedimented bacteria at intervals, during several hours, mycolate synthesis resumes when the cell concentration becomes lighter. In these conditions the sequence of radiolabeling of mycolates and of their potential precursors (tetracosanoate and meromycolates) can be observed. In spite of their low accumulation, tetracosanoate and meromycolates were isolated and purified and their specific radioactivity, after different incubation times, could be measured. The results are in agreement with the hypothesis that meromycolates are condensed with tetracosanoate to produce mycolates. However, because of the large differences of isotopic dilution of these two precursors inside the mycolate molecule, this hypothesis, generally taken as evidence, has to be modified. A hypothetical pathway of the mycolate synthesis is proposed, taking into account all these observations.

[Taxonomic value of mycobacterial fatty acids: proposal for a method of analysis]. / Intérêt taxonomique des acides gras des mycobactéries: proposition d'une méthode d'analyse.

A simple and quantitative method for the alkaline hydrolysis of fatty acid derivatives occurring in the lipids of mycobacteria is described. After methylation, the lipidic mixtures were chromatographed on a thin layer of silicagel. The contents in mycolates and secondary alcohols allowed the distinction of 9 groups among the 27 species studied. Such an analysis is generally insufficient to identify a species, but its discriminating power differs from that of other commonly-used methods. Complementary tests chosen according to each particular situation are necessary, including vapour phase chromatography applied to the same lipidic mixture as that used for thin-layer chromatography. Coupling of the two chromatographic methods would allow the recognition of 22 groups among the 27 species of mycobacteria studied.

Lipids as taxonomic markers for bacteria derived from leprosy infections.

Lipid analysis allows the specific detection of M. leprae among various other bacteria isolated from leprosy lesions. In this report mycolates and glycolipid compositions were used for such a discrimination. Comparative studies of the lipid composition of tissue fragments from different organs of experimentally infected armadillos, and of cultivable strains isolated from these tissues showed that the last ones did not multiply extensively in the tissues of the animals.

Taxonomy of mycobacterial strains isolated from the tissues of leprosy patients.

Thirty-six slowly growing mycobacteria isolated from the tissues of leprosy patients were studied using 40 characteristics as well as susceptibility to 27 distinct mycobacteriophages. The composition in mycolic acids of selected strains was also studied. According to the data, the strains formed 5 clusters. Some of the clusters were possibly as yet undescribed species; however, comparison of the data with the known properties of Mycobacterium leprae leads to the conclusion that none of the strains were identical to the leprosy bacillus.

[Lipidic constituents of "Mycobacterium leprae" isolated from experimentally infected armadillo (author's transl)]. / Constituants lipidiques de Mycobacterium leprae isolé de tatou infecté expérimentalement.

Mycobacterium leprae (obtained from experimentally infected armadillo) was submitted to saponification. The liposoluble part was methylated and fractionated by chromatographic methods. Each fraction was studied by gas-liquid chromatography. Cholesterol (from the infected host) and the main fatty acids were identified. Mycolic acids were isolated, and their structures determined, using mass spectrometry. These structures are useful to make a comparison of M. leprae with some other mycobacteria. Some of these comparisons are discussed here. The absence-or, at least, the very low level-of tuberculostearate suggests comparative studies of M. leprae and M. gordonae.

Identification of Mycobacterium fortuitum and Mycobacterium chelonei.

The study of 52 strains of rapidly growing mycobacteria showed that Mycobacterium fortuitum and M. chelonei were clearly distinguished by the aid of seven key tests (nitrate reductase, iron uptake, beta-glucosidase, penicillinase, growth on fructose, resistance to pipemidic acid, and resistance to capreomycin) and by analysis of their respective mycolic acids. However, the subdivision of these species into M. fortuitum var. fortuitum and M. fortuitum var. peregrinum and M. chelonei subsp. chelonei and M. chelonei subsp. abscessus was not satisfactorily accomplished.

A cooperative taxonomic study of mycobacteria isolated from armadillos infected with Mycobacterium leprae.

Seventeen strains of mycobacteria, recovered from six armadillos experimentally infected with Mycobacterium leprae, were examined in ten different laboratories. This collaborative study included use of conventional bacteriological tests, lipid analyses, determination of mycobactins and peptidoglycans, characterization by Py-MS, and immunological, metabolic, pathological and DNA studies. These armadillo-derived mycobacteria (ADM) formed five homogeneous groups (numbered ADM 1 to 5) on the basis of phenetic analyses. However, DNA studies revealed only four homogeneous groups since group ADM 1 and one of the two strains in group ADM 3 showed a high level of DNA relatedness. The phenetic and DNA studies confirmed that the ADM strains differed from all other known mycobacteria. Cultural, biochemical, metabolic and pathogenic properties as well as DNA-DNA hybridizations clearly differentiated these ADM from M. leprae.