Description of Corynebacterium tuberculostearicum sp. nov., a leprosy-derived Corynebacterium.

Leprosy-derived corynebacteria (LDC) have been extensively studied over the past decade. A composite of their biological properties (cell morphology, staining reactions, cellular inclusions and guanine-plus-cytosine content of their deoxyribonucleic acid; 16 strains studied) and their chemical structures (peptidoglycan type, major cell wall polysaccharide, major glycolipid as well as characteristic mycolic acids) appears to define them as members of the genus Corynebacterium. In relation to other corynebacteria found in humans, including "JK corynebacteria", they seem to be distinct. They are here named Corynebacterium tuberculostearicum sp. nov. because they produce a 10-methyloctadecanoic (tuberculostearic) acid (8 strains studied). This and some of their other attributes are considered in relation to properties of leprosy bacilli and Mycobacterium leprae.

Biosynthesis of the novel fatty acid, 17-methyl-cis-9,10-methyleneoctadecanoic acid, by the parasitic protozoan, Herpetomonas megaseliae.

Herpetomonas megaseliae, a flagellate protozoan parasite of the gut of a dipteran, Megaselia scalaris, is shown by chromatographic, spectrometric and radiotracer methods to synthesize de novo an iso-branched chain cyclopropane fatty acid, 17-methyl-cis-9,10-methyleneoctadecanoic acid.

Propionyl-Coa induced synthesis of even-chain-length fatty acids by fatty acid synthetase from Brevibacterium ammoniagenes.

The product distribution of Brevibacterium ammoniagenes fatty acid synthetase has been investigated using propionyl-CoA instead of acetyl-CoA as the primer. The synthetase produces not only an odd-numbered fatty acid (heptadecanoic acid) but also even-numbered fatty acids (stearic and oleic acids) in the presence of propionyl-CoA. The amounts of heptadecanoic, stearic and oleic acids increased with increasing concentration of propionyl-CoA. However, the formation of heptadecenoic acid (C17:1) was not observed under any conditions tested. The failure of C17:1 synthesis suggested that the enzyme component catalyzing the beta, gamma-dehydration, which is responsible for the synthesis of unsaturated fatty acids, has a high degree of chain length specificity. Under standard assay conditions, stearic acid predominated and heptadecanoic and oleic acids were found in lesser amounts. Mass spectrometric analyses of fatty acids synthesized either from [2H]propionyl-CoA or in 2H2O revealed that propionyl-CoA is utilized as the priming substrate for the synthesis of heptadecanoic acid and that an acetyl residues, which is formed by the decarboxylation of malonyl-CoA, served as the priming substrate for the syntheses of stearic and oleic acids. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. It is concluded that the decarboxylation of the malonyl moiety bound to the synthetase occurs efficiently only in the course of fatty acid synthesis. A hypothetical scheme is presented to explain the propionyl-CoA-dependent decarboxylation of the malonyl moiety.

Use of selected ion monitoring for detection of tuberculostearic and C32 mycocerosic acid in mycobacteria and in five-day-old cultures of sputum specimens from patients with pulmonary tuberculosis.

Gas chromatography/mass spectrometry and selected ion monitoring (SIM), employing both electron (EI) and chemical ionization (CI), was used to detect 10-methyloctadecanoic (tuberculostearic) and 2, 4, 8, 8-tetramethyloctacosanoic (C32 mycocerosic) acids in bacteria of 14 species of Mycobacterium and 3 species of Nocardia. Tuberculostearic acid was found in all species studied, while C32 mycocerosic acid was demonstrated only in M. africanum, M bovis, M. bovis strain BCG, M. kansasii and M. tuberculosis. The relative amounts of these acids in the organisms of these five species varied, thereby constituting a presumptive diagnostic technique. The lowest detectable amount of C32 mycocerosic acid was approximately 5 pg when using EI-SIM, monitoring at m/zz 88 and m/z 101. When using CI, employing isobutane as reactant gas, and focusing at m/z 495, 2 pg could be detected, and when ammonia was the reactant gas, the corresponding figure was 1 pg, monitoring at m/z 512. Tuberculostearic acid was demonstrated in 5-day incubated sputum specimens from 6 patients with pulmonary tuberculosis, including 5 patients infected with M tuberculosis and 1 patient infected with M. avium. C32 mycocerosic acid was detected in 4 of the 5 patients with M. tuberculosis infection. None of the acids was found in a further 8 patients who had viral or bacterial (non-mycobacterial) pneumonia. Tuberculostearic acid could be demonstrated in 10 of another 12 sputum specimens from patients with tuberculosis, when the samples were analyzed directly, viz prior to culturing. The possibility of using SIM for the rapid diagnosis of pulmonary tuberculosis is thus worth consideration.

Origin of hydrogen atoms in the fatty acids synthesized with yeast fatty acid synthetase.

The mechanism of hydrogen incorporation into fatty acids was investigated with an enzyme preparation from baker's yeast. Fatty acids synthesized from malonyl-CoA and acetyl-CoA in the presence of D2O or stereospecifically deuterium-labeled NADPH were isolated and analyzed by mass chromatography to examine the localization of deuterium atoms in the molecule. The following results were obtained: 1. Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom). The second hydrogen atom was incorporated as the result of hydrogen exchange phenomenon between the methylene group of malonyl CoA and water. 2. HB hydrogen of NADPH was used for beta-ketoacyl reductase. 3. HB hydrogen of NADPH was also used for enoyl reductase. 4. Hydrogen atoms from HB position of NADPH were found on the odd-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom).

