Distribution of a novel mycolic acid in species of the genus Mycobacterium

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.     

Lipid Composition in the Classification of Nocardiae and Mycobacteria

Ninety-six strains of aerobic actinomycetes with a type IV cell wall (major amounts of meso-diaminopimelic acid, arabinose, and galactose) were analyzed for the presence of mycolic acids and nocardomycolic acids. The method used was comparatively simple and permits the separation of these organisms into two groups: the mycobacteria and the nocardiae. In general, strains received as mycobacteria contained mycolic acids, confirming the generic assignment made by other methods. On the basis of nocardomycolic acid content, Mycobacterium brevicale, M. rhodochrous, and M. thamnopheos should be placed in the genus Nocardia, and on the basis of mycolic acid content, strains recently isolated from bovine farcy should be placed in the genus Mycobacterium. Nocardia farcinica should be considered a nomen dubium and N. asteroides should be considered the type species of the genus.     

Identification of Mycobacterium leprae: use of wall-bound mycolic acids

A simple method for extraction and analysis of wall-bound mycolic acids from small samples of mycobacteria is described. Separation of mycolic acid classes according to their functional groups by thin-layer chromatography showed a difference between Mycobacterium leprae and a number of strains of acid-fast bacilli cultured from leprosy biopsies in vitro. This technique is proposed as a convenient preliminary test in the identification of possible cultures of M. leprae.     

Deoxyribonucleic acid methylation in mycobacteria.

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.     

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria

Background

Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/Principle Findings

We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the α- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/Significance

This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.     

Proposed minimal standards for the genus Mycobacterium and for description of new slowly growing Mycobacterium species.

In accordance with Recommendation 30b of the International Code of Nomenclature of Bacteria, which calls for the development of recommended minimal standards for describing new species, we propose minimal standards for describing the genus Mycobacterium and new slowly growing species of this genus. The minimal standards for assignment of a strain to the genus Mycobacterium include acid-alcohol fastness, a DNA G+C content in the range from 61 to 71 mol%, and mycolic acid detection with characterization of C22 to C26 pyrolysis esters. The recommended minimal standards for describing a new slowly growing Mycobacterium species are based on the results of phenotypic and genomic studies and include the results of the following conventional tests: growth at 25, 30, 33, 37, 42, and 45 degrees C; pigmentation; resistance to isoniazid, thiophene-2-carboxylic acid hydrazide, hydroxylamine, p-nitrobenzoic acid, sodium chloride, thiacetazone, picrate, and oleate; catalase activity; Tween hydrolysis; urease activity; niacin detection; and nitrate reductase, acid phosphatase, arylsulfatase, pyrazinamidase, and alpha-esterase activities. In addition, a mycolic acid profile should be determined, and DNA-DNA hybridization experiments in which the difference between the denaturation temperature of the homologous reaction and the denaturation temperature of the heterologous reaction is determined should be performed. This proposal has been endorsed by the members of the Subcommittee for Taxonomy of the Mycobacteria of the International Committee on Systematic Bacteriology.     

Mycobacterium paraterrae sp. nov. recovered from a clinical specimen: novel chromogenic slow growing mycobacteria related to Mycobacterium terrae complex

A previously unidentified, slowly growing scotochromogenic Mycobacterium was isolated from a Korean patient with symptomatic pulmonary infection. Phenotypically, this strain was generally similar to Mycobacterium terrae complex strains, however it uniquely produced orange pigmentation. Unique mycolic acid profiles and phylogenetic analyses based on three alternative chronometer molecules, 16S rRNA gene, hsp65 and rpoB , confirmed the taxonomic status of this strain as a novel species. These results support that this strain represents a novel Mycobacterium species. The name Mycobacterium paraterrae sp. nov. is proposed. The type strain is 05-2522 (= DSM 45127 = KCTC 19556).     

Classification of Mycobacterium farcinogenes and Mycobacterium senegalense by immunodiffusion and thin-layer chromatography of long-chain components

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.     

Mycobacterium confluentis sp. nov.

