Mycolic Acid Patterns of Representative Strains of Mycobacterium fortuitum, "Mycobacterium peregrinum" and Mycobacterium smegmatis

Minnikin, David E.; Minnikin, S. M.; Hutchinson, I; Goodfellow, Michael; Grange, John M.

doi:10.1099/00221287-130-2-363

Cited by 32 publications

(26 citation statements)

References 17 publications

(8 reference statements)

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“…In separate experiments, the filters with cells were immersed in 2 ml of benzene-methanol-20% methanolic tetramethylammonium hydroxide (2:1:1, vol/vol/vol) and left overnight at 37°C, in order to disrupt the structure of mycobacterial cell envelope by alkaline methanolysis (28). The supernatant was collected, and the residue on the filter was washed three times in 2 ml of ethyl acetate.…”

Section: Methodsmentioning

confidence: 99%

Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

Miyata

Iwahori

Foght

et al. 2004

Appl Environ Microbiol

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The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbondegrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-14 C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state 14 C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K t ) of 26 ؎ 3 nM (mean ؎ standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated 14 C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K d ) of 41 ؎ 21 nM (mean ؎ standard deviation). Given the low values of K t and K d , Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.Many polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, phenanthrene, fluoranthene, anthracene, pyrene, benz [a]anthracene, and benzo [a]pyrene are known to be utilized as sole carbon and energy sources, or are degraded cometabolically, by a diverse group of bacteria (11,22). Biodegradation is a significant tool for the removal of these contaminants from environments, but their low bioavailability, especially for high-molecular-weight PAHs, has often been observed in the environment and bioremediation systems (2,22). Due to their extremely hydrophobic nature, PAHs partition readily into solid and liquid organic phases and exist in aqueous phase only at limited concentrations. This makes uptake of PAHs by bacterial cells difficult. In bacterial cultures where only the compounds dissolved in aqueous solution are utilized, mass transfer or the rate of desorption controls the rates of degradation and culture growth (2).Nevertheless, several studies have reported that degradation kinetics for the PAHs vary among different bacteria and that some bacteria apparently show rapid degradation of such PAHs with concomitant growth (16,18,37). Thus, some bacteria seem to have adapted to the low availa...

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Section: Methodsmentioning

confidence: 99%

Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

Miyata

Iwahori

Foght

et al. 2004

Appl Environ Microbiol

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“…These functional groups are either nonpolar moieties, such as olefins, methyl branches, and cyclopropanes, or polar moieties, such as ketones, methoxy groups, epoxides, and esters. The distribution of functional groups among species is distinct and useful for taxonomic identification of species (9,10). In M. tuberculosis, three distinct mycolate species are produced, ␣ mycolates containing two cis-cyclopropane rings and a methoxy and keto series, each of which contains a cis-or trans-cyclopropane in the proximal position, in addition to the distal oxygen function with an adjacent methyl branch (8,11).…”

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confidence: 99%

“…Ketomycolates occur in a variety of mycobacterial species (8), while methoxymycolates have only been isolated from M. tuberculosis (11) (10,15). These studies were undertaken to explore the biosynthetic pathway and function of methoxymycolates in M. tuberculosis.…”

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confidence: 99%

A common mechanism for the biosynthesis of methoxy and cyclopropyl mycolic acids in Mycobacterium tuberculosis

Yuan

Barry

1996

Proc. Natl. Acad. Sci. U.S.A.

131

117

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Mycobacterium tuberculosis produces three classes of mycolic acids that differ primarily in the presence and nature of oxygen-containing substituents in the distal portion of the meromycolate branch. The methoxymycolate series has a methoxy group adjacent to a methyl branch, in addition to a cyclopropane in the proximal position. Using the gene for the enzyme that introduces the distal cyclopropane (cma1) as a probe, we have cloned and sequenced a cluster of genes coding for four highly homologous methyl transferases (mma1-4). When introduced into Mycobacterium smegmatis, this gene cluster conferred the ability to synthesize methoxymycolates. By determining the structure of the mycolic acids produced following expression of each of these genes individually and in combination, we have elucidated the biosynthetic steps responsible for the production of the major series of methoxymycolates. The mma4 gene product (MMAS-4) catalyzes an unusual S-adenosyl-L-methionine-dependent transformation of the distal cis-olefin into a secondary alcohol with an adjacent methyl branch. MMAS-3 O-methylates this secondary alcohol to form the corresponding methyl ether, and MMAS-2 introduces a cis-cyclopropane in the proximal position of the methoxy series. The similarity of these reactions and the enzymes that catalyze them suggests that some of the structural diversity of mycolic acids results from different chemical fates of a common cationic intermediate, which in turn results from methyl group addition to an olefinic mycolate precursor.Mycobacterium tuberculosis is directly responsible for more human deaths than any other single infectious agent (1). The intrinsic resistance of this bacterium to many standard chemotherapeutic agents severely limits options for treatment (2). Isoniazid, which targets cell wall biosynthesis, is one of the most effective therapies available, yet it requires prolonged treatment regimens, lowering patient compliance and fostering drug resistance (3). The mycobacterial cell wall is the primary permeability barrier responsible for the intrinsic resistance to many hydrophobic antibiotic substances (4, 5). Understanding the biosynthesis, structure, and function of the mycobacterial cell wall is important, both from the perspective of providing novel drug targets and designing strategies for circumventing intrinsic resistance.The cell wall contains a very high proportion of lipid in the form of mycolic acid, whose physical organization has recently been explored in detail (6, 7). Mycolic acids are long-chain, two-branched, 3-hydroxy fatty acids that range in size in mycobacteria from Ϸ60 to 80 carbons and contain a variety of functional groups on the long (mero) chain (8). These functional groups are either nonpolar moieties, such as olefins, methyl branches, and cyclopropanes, or polar moieties, such as ketones, methoxy groups, epoxides, and esters. The distribution of functional groups among species is distinct and useful for taxonomic identification of species (9, 10). In M. tuberculosis, three...