Biosynthesis of saturated and unsaturated fatty acids by a T-strain mycoplasma (Ureaplasma).

A human T mycoplasma (Ureaplasma urealyticum) incorporated radioactivity into its lipids from [1-14C]acetate in the growth medium. Methanolysis of the lipids showed the label to be confined almost entirely to the methyl esters of the fatty acids. About 80% of the label was associated with the methyl esters of the saturated fatty acids, and the rest was found in the unsaturated methyl ester fraction. Gas-liquid chromatography of the saturated methyl esters showed the label to be present in the peaks of palmitate, myristate, and stearate, whereas in the unsaturated methyl ester fraction most of the radioactivity emerged in the peak of palmitoleate. The addition of either oleic or palmitic acid to the growth medium markedly decreased the organisms' incorporation of radioactivity from acetate. It is concluded that the T mycoplasma strain is capable of de novo synthesis of both saturated and unsaturated fatty acids, in this respect differing from all of the Mycoplasma and Acholeplasma strains investigated to date.

The occurrence of cis-octadec-15-enoic acid as a major biohydrogenation product from methyl linolenate in bovine rumen liquor.

The biohydrogenation products from methyl linolenate varied according to its concentration in the rumen. At low concentrations, over 60% of the biohydrogenated products was stearic acid, but when the concentration was raised above 100 mg/100 ml of rumen liquor, monoenoic acids, including cis-octadec-15-enoic acid, were the major products.

Intestinal microflora in rats: isolation and characterization of strictly anaerobic bacteria requiring long-chain fatty acids.

Three strains of strictly anaerobic bacteria, isolated from the cecal contents of rats, have strict requirements for long-chain fatty acids. The effect of exogenous fatty acids on the growth and fatty acid composition of the bacteria was examined. Biohydrogenation of linoleic acid into octadecenoic acid was observed. These observations suggest that long-chain fatty acids in the intestine are factors in controlling the localization and the population levels of indigenous bacteria in vivo in rats.

The hydrogenation of unsaturated fatty acids by five bacterial isolates from the sheep rumen, including a new species.

Five strictly anaerobic bacteria able to hydrogenate unsaturated fatty acids were isolated from sheep rumen. One was characterized as Ruminococcus albus, two as Eubacterium spp. and two as Fusocillus spp., one of which is named as a new species. The Fusocillus organisms were able to hydrogenate oleic acid and linoleic acid to stearic acid, and linolenic acid to cis-octadec-15-enoic acid. The R. albus and the two Eubacteria did not hydrogenate oleic acid but converted linoleic and linolenic acids to a mixture of octadecenoic acids; trans-octadec-II-enoic acid predominated but several isomeric cis and trans octadecenoic acids were produced together with isomers of non-conjugated octadecadienoic acids. The intermediate and final products of hydrogenation by each organism were compatible with the results from mixed rumen bacteria.

Distribution of newly formed palmitate and stearate among molecular species of choline and ethanolamine phosphatides.

The choline and ethanolamine phosphatides derived from isolated rat livers during perfusion with 75 percent deuterated water (Kuksis, A., Myher, J.J., Marai L., Yeung, S.K.F., Steiman, I. & Mookerjea, S. (1975) Can. J. Biochem. 53, 509-518) were resolved into molecular species by argentation thin-layer chromatography. The time course of percentage replacement of the newly synthesized fatty acids in each molecular species was determined by gas chromatography with mass spectrometry. The results confirmed the earlier postulated differential utilization of palmitic and stearic acids in glycerolipid biosynthesis as well as supported the hypothesis of a precursor-product relationship between the oligoenoic and tetranoic species of both phosphatides. Calculations of half-lives gave values of 14-19 h for palmitoyl oligoenes, 40-50 h for palmitoyl tetraenes, and 22-28 h for palmitoyl hexaenes of both choline and ethanolamine phosphatides. The corresponding stearoyl species had half-lives which ranged from 89 to 200 h. Evidence was obtained for a metabolic heterogeneity among subsets of molecular species recognized on the basis of combinations of new and old glycerol and fatty acids in the same glycerolipid molecule.

The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids.

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.

Metabolism of trans-3-hexadecenoic acid in broad bean.

1. Broad bean (Vicia faba) leaves contain rather high concentrations (about 4% of total fatty acids) of the trans-3-hexadecenoic acid. 2. Amounts of the acid increase with the age of the leaves and are absent from etiolated tissue. 3. Changes in the levels of trans-delta-4-hexadecenoic acid can be produced by subjecting the intact plants to various light/dark periods. 4. Chloroplasts isolated from broad-bean leaves show high rates of fatty acid synthesis from [1-14C]acetate. Synthesis is dependent on coenzyme A and ATP but is insensitive to the addition of exogenous acyl carrier protein. 5. The pattern of acids made includes about 20% palmitic, 5% hexadeconoic, 10% stearic and 60% oleic. trans-3-Hexadecenoic acid synthesis was most active in chloroplasts from plants exposed to the dark for 5 days and light for 3 days. 6. Arsenite addition inhibited stearate formation by isolated chloroplasts but resulted in a two-fold stimulation of overall synthesis. 7. The rate of fatty acid synthesis by isolated chloroplasts paralleled the changes in endogenous trans-3-hexadecenoic acid levels in the leaves from which they were isolated.