A new rapidly growing mycobacterium was isolated from human sputum. This organism grew at 22, 31, 37, and 41 degrees C and possessed catalase, acid phosphatase, acetamidase, urease, nicotinamidase, pyrazinamidase, and nitrate reductase activities. It did not produce nicotinic acid, hydrolyze Tween, or have benzamidase, isonicotinamidase, succinidamidase, and arylsulfatase activities. A mycolic acid analysis revealed a simple, unique pattern. The organism is susceptible to antituberculotic drugs. A comparative 16S rRNA sequence analysis placed this organism within the confines of the genus Mycobacterium, most closely related to the thermotolerant rapidly growing species. On the basis of the pattern of enzymatic activities and metabolic properties, as well as the unique 16S rRNA sequence, we propose that our single strain represents a new species, for which we propose the name Mycobacterium confluentis. The type strain is strain 1389/90; a culture of this strain has been deposited in the German Collection of Microorganisms and Cell Cultures as strain DSM 44017.     

Vibrio navarrensis sp. nov., a species from sewage.

A group of 11 strains, mostly isolated from sewage water in the Province of Navarra, Spain, were found to constitute a DNA relatedness group which is 2 to 39% related to 23 species of the genus Vibrio and 2 to 3% related to two Aeromonas species. Phenotypically, these strains have all of the properties that define the genus Vibrio. However, they differ from the previously described species by three or more properties. The strains are negative for arginine, ornithine, and lysine decarboxylase activities and the Voges-Proskauer test and are unable to utilize putrescine, gluconate, glucuronate, and histidine. They utilize and produce acid from sucrose and grow at 40 degrees C. All strains grow in the presence of 0.5% (wt/vol) NaCl, and seven strains grow weakly in peptone water lacking NaCl. The group of strains which we studied can also be differentiated from other Vibrio species by fatty acid content. The G+C ratio of the DNA is 45 to 47 mol%. The name Vibrio navarrensis sp. nov. is proposed for these strains; strain 1397-6 (= CIP 103381) is the type strain.     

Three new species of the genus Peptostreptococcus isolated from humans: Peptostreptococcus vaginalis sp. nov., Peptostreptococcus lacrimalis sp. nov., and Peptostreptococcus lactolyticus sp. nov.

We describe three new species of the genus Peptostreptococcus which were isolated from human specimens and were tentatively identified as Peptostreptococcus prevotii. These three organisms were not homologous with previously described type strains of the genus Peptostreptococcus. A total of 12 strains that were identified biochemically as P. prevotii were divided into five independent DNA similarity groups; 10 of these strains were divided into three similarity groups which exhibited significant phenotypic differences from previously described species. Therefore, we propose the following new species: Peptostreptococcus vaginalis for group 1 strains, Peptostreptococcus lacrimalis for group 2 strains, and Peptostreptococcus lactolyticus for group 3 strains. The type strain of P. vaginalis is strain GIFU 12669 (= JCM 8138), the type strain of P. lacrimalis is strain GIFU 7667 (= JCM 8139), and the type strain of P. lactolyticus is strain GIFU 8586 (= JCM 8140).     

Fatty acid composition and mycolic acid pattern of some chromogenic mycobacteria

Twenty-nine strains of chromogenic mycobacteria belonging to the species Mycobacterium aurum (5 strains), M. duvalii (2), M. flavescens (1), M. gordonae (6), M. kansasii (3), M. obuense (1), M. parafortuitum (3), M. phlei (2), M. rhodesiae (1), M. vaccae (2) and Mycobacterium spp. (3) were studied for fatty acid composition and mycolic acid patterns by gas-liquid chromatography and thin-layer chromatography respectively. Fatty acids found ranged from those with 12-24 carbon atoms and were saturated and monounsaturated straight chain fatty acids, along with 10-methyl branched of 16, 17 and 18 (tuberculostearic acid) carbon atoms. Moreover, 2-methyl tetradecanoic acid was found in M. gordonae, M. kansasii and Mycobacterium spp. (2 strains), and 2,4-dimethyl tetradecanoic acid in M. kansasii and Mycobacterium spp. (2 strains). Nonadecenoic acid was found only in M. flavescens and tuberculostearic acid was not detected in M. gordonae. Three patterns of mycolic acids were obtained: the first, found in M. aurum, M. flavescens, M. phlei, M. rhodesiae and Mycobacterium spp. (1 strain), was characterized by the presence of several spots assigned to alpha-mycolates, keto-mycolates and wax-ester mycolates (omega-carboxy-mycolates and 2-eicosanol and related alcohols); the second, found in M. duvalii, M. obuense, M. parafortuitum and M. vaccae was similar to the first, but it contained an additional spot of alpha'-mycolates; the third pattern, found in M. gordonae, M. kansasii and Mycobacterium spp. (2 strains) contained three spots considered to be alpha-mycolates, methoxy-mycolates and keto-mycolates. The results obtained confirm previously reported data on the fatty and mycolic acid composition of the species studied.     

