Mycocerosic Acids Research Articles

Lipid biomarkerek HPLC-HRMS vizsgálata történeti embertani minták tbc diagnosztikája során

Tuberculosis (TB) is not only an infectious disease but one of the top 10 causes of death, spreading mainly with aerosol transmission and accompanying the history of humankind for several millennia. TB is caused in humans and animals by members of the Mycobacterium tuberculosis complex. For better understanding of the disease and the evolutional background of its causative agent, involvement of palaeopathological investigations is surpassingly important. Traditionally, palaeopathology is using a broad variety of markers, which are observable by macroscopic investigations. These markers are mainly related to different extra-pulmonary forms of TB. However, these manifestations develop only in a few cases. Moreover, many markers are not pathognomonic of TB. Therefore, to avoid underestimation of TB prevalence in paleopathological studies, the diagnosis on archaeological material requires a multidisciplinary approach. For better estimation of TB incidence in past populations, an array of specific biomarkers can be brought into play to confirm initial, macromorphology-based skeletal diagnoses, namely aDNA and lipid biomarkers. The three lipid biomarker groups, which are mainly involved in such investigations are the mycolic acids (MAs), the mycocerosic acids (MCs) and the C27 mycolipenic acid (ML). These unique lipids can be located in the lipid-rich mycobacterial cell wall. The application of lipid-based TB diagnostic approach has been proved to be robust and reliable through many examples. A variety of analytical methods have been employed for mycobacterial lipid biomarker profiling. Fluorescence HPLC is well-developed for the analysis of mycolic acids and phthiocerols. Furthermore, one isolated case of direct mass spectrometric detection of M. tuberculosis free mycolic acids has been also introduced. Our aim was to establish a lipid-biomarker-based HPLC-MS method for TB diagnosis in historical human samples, as this instrumentation is available in many laboratories, has the potential of a quick and sensitive and at the same time an affordable measurement protocol. We successfully developed and optimised a method, which is capable to separate and detect MAs and MCs. Moreover, we optimised the sample pre-treatment process. We started to build an MA and MC lipid profile library, which can serve as a comparison to diagnose TB. For the verification of new methods and the estimation of reliability of new markers in macroscopic analysis, palaeopathologists usually use well-documented collections of skeletons and mummies from the pre-antibiotic era. For this purpose, we chose to test our method on the Vác Mummy Collection. In the case of four out of six mummified individuals, MC profiles of characteristic M. tuberculosis MCs were recorded. The HPLC-ESI-MS method, developed for the detection of MCs, opens a new avenue for the detection of ancient mycobacterial disease, encompassing both tuberculosis, leprosy and joint cases.

Novel hydrocarbon-utilizing soil mycobacteria synthesize unique mycocerosic acids at a Sicilian everlasting fire

Abstract. Soil bacteria rank among the most diverse groups of organisms on Earth and actively impact global processes of carbon cycling, especially in the emission of greenhouse gases like methane, CO2 and higher gaseous hydrocarbons. An abundant group of soil bacteria are the mycobacteria, which colonize various terrestrial, marine and anthropogenic environments due to their impermeable cell envelope that contains remarkable lipids. These bacteria have been found to be highly abundant at petroleum and gas seep areas, where they might utilize the released hydrocarbons. However, the function and the lipid biomarker inventory of these soil mycobacteria are poorly studied. Here, soils from the Fuoco di Censo seep, an everlasting fire (gas seep) in Sicily, Italy, were investigated for the presence of mycobacteria via 16S rRNA gene sequencing and fatty acid profiling. The soils contained high relative abundances (up to 34 % of reads assigned) of mycobacteria, phylogenetically close to the Mycobacterium simiae complex and more distant from the well-studied M. tuberculosis and hydrocarbon-utilizing M. paraffinicum. The soils showed decreasing abundances of mycocerosic acids (MAs), fatty acids unique for mycobacteria, with increasing distance from the seep. The major MAs at this seep were tentatively identified as 2,4,6,8-tetramethyl tetracosanoic acid and 2,4,6,8,10-pentamethyl hexacosanoic acid. Unusual MAs with mid-chain methyl branches at positions C-12 and C-16 (i.e., 2,12-dimethyl eicosanoic acid and 2,4,6,8,16-pentamethyl tetracosanoic acid) were also present. The molecular structures of the Fuoco di Censo MAs are different from those of the well-studied mycobacteria like M. tuberculosis or M. bovis and have relatively δ13C-depleted values (−38 ‰ to −48 ‰), suggesting a direct or indirect utilization of the released seep gases like methane or ethane. The structurally unique MAs in combination with their depleted δ13C values identified at the Fuoco di Censo seep offer a new tool to study the role of soil mycobacteria as hydrocarbon gas consumers in the carbon cycle.