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“…Differentiation at the subspecific level is more difficult. Electrophoresis of cell proteins (12), immunodiffusion and immunoelectrophoresis of soluble antigens (5,21,27), bacteriocin typing (28), in vitro antibiotic susceptibility tests (4, 18, 32), the lipid patterns obtained by thin-layer chromatography (15), and expanded mycolic acid analyses (7, 8,19,23,24) The purpose of the present work was to evaluate the validity of subdivisions in the M . fortuitum-M. chelonae complex by using precise DNA relatedness methods.…”

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confidence: 99%

Deoxyribonucleic Acid Relatedness Study of the Mycobacterium fortuitum-Mycobacterium chelonae Complex

Lévy-Frébault¹,

Grimont

et al. 1986

International Journal of Systematic Bacteriology

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A deoxyribonucleic acid relatedness study of 13 Mycobacterium chelonae strains indicated that M . chelonae subsp. cheZonae and M. cheZonae subsp. abscessus are two different genomic species, both distinct from Mycobacterium fortuitum and "Mycobacterium peregrinum." One strain of M . chelonae constitutes a third genomic species. The type strain of M. fortuitum and the reference strain of "M. peregrinum'' (strain ATCC 14467) belong to two different genomic groups. (17) demonstrated that there are two distinct phena, which correspond to M . chelonae and M . fortuitum, and the following subspecies were recognized: M . chelonae subsp. chelonae (type strain, strain NCTC 946) and M . chelonae subsp. abscessus (type strain, strain ATCC 19977). In a previous deoxyribonucleic acid (DNA) relatedness study in which a spectrophotometric method was used (l), Baess claimed that M . chelonae subsp. chelonae ATCC 19235 (=SN281S) (the proposed reference strain of " M . borstelense") and M . chelonae subsp. abscessus ATCC 19977T (T = type strain) belong to the same DNA relatedness group. In the same study, little relatedness was found between M . chelonae and M . fortuitum ATCC 6841T or "M. peregrinum" reference strain ATCC 14467 (1). The results of additional phenotypic studies in which newly proposed tests were used indicated that M . chelonae and M . fortuitum could be unambiguously differentiated (4,19,26). Differentiation at the subspecific level is more difficult. Electrophoresis of cell proteins (12), immunodiffusion and immunoelectrophoresis of soluble antigens (5,21,27), bacteriocin typing (28), in vitro antibiotic susceptibility tests (4, 18, 32), the lipid patterns obtained by thin-layer chromatography (15), and expanded mycolic acid analyses (7, 8,19,23,24) The purpose of the present work was to evaluate the validity of subdivisions in the M . fortuitum-M. chelonae complex by using precise DNA relatedness methods. Strains of the MATERIALS AND METHODSBacterial strains. The strains selected for this study are listed in Table 1. The strains were identified by using previously described methods (19), and identification at the subspecific level was performed as recommended by the Centers for Disease Control, Atlanta, Ga. (26).Extraction and purification of DNA. DNA was extracted as described previously (20). Briefly, the bacteria were converted to spheroplasts and subsequently lysed by adding sodium dodecyl sulfate and pronase. DNA was then purified by using the method of Brenner (3).DNA-DNA hybridization. DNAs from M . chelonae NCTC 946* and CIPT 810192 and M . fortuitum ATCC 6841T and ATCC 14467 were labeled in vitro by nick translation (ll), with minimal adaptations (20). The S1 nuclease method (6) was used by following the S1 nuclease-trichloroacetic acid procedure (ll), except that all volumes were reduced by one-half and the hybridization temperature was 75°C. The temperature at which 50% of the reassociated DNA became hydrolyzable by S1 nuclease (T,) was determined by using the method of Crosa et al. (6), modified ...

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Mycolic Acid Patterns of Representative Strains of Mycobacterium fortuitum, "Mycobacterium peregrinum" and Mycobacterium smegmatis

Cited by 32 publications

References 17 publications

Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

A common mechanism for the biosynthesis of methoxy and cyclopropyl mycolic acids in Mycobacterium tuberculosis

Deoxyribonucleic Acid Relatedness Study of the Mycobacterium fortuitum-Mycobacterium chelonae Complex

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