Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis?

Fatty acid synthetase activity in extracts of Mycobacterium leprae was equivalent to 1.7 pmol malonyl-CoA incorporated into fatty acid min-1 (mg protein)-1. This activity--if representative of living M. leprae organisms--is insufficient to enable them to synthesize their lipid requirements rapidly enough to support growth. The major activity for scavenging fatty acids in extracts of Mycobacterium microti and Mycobacterium avium, as well as in extracts of M. leprae, was acetyl-CoA-dependent fatty acyl-CoA 'elongase'. This activity was about four times higher in M. avium and M. microti grown in a medium which contained lipids, or when grown in mice, than in medium without added lipids. In contrast, the de novo fatty acid synthetase activity was repressed in M. avium and M. microti when grown in medium that contained lipids, or when grown in mice. These results are consistent with the hypothesis that mycobacteria grown in vivo preferentially scavenge lipids from the host cells, and suggest that a source of lipid should be included in media for attempted axenic isolation of M. leprae.     

Fatty acid oxidation and the beta-oxidation complex in Mycobacterium leprae and two axenically cultivable mycobacteria that are pathogens

Intact, non-growing Mycobacterium leprae, M. avium and M. microti oxidized a wide range of 1-14C-labelled fatty acids (C8 to C24) to 14CO2. Laurate (C12) was oxidized most rapidly, and its oxidation by M. leprae was inhibited by the antileprosy agents Dapsone, clofazamine and rifampicin. Key enzymes of beta-oxidation were detected in extracts from all three mycobacteria. All these activities (both in intact mycobacteria and the enzymes) were stimulated in M. avium grown in Dubos medium plus palmitate but activities in M. microti or M. avium grown either in Dubos medium with added liposomes or triolein, or in vivo were similar to those detected in the same strain grown in Dubos medium alone. M. avium could be grown in medium in which 95% of its fatty acyl elongase activity is acetyl-CoA dependent. In this medium growing M. avium organisms oxidized [1-14C]palmitate to 14CO2 but simultaneously elongated palmitate to C24 acids and even longer. Acetyl-CoA-dependent elongase activity is similar but clearly not identical to reversed beta-oxidation, but the exact point(s) of difference have not yet been identified.     

Biosynthesis and scavenging of pyrimidines by pathogenic mycobacteria

Mycobacterium microti incorporated a wide range of exogenously supplied pyrimidines into its nucleic acids. M. avium incorporated a relatively narrow range of pyrimidines but both M. avium and M. microti when recovered after growth in vivo incorporated a slightly wider range of pyrimidines than the same strains grown in vitro. M. microti and M. leprae could not take up uridine nucleotides directly but could utilize the pyrimidines by hydrolysing them to uridine and then taking up the uridine. Pyrimidine biosynthesis, judged by the ability to incorporate carbon from CO2 or aspartate into pyrimidines was readily detected in non-growing suspensions of M. microti and M. avium harvested from Dubos medium, which does not contain pyrimidines. The biosynthetic activity was diminished in mycobacteria grown in vivo when there is likely to be a source of pyrimidines which they might use. Relative activities for pyrimidine biosynthesis de novo in M. microti were 100 for cells isolated from Dubos medium, 6 for cells isolated from Dubos medium containing the pyrimidine cytidine and 11 from cells recovered after growth in mice. In contrast, relative activities for a scavenging reaction, uracil incorporation, were 100, 71 and 59, respectively. Three key enzymes in the pathway of pyrimidine biosynthesis de novo were detected in M. microti and M. avium. Two, dihydroorotate synthase and orotate phosphoribosyltransferase appeared to be constitutive in M. microti and M. avium. Aspartate transcarbamoylase activity was higher in these mycobacteria grown in vivo than in Dubos medium but it was repressed in M. microti or M. avium grown in Dubos medium in the presence of 50 microM-pyrimidine. Aspartate transcarbamoylase was strongly inhibited by the feedback inhibitors ATP, CTP and UTP. Enzymes for scavenging pyrimidines were detected at low specific activities in all mycobacteria studied. Activities of phosphoribosyltransferases, enzymes that convert bases directly to nucleotides, were not related to the ability of intact mycobacteria to take up pyrimidine bases while activities of pyrimidine nucleoside kinases were generally related to the ability of intact mycobacteria to take up nucleosides. Phosphoribosyltransferase activity for uracil, cytosine, orotic acid and--in organisms grown in Dubos medium with 50 microM-uridine-thymine, as well as kinases for uridine, deoxyuridine, cytidine and thymidine were detected in M. microti. However, M. avium only contained uracil and orotate phosphoribosyltransferase, uridine, cytidine and thymidine kinase, and additionally deoxyuridine kinase when grown axenically with 50 microM-uracil, reflecting its more limited abilities in pyrimidine scavenging.     