Verification of tuberculosis infection among Vác mummies (18th century CE, Hungary) based on lipid biomarker profiling with a new HPLC-HESI-MS approach

Tuberculosis (TB) was a large burden of infections that peaked during the 19th century in Europe. Mummies from the 18th century CE, discovered in the crypt of a church at Vác, Hungary, had high TB prevalence, as revealed by amplification of key fragments of TB DNA and genome-wide TB analysis. Complementary methods are needed to confirm these diagnoses and one approach uses the identification of specific lipid biomarkers, such as TB mycocerosic acids (MCs). Previously, MC derivatives were profiled by specialised gas chromatography-mass spectrometry (GC-MS), so an alternative more direct approach has been developed. Underivatized MCs are extracted and analysed by high-performance liquid chromatography linked to a mass spectrometer, in heated electrospray ionisation mode (HPLC-HESI-MS). The method was validated using representatives of the Mycobacterium tuberculosis complex and other mycobacteria and tested on six Vác mummy cases, previously considered positive for TB infection. Analysing both rib and soft tissue samples, four out of six cases gave profiles of main C32 and major C29 and C39 mycocerosates correlating well with those of M. tuberculosis. Multidisciplinary methods are needed in the diagnosis of ancient tuberculosis; this new protocol accesses important confirmatory evidence, as demonstrated by the confirmation of TB in the Vác mummies.

Characterization of phthiocerol and phthiodiolone dimycocerosate esters of M. tuberculosis by multiple-stage linear ion-trap MS

Both phthiocerol/phthiodiolone dimycocerosate (PDIM) and phenolic glycolipids are abundant virulent lipids in the cell wall of various pathogenic mycobacteria, which can synthesize a wide range of complex high-molecular-mass lipids. In this article, we describe linear ion-trap MS(n) mass spectrometric approach for structural study of PDIMs, which were desorbed as the [M + Li](+) and [M + NH(4)](+) ions by ESI. We also applied charge-switch strategy to convert the mycocerosic acid substituents to their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives and analyzed them as M (+) ions, following alkaline hydrolysis of the PDIM to release mycocerosic acids. The structural information from MS(n) on the [M + Li](+) and [M + NH(4)](+) molecular species and on the M (+) ions of the mycocerosic acid-AMPP derivative affords realization of the complex structures of PDIMs in Mycobacterium tuberculosis biofilm, differentiation of phthiocerol and phthiodiolone lipid families and complete structure identification, including the phthiocerol and phthiodiolone backbones, and the mycocerosic acid substituents, including the locations of their multiple methyl side chains, can be achieved.

Morphological and biomolecular evidence for tuberculosis in 8th century AD skeletons from Bélmegyer-Csömöki domb, Hungary

Macromorphological analysis of skeletons, from 20 selected graves of the 8th century AD Bélmegyer-Csömöki domb, revealed 19 cases of possible skeletal tuberculosis. Biomolecular analyses provided general support for such diagnoses, including the individual without pathology, but the data did not show coherent consistency over the range of biomarkers examined. Amplification of ancient DNA fragments found evidence for the Mycobacterium tuberculosis complex DNA only in five graves. In contrast, varying degrees of lipid biomarker presence were recorded in all except two of the skeletons, though most lipid components appeared to be somewhat degraded. Mycobacterial mycolic acid biomarkers were absent in five cases, but the weak, possibly degraded profiles for the remainder were smaller and inconclusive for either tuberculosis or leprosy. The most positive lipid biomarker evidence for tuberculosis was provided by mycolipenic acid, with 13 clear cases, supported by five distinct possible cases. Combinations of mycocerosic acids were present in all but three graves, but in one case a tuberculosis-leprosy co-infection was indicated. In two specimens with pathology, no lipid biomarker evidence was recorded, but one of these specimens provided M. tuberculosis complex DNA fragments.

Inactivation of tesA Reduces Cell Wall Lipid Production and Increases Drug Susceptibility in Mycobacteria

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.

A Lipid Profile Typifies the Beijing Strains of Mycobacterium tuberculosis: IDENTIFICATION OF A MUTATION RESPONSIBLE FOR A MODIFICATION OF THE STRUCTURES OF PHTHIOCEROL DIMYCOCEROSATES AND PHENOLIC GLYCOLIPIDS