A phylogeny of the genus Nocardia deduced from the analysis of small-subunit ribosomal DNA sequences, including transfer of Nocardia amarae to the genus Gordona as Gordona amarae comb. nov.

Abstract According to phylogenetic analyses of nearly complete small-subunit ribosomal DNA sequences, the genus Nocardia should not comprise the two species Nocardia petroleophila and Nocardia amarae. N. amarae should be reassigned to the genus Gordona as Gordona amarae . All of the other Nocardia species form a monophyletic unit, closely related to species of the genus Rhodococcus . It is proposed to revive the name 'CMN' to comprise the genera Corynebacterium, Tsukamurella, Mycobacterium, Gordona, Rhodococcus and Nocardia that form a well identified and monophyletic unit. They are all characterized by a cell wall chemotype IV with mycolic acids.     

Gaschromatography of Constitutive Fatty Acids in Mycobacterium leprae

A constitutive saturated and monounsaturated fatty acid pattern of Mycobacterium leprae, isolated from the liver of a nine-banded armadillo with experimental leprosy, was analyzed gaschromatographically and compared with that of cultured M. lepraemurium, M. avium, M. bovis, strain BCG and M. smegmatis. In comparing the fatty acid pattern thus obtained and the known structure of mycolic acids in these mycobacteria, an experiential rule that each species of mycobacteria has a relatively high content of normal (straight-chained) saturated fatty acid having two more carbons than those of the α-branch in this species' mycolic acids, coincided well for all mycobacteria tested. In particular, M. leprae was found to contain a relatively high content of behenic acid (n-C_22:0) and the carbon-number of the α-branch in this species' mycolic acids is 20 as we previously reported. These data suggested the possibility of simple detection of M. leprae by gaschromatography, and results sustaining this possibility were obtained.     

Tsukamurella spumae sp. nov., a novel actinomycete associated with foaming in activated sludge plants

A polyphasic taxonomic study was undertaken to establish the taxonomic position of six representative strains isolated from activated sewage sludge foam. The organisms were found to have chemical and morphological properties consistent with their assignment to the genus Tsukamurella. DNA:DNA relatedness studies showed that five out of the six isolates formed a distinct genomic species, the remaining strain was most closely associated with this taxon. The five isolates had a unique phenotypic profile that served to distinguish them from representatives of the validly described species of Tsukamurella. The combination of the genotypic and phenotypic data indicated that the five strains should be classified as a new species in the genus Tsukamurella. The name proposed for this taxon is Tsukamurella spumae, the type strain is N1171T (= DSM 44.113T = NCIMB 13947T). It was also shown that some of the reference strains were misclassified as Tsukamurella paurometabola.     

The role played the lipid fractions of Mycobacterium bovis BCG in the development of antituberculosis immunity in an animal experiment.

The authors investigated the antituberculosis and antitumour immunogenicity as well as tuberculin allergenicity of the lipid fractions from Mycobacterium bovis BCG strains of Danish, French, Japanese origin and of Czechoslovak 725. The fractions explored included phospholipids, Cord factor, ethanol-extractable lipids, waxes A, B, C + D and fats. The fractions were divided into three groups according to their effectiveness. 1. The Cord factor and phospholipids from all the studied strains were effective in the antituberculosis and antitumour models with the only exception of strain 725 phospholipids. Phospholipids from all strains were capable of inducing tuberculin allergy. 2. In the second group (waxes A, C + D and lipids extractable by ethanol) a variance was observed in the antigenic properties of identical fractions from different strains suggesting differing metabolism in the strains producing these fractions. A mixture of waxes C + D from the French and Danish strains showed a degree of suppression in its antituberculosis effectiveness. 3. Waxes B and fats were entirely ineffective in the antitumour model and, with the exception of waxes B from strain 725 and fats from the Japanese strain, in the antituberculosis model. The antituberculosis and antitumour effectiveness directly depended on the content of a mycolic acid complex in fractions. Tuberculin allergenicity was associated with the intensity of phospholipid production by mycobacteria.     