Enzymes for purine synthesis and scavenging in pathogenic mycobacteria and their distribution in Mycobacterium leprae

Three enzymes catalysing the synthesis of four intermediates (phosphoribosylglycinamide, phosphoribosylaminoimidazole-succinocarboxamide, phosphoribosylaminoimidazole-carboxamide and AMP) in the purine biosynthetic pathway were detected in extracts of Mycobacterium microti and M. avium, even when the organisms had been grown in mice. However only one of the three enzymes, adenylosuccinate AMP-lyase (catalysing the synthesis of the last two of the four intermediates listed above) was detected in M. leprae. Phosphoribosyltransferases, which convert adenine, guanine and hypoxanthine to the corresponding nucleoside monophosphates, and adenosine kinase were the major enzymes for purine scavenging in all mycobacteria studied. In contrast to enzymes in the synthetic pathway, evidence for metabolic regulation of the purine-scavenging enzymes was obtained. In particular, 20-80-fold differences in the activities of guanine phosphoribosyltransferase and adenosine kinase were observed when M. microti was grown in media with or without purines, or in mice. In M. leprae, activities of all phosphoribosyltransferases were low in comparison with activities in M. microti and M. avium (specific activity less than 2% when comparisons were made between extracts of host-grown mycobacteria). However, activity of adenosine kinase was higher in host-grown M. leprae than in host-grown M. microti or M. avium.     

Aspartate metabolism in Mycobacterium avium grown in host tissue and axenically and in Mycobacterium leprae

Aspartokinase activity was detected in extracts from Mycobacterium leprae (recovered from armadillo liver) and in Mycobacterium avium grown axenically and in vivo. Homoserine dehydrogenase activity was only detected in M. leprae and in M. avium grown axenically. Activities, when detected, were 50 to 70% lower in M. leprae or M. avium grown in vivo than in axenically grown M. avium. In these two pathogenic mycobacteria, aspartokinase and homoserine dehydrogenase are subject to feedback inhibition by methionine - an additional regulator over those observed for the enzymes from Mycobacterium smegmatis. Intact mycobacterium incorporated carbon from [U-14C]aspartate into the aspartate family of amino acids (threonine, isoleucine, methionine and lysine) though the rate of incorporation in M. avium grown in vivo was about half that in M. avium grown axenically.     

Control and location of acyl-hydrolysing phospholipase activity in pathogenic mycobacteria.

Phospholipase activities releasing fatty acyl moieties from phosphatidylcholine and phosphatidylethanolamine and lysophospholipase activity releasing fatty acid from lyso-phosphatidylcholine were detected in both Mycobacterium microti and Mycobacterium avium. Fatty acyl groups were released from both the 1- and 2-positions of phosphatidylcholine. Generally, phospholipase activities of M. avium were cryptic while phospholipase activities of M. microti were located on the bacterial surface. However, intact M. microti did not release fatty acids from phospholipids faster than M. avium. Neither Mycobacterium secreted acyl-hydrolysing phospholipase activity. All phospholipase activities were stimulated by including phospholipids in growth media: generally, cell extracts contained 6- to 15-fold higher specific activities than extracts from mycobacteria grown in media without added phospholipid. However, not all phospholipase activities were stimulated to the same degree in any given set of conditions, suggesting the existence of more than one phospholipase gene in each Mycobacterium.     

Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria.

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.     