The Mycobacterium tuberculosis Beijing strains are a family highly prevalent in Asia and have recently spread worldwide, causing a number of epidemics, suggesting that they express virulence factors not found in other M. tuberculosis strains. Accordingly, we looked for putative characteristic compounds by comparing the lipid profiles of several Beijing and non-Beijing strains. All the Beijing strains analyzed were found to synthesize structural variants of two well known characteristic lipids of the tubercle bacillus, namely phthiocerol dimycocerosates (DIM) and eventually phenolglycolipids (PGL). These variants were not found in non-Beijing M. tuberculosis isolates. Structural elucidation of these variants showed that they consist of phthiotriol and glycosylated phenolphthiotriol dimycocerosates, eventually acylated with 1 mol of palmitic acid, in addition to the conventional acylation of the beta-diol by mycocerosic acids. We demonstrated that this unusual lipid profile resulted from a single point mutation in the Rv2952 gene, which encodes the S-adenosylmethionine-dependent methyltransferase participating to the O-methylation of the third hydroxyl of the phthiotriol and phenolphthiotriol in the biosynthetic pathway of DIM and PGL. Consistently, the mutated enzyme exhibited in vitro a much lower O-methyltransferase activity than did the wild-type Rv2952. We finally demonstrated that the structural variants of DIM and PGL fulfill the same function in the cell envelope and virulence than their conventional counterparts.

Mycocerosic acid biomarkers for the diagnosis of tuberculosis in the Coimbra Skeletal Collection

Tuberculosis has been a scourge of humans over many millennia, but questions remain regarding its evolution and epidemiology. Fossil biomarkers, such as DNA and long-chain mycolic acids, can be detected in ancient skeletal and other materials. The phthiocerol dimycocerosate waxes are also robust biomarkers for tuberculosis and sensitive methods are available for the detection of their mycocerosic acid components. The presence of mycocerosic acids was investigated in 49 individuals from the 1837-1936 Coimbra Identified Skeletal Collection (Portugal), half with documentary data indicating tuberculosis as a cause of death. Samples were hydrolysed, acidic components converted to pentafluorobenzyl esters, the non-hydroxylated long-chain esters isolated, and this fraction separated into multimethyl-branched and other esters by normal phase high performance liquid chromatography. Negative ion chemical ionisation gas chromatography mass spectrometry was used to detect diagnostic C29, C30 and C32 mycocerosic acids. Mycocerosic acids were detected in archaeological material for the first time, illustrating that they are valuable biomarkers for the diagnosis of ancient tuberculosis. A 72% correlation with the Coimbra burial record supported TB as the major cause of death. In addition, 30% of the skeletons, positive for mycocerosates, showed the presence of related long-chain mycolipenic acids.

A Novel Interaction Linking the FAS-II and Phthiocerol Dimycocerosate (PDIM) Biosynthetic Pathways

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.

Structure-based inhibitor design of AccD5, an essential acyl-CoA carboxylase carboxyltransferase domain of Mycobacterium tuberculosis

Mycolic acids and multimethyl-branched fatty acids are found uniquely in the cell envelope of pathogenic mycobacteria. These unusually long fatty acids are essential for the survival, virulence, and antibiotic resistance of Mycobacterium tuberculosis. Acyl-CoA carboxylases (ACCases) commit acyl-CoAs to the biosynthesis of these unique fatty acids. Unlike other organisms such as Escherichia coli or humans that have only one or two ACCases, M. tuberculosis contains six ACCase carboxyltransferase domains, AccD1-6, whose specific roles in the pathogen are not well defined. Previous studies indicate that AccD4, AccD5, and AccD6 are important for cell envelope lipid biosynthesis and that its disruption leads to pathogen death. We have determined the 2.9-Angstroms crystal structure of AccD5, whose sequence, structure, and active site are highly conserved with respect to the carboxyltransferase domain of the Streptomyces coelicolor propionyl-CoA carboxylase. Contrary to the previous proposal that AccD4-5 accept long-chain acyl-CoAs as their substrates, both crystal structure and kinetic assay indicate that AccD5 prefers propionyl-CoA as its substrate and produces methylmalonyl-CoA, the substrate for the biosyntheses of multimethyl-branched fatty acids such as mycocerosic, phthioceranic, hydroxyphthioceranic, mycosanoic, and mycolipenic acids. Extensive in silico screening of National Cancer Institute compounds and the University of California, Irvine, ChemDB database resulted in the identification of one inhibitor with a K(i) of 13.1 microM. Our results pave the way toward understanding the biological roles of key ACCases that commit acyl-CoAs to the biosynthesis of cell envelope fatty acids, in addition to providing a target for structure-based development of antituberculosis therapeutics.

Biochemical and Structural Characterization of an Essential Acyl Coenzyme A Carboxylase from Mycobacterium tuberculosis

Pathogenic mycobacteria contain a variety of unique fatty acids that have methyl branches at an even-numbered position at the carboxyl end and a long n-aliphatic chain. One such group of acids, called mycocerosic acids, is found uniquely in the cell wall of pathogenic mycobacteria, and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl coenzyme A (CoA), represents an attractive target for developing new antituberculous drugs. Heterologous protein expression and purification of the individual subunits allowed the successful reconstitution of an essential acyl-CoA carboxylase from Mycobacterium tuberculosis, whose main role appears to be the synthesis of methylmalonyl-CoA. The enzyme complex was reconstituted from the alpha biotinylated subunit AccA3, the carboxyltransferase beta subunit AccD5, and the epsilon subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant fivefold higher), indicating that the main physiological role of this enzyme complex is to generate methylmalonyl-CoA for the biosynthesis of branched-chain fatty acids. The alpha and beta subunits are capable of forming a stable alpha6-beta6 subcomplex but with very low specific activity. The addition of the epsilon subunit, which binds tightly to the alpha-beta subcomplex, is essential for gaining maximal enzyme activity.