Lipoarabinomannan of Mycobacterium tuberculosis. Capping with mannosyl residues in some strains.

Previously we had demonstrated that the termini of the arabinan component of mycobacterial cell wall arabinogalactan, the site of mycolic acid location, consists mostly of clusters of a pentaarabinofuranoside, [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2----(3 and 5)-alpha-D-Araf. Subsequently, the same arrangement was shown to dominate the non-reducing ends of lipoarabinomannan (LAM), a key component in the interaction of mycobacteria with host cell. Accordingly, we had proposed that mycobacteria universally elaborate the same Araf-containing motifs in two settings for different pathophysiological purposes. However, we now report that the termini of LAM from the virulent, Erdman, strain of Mycobacterium tuberculosis, unlike those from the attenuated H37Ra strain, are extensively capped with mannosyl (Manp) residues, either a single alpha-D-Manp, a dimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp), or a trimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp ). The use of monoclonal antibodies demonstrates a clear difference in the antigenicity of the basic and mannose-capped LAM. The possibility that these structures are a factor in the virulence of some strains of M. tuberculosis and represent an example of carbohydrate mimicry in mycobacterial infections is discussed.     

Fatty acid composition and mycolic acid pattern of some chromogenic mycobacteria

Twenty-nine strains of chromogenic mycobacteria belonging to the species Myco-bacterium aurum (5 strains), M. duvalii (2), M. flavescens (1), M. gordonae (6), M. kansasii (3), M. obuense (1), M. parafortuitum (3), M. phlei (2), M. rhodesiae (1), M. vaccae (2) and Mycobacterium spp. (3) were studied for fatty acid composition and mycolic acid patterns by gas-liquid chromatography and thin-layer chromatog-raphy respectively. Fatty acids found ranged from those with 12–24 carbon atoms and were saturated and monounsaturated straight chain fatty acids, along with 10-methyl branched of 16, 17 and 18 (tuberculostearic acid) carbon atoms. Moreover, 2-methyl tetradecanoic acid was found in M. gordonae, M. kansasii and Mycobacterium spp. (2 strains), and 2,4-dimethyl tetradecanoic acid in M. kansasii and Mycobacterium spp. (2 strains). Nonadecenoic acid was found only in M. flavescens and tuberculostearic acid was not detected in M. gordonae . Three patterns of mycolic acids were obtained: the first, found in M. aurum, M. flavescens, M. phlei, M. rhodesiae and Mycobacterium spp. (1 strain), was characterized by the presence of several spots assigned to α-mycolates, keto-mycolates and wax-ester mycolates (ω-carboxy-rnycolates and 2-eicosanol and related alcohols); the second, found in M. duvalii, M. obuense, M. parafortuitum and M. vaccae was similar to the first, but it contained an additional spot of α-mycolates; the third pattern, found in M. gordonae, M. kansasii and Mycohacterium spp. (2 strains) contained three spots considered to be α-mycoiates, methoxy-mycolates and keto-mycolates. The results obtained confirm previously reported data on the fatty and mycolic acid composition of the species studied.     

Nucleic Acid Homology in the Genus Mycobacterium

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.     

Mechanism of mycobacteriocin and Tween 80 mediated antimycobacterial activity

Rapidly growing mycobacteria (14 species, 67 strains) showed a highly significant correlation between their susceptibility to smegmatocin (a Tween-hydrolysing esterase of Mycobacterium smegmatis exhibiting antimicrobial activity in the presence of Tween 80) and sensitivity to oleic acid (a hydrolysis product of Tween 80). Polyoxyethylenesorbitan ether, the other product of Tween 80 hydrolysis, also showed some toxic effect on smegmatocin-sensitive mycobacteria. From Mycobacterium diernhoferi ATCC 19340, a representative smegmatocin-sensitive strain, mutant strains with smegmatocin-resistant phenotypes were isolated. These mutants were more resistant to oleic acid than the parent strain, although their oleic acid resistance was somewhat lower than their smegmatocin resistance.