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U-¹⁴C]glucose and [1-¹⁴C]acetate, with lipid synthesis from exogenous albumin-bound [1-¹⁴C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain-length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non-esterified fatty acid pool in the two situations. At the period of their most active formation, the very long-chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C₁₈ acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.     

Biosynthesis and scavenging of purines by pathogenic mycobacteria including Mycobacterium leprae

Purine biosynthesis de novo could not be detected in suspensions of Mycobacterium leprae isolated from armadillo tissue. In contrast, non-growing suspensions of other pathogenic mycobacteria, also isolated from infected host tissue did synthesize purines. Rates of synthesis, judged by incorporation of [2-14C]glycine or [3-14C]serine into nucleic acid purines were 600 times higher in M. microti and 110 times higher in M. avium--both isolated from infected mouse tissue--than the lowest possible rate detectable and therefore the highest possible rate in M. leprae. The rate of purine synthesis relative to purine scavenging (judged by comparing incorporation of [3-14C]serine and [8-14C]hypoxanthine into nucleic acid purines in suspensions of mycobacteria) varied only slightly--4-fold in M. microti and 6-fold in M. avium--whether organisms were harvested from media with or without purines, from media with a low nitrogen content but containing a purine, from mice or even with starved organisms. Thus, the failure of M. leprae to synthesize purines could not be explained as either a result of using non-growing mycobacteria in the incubations with 14C-labelled precursors or as repression or inhibition of synthesis de novo. It appears that M. leprae requires a supply of the purine ring from its environment. Nucleotides, which may be the major source of the purine ring in the intracellular environment, were not taken up directly by M. leprae but could be hydrolysed first to nucleosides and then taken up.     

Identification and characterization of a gene encoding a 35-kDa protein from Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. A gene homologous to that of 35-kDa antigen of Mycobacterium leprae was cloned and sequenced from Mycobacterium paratuberculosis. The database searches revealed 82.79% and 95.67% similarities of its nucleotide sequence, with those of immunodominant 35-kDa protein of M. leprae and M. avium, respectively.     

The incorporation of [14C]acetate into the constituent fatty acids of monogalactosyldiglyceride by isolated spinach chloroplasts

The incorporation into diglycerides of the acyl products synthesized from acetate by spinach chloroplasts was greatly stimulated by the addition of glycerol 3-phosphate. When UDP-galactose was added also, monogalactosyldiglycerides became the major products. Palmitate biosynthesis was stimulated about twofold by these additions, while oleate biosynthesis decreased slightly, so that oleate:palmitate ratios were in the range 0.6 to 0.8 rather than about 1.6 when glycerol 3-phosphate and UDP-galactose were not added. On the other hand, Triton X-100 greatly stimulated both oleate and palmitate biosynthesis to give oleate:palmitate ratios of about 2.0. The proportions of oleate and palmitate in the newly synthesized diglycerides, or in monogalactosyldiglycerides when exogenous UDP-galactose was added, did not always reflect the proportions of these two fatty acids synthesized from acetate. When oleate:palmitate ratios were ?1, equal amounts were incorporated into diglycerides or into monogalactosyldiglycerides. When oleate:palmitate ratios were <1, incorporation of palmitate into diglycerides and monogalactosyldiglycerides exceeded that of oleate. 1-Oleoyl, 2-palmitoyl glycerol compounds were the principal products under all conditions but 1,2-dipalmitoyl compounds were also quantitatively important when glycerol 3-phosphate alone, or glycerol 3-phosphate together with UDP-galactose, was added. The distribution of label in the constituent glycerol and fatty acid moieties when monogalactosyldiglycerides were synthesized from diglycerides is consistent with galactosylation occurring without modification or exchange of fatty acids. The distribution of 16- and 18-carbon acyl residues between the 1 and 2 stereospecific positions of newly synthesized monogalactosyldiglyceride was typical of the endogenous polyene monogalactosyldiglycerides. However when palmitate synthesis was in excess of oleate synthesis some palmitate was esterified in position 1, whereas in the endogenous monogalactosyldiglycerides hexadecatrienoate is confined to position 2.     

In vitro lipid metabolism in the rat pancreas. I. Basal lipid metabolism.

1. The in vitro basal lipid metabolism of rat pancreatic fragments was compared with that in adipose tissue fragments and liver slices. 2. [1-14C]Acetate added to the media was mostly incorporated into palmitic acid and to a lesser extent into oleic acid. In addition, pancreatic tissue exhibited a marked capacity for elongation of polyunsaturated fatty acids by [1-14C]acetate and resulting desaturation when compared to adipose tissue and liver. 3. Data obtained in the presence of [U-14C]glucose, [1-14C]palmitate and 3H20 indicate that acetyl-CoA derived from glucose and from beta-oxidation of fatty acids contributed to de novo lipogenesis. 4. Oxidation of [1-14C]palmitic acid was 9-13 times higher in the pancreas than in adipose tissue or liver when expressed on a wet weight basis. 5. The fatty acid moiety of pancreatic glycerolipids could be derived from de novo synthesis, fatty acids added to the medium, or from fatty acids formed from the hydrolysis of endogenous lipids. The glycerol moiety could be derived either from glucose, or directly from glycerol through participation of glycerol kinase.     

Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.     

Phospholipid aliphatic chain composition modulates lipid class composition, but not lipid asymmetry in Clostridium butyricum

The phospholipid composition of the butyric acid-producing clostridia is responsive to the degree of enrichment of the lipids with cis-unsaturated fatty acids. When Clostridium butyricum and Clostridium beijerinckii are grown on oleic acid in media devoid of biotin, the acyl and alk-1-enyl chains of the phospholipids become highly enriched with 18:1 and C19-cyclopropane. Under these conditions there is a marked increase in the glycerol acetals of the major plasmalogens of these organisms. We have grown both species on mixtures of palmitate and oleate in the absence of biotin. The alk-1-enyl chains were highly enriched with C18-unsaturated and C19-cyclopropane residues at all but the highest ratios of palmitate to oleate (80:20, w/w) added to the medium. At ratios of palmitate to oleate greater than or equal to 40:60, the saturated acid was incorporated predominantly into the phospholipid acyl chains in both organisms. The effects of increasing unsaturation of the acyl chains as the ratio of oleate to palmitate was increased was examined in C. butyricum. In cells grown on mixtures of palmitate and oleate equal to or exceeding 40% palmitate, the ratio of glycerol acetal lipid to total phosphatidylethanolamine (PE) was relatively constant. As the proportion of oleic acid added to the medium was increased, the ratio of glycerol acetal lipid to PE increased from 0.7 to 2.0. Thus the ratio of the polar lipids appears to respond to the content of phospholipids that contain two unsaturated chains. The fraction of PE present as plasmalogen remained relatively stable (0.82 +/- 0.05) at varying ratios of medium oleic and palmitic acids. Both the glycerol acetal of ethanolamine plasmalogen, and ethanolamine plasmalogen, are shown to be 80% or more in the outer monolayer of the cell membrane. These two polar lipids represent approx. 50% of the phospholipids in cells grown on exogenous fatty acid. The bulk of the remainder is polyglycerol phosphatides. We suggest that the ability of both species to grow with highly unsaturated membranes is related to their ability to modulate their polar lipid composition.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth

Through examination of one of the fundamental in vitro characteristics of Mycobacterium bovis--its requirement for pyruvate in glycerol medium--we have revealed a lesion in central metabolism that has profound implications for in vivo growth and nutrition. Not only is M. bovis unable to use glycerol as a sole carbon source but the lack of a functioning pyruvate kinase (PK) means that carbohydrates cannot be used to generate energy. This disruption in sugar catabolism is caused by a single nucleotide polymorphism in pykA, the gene which encodes PK, that substitutes glutamic acid residue 220 with an aspartic acid residue. Substitution of this highly conserved amino acid residue renders PK inactive and thus blocks the ATP generating roles of glycolysis and the pentose phosphate pathway. This mutation was found to occur in other members of the M. tuberculosis complex, namely M. microti and M. africanum. With carbohydrates unable to act as carbon sources, the importance of lipids and gluconeogenesis for growth in vivo becomes apparent. Complementation of M. bovis with the pykA gene from M. tuberculosis H37Rv restored growth on glycerol. Additionally, the presence of a functioning PK caused the colony morphology of the complemented strain to change from the characteristic dysgonic growth of M. bovis to eugonic growth, an appearance normally associated with M. tuberculosis. We also suggest that the glycerol-soaked potato slices used for the derivation of the M. bovis bacillus Calmette and Guérin (BCG) vaccine strain selected for an M. bovis PK+ mutant, a finding that explains the alteration in colony morphology noted during the derivation of BCG. In summary, the disruption of a key step in glycolysis divides the M. tuberculosis complex into two groups with distinct carbon source utilization.     

Study of the biosynthesis of phleic acids, polyunsaturated fatty acids synthesised by Mycobacterium phlei (author's transl)]

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.     

Mycobactins and exochelins of Mycobacterium tuberculosis, M. bovis, M. africanum and other related species

Following iron-deficient growth, mycobactins and exochelins were isolated from 11 strains of Mycobacterium tuberculosis (including type strains of the virulent H37Rv and avirulent H37Ra organisms) as well as from M. bovis (one strain), M. bovis BCG (two strains), M. africanum (eight strains) and M. xenopi (one strain) but not from M. microti (one strain). The mycobactins from the tuberculosis group (M. tuberculosis, M. bovis and M. africanum) were identical and could each be resolved into four compounds by thin-layer chromatography (TLC) and into several fractions by high-performance liquid chromatography, supporting previous claims that this group is a single taxon. Exochelins were all chloroform-soluble and showed no species specificity; no single exochelin was recognized by TLC that had not been previously seen in M. avium or a related species.     

Radiometric Measurement of Metabolic Activity of Mycobacterium lepraemurium

A sensitive and nondestructive radiometric method has been applied to the detection of metabolism of Mycobacterium lepraemurium, as a model for the study of the metabolism and substrate requirements of M. leprae. The method is based on the measurement of the (14)CO(2) produced through the bacterial conversion of [U-(14)C]acetate or [U-(14)C]glycerol by 7 x 10(9) bacteria suspended in 10 ml of either a simple buffer system (K-36) or a complex medium (NC-5). Metabolism of the bacilli was easily detected within 3 days after inoculation and was measured daily. NC-5 medium supported metabolism of M. lepraemurium for several weeks longer than the simple K-36 buffer. The radiometric technique shows promise as a rapid and efficient system for evaluating the metabolism of mycobacteria without introducing any changes in the physiologic state of the organisms, studying their metabolic pathways, determining conditions potentially favorable for multiplication of these organisms in vitro, and studying their susceptibility to inhibition by drugs.     

Wild Animal Mycobacterial Isolates

Thirteen strains of mycobacteria isolated from deer and various species of wild birds were analysed by gas chromatography (GG) for cellular fatty acids and by thin-layer chromatography (TLG) for polar lipids. These strains were compared to reference strains of Mycobacterium avium, M. para tuberculosis and M. mal-moense. All the examined strains exhibited a generally similar fatty acid pattern characterized by relatively large amounts of hexadenca-noate (16:0), octadecenoate (18:1), octadecanoate (18:0) and 10-me-thyl-octadecanoate (tuberculostearic acid, 10-Me-18:0). Several additional acids were also generally present but in smaller amounts. By means of small but distinct differences in fatty acid composition, the wild animal isolates could be distinguished from both M. paratuber-culosis and M. malmoense but not from M. avium. The TLG polar lipid patterns on the other hand separated the wild animal isolates into 2 distinct groups of complex and simple polar lipid composition which corresponded to the morphologically smooth and rough types, respectively. The complex patterns of the smooth strains were comparable to those of the M. avium serovars whereas both the rough wild animal isolates and all the M. paratuber-culosis strains showed a simple pattern of polar lipids. Both fatty acid profiles and TLG polar lipid patterns support allocation of the wild animal isolates to the MAIS complex. Moreover, the 2 chemical techniques, particularly the GC procedure, are very useful for a more rapid and precise identification of the slow-growing wild animal mycobacterial isolates which have hitherto been characterized on basis of vague criteria.