Virulence attenuation of two Mas-like polyketide synthase mutants of Mycobacterium tuberculosis.

The cell envelope of pathogenic mycobacteria is highly distinctive in that it contains a large number of structurally related very long multiple methyl-branched fatty acids. These complex molecules are thought to play important roles in cell envelope organization and virulence. The genetic and enzymic characterization of the polyketide synthase Mas, which is responsible for the synthesis of one such family of fatty acids (the mycocerosic acids), paved the way towards the identification of other enzymes involved in the synthesis of methyl-branched fatty acids in M. tuberculosis. In an effort to elucidate the origin of these complex fatty acids and their possible involvement in pathogenesis, the two mas-like polyketide genes pks5 and pks7 were disrupted in M. tuberculosis and the effects of their inactivation on fatty acid composition and virulence were analysed. While the disruption of pks7 resulted in a mutant deficient in the production of phthiocerol dimycocerosates, the cell envelope composition of the pks5 mutant was found to be identical to that of the wild-type parental strain M. tuberculosis H37Rv. Interestingly, both the pks5 and pks7 mutants displayed severe growth defects in mice.

The largest open reading frame (pks12) in the Mycobacterium tuberculosis genome is involved in pathogenesis and dimycocerosyl phthiocerol synthesis.

The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-(14)C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

Attenuation of Mycobacterium tuberculosis by disruption of a mas-like gene or a chalcone synthase-like gene, which causes deficiency in dimycocerosyl phthiocerol synthesis.

Tuberculosis is one of the leading preventable causes of death. Emergence of drug-resistant tuberculosis makes the discovery of new targets for antimycobacterial drugs critical. The unique mycobacterial cell wall lipids are known to play an important role in pathogenesis, and therefore the genes responsible for their biosynthesis offer potential new targets. To assess the possible role of some of the genes potentially involved in cell wall lipid synthesis, we disrupted a mas-like gene, msl7, and a chalcone synthase-like gene, pks10, with phage-mediated delivery of the disruption construct, in which the target gene was disrupted by replacement of an internal segment with the hygromycin resistance gene (hyg). Gene disruption by allelic exchange in the case of each disruptant was confirmed by PCR and Southern blot analyses. Neither msl7 nor pks10 mutants could produce dimycocerosyl phthiocerol, although both could produce mycocerosic acids. Thus, it is concluded that these gene products are involved in the biosynthesis of phthiocerol. Both mutants were found to be attenuated in a murine model, supporting the hypothesis that dimycocerosyl phthiocerol is a virulence factor and thus the many steps involved in its biosynthesis offer potential novel targets for antimycobacterial therapy.

A Newly Identified Methyl-branched Chain Fatty Acid Synthesizing Enzyme from Mycobacterium tuberculosis var. bovis BCG

Mycobacterium bovis BCG produces a variety of methyl-branched fatty acids. They include C28 to C32 mycocerosic acids esterified to phthiocerol and phenolphthiocerol and the shorter (C22 to C26) mycocerosic acids esterified to phthiocerol. A mycocerosic acid synthase gene-disrupted mutant was still able to produce the shorter mycocerosic acids. The enzyme short chain mycocerosic acid synthase (SMAS), that catalyzes the synthesis of such acids, was purified using anion exchange and red-agarose chromatography. Gel filtration showed the native enzyme to be a 537-kDa protein. Since SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a 280-, 170-, and 100-kDa protein and they cross-reacted with antibodies prepared against the 280- or 100-kDa protein, this enzyme is composed of the three subunits or two 280-kDa monomers with an unusual susceptibility to a proteolytic nick. SMAS utilizes methylmalonyl-CoA with C12 to C20 acyl-CoA as primers and with either NADH or NADPH as the reductant to synthesize the short mycocerosic acids. The Km values for NADH and NADPH were 93 and 90 microM, respectively. Antibodies raised against either the 280- or 100-kDa protein inhibited the incorporation of methylmalonyl-CoA into fatty acids by SMAS. The enzyme is not immunologically closely related to mycocerosic acid synthase or fatty acid synthase.

Gas chromatography–mass spectrometry methods for the analysis of mycocerosic acids present in Mycobacterium tuberculosis

Mycocerosic acids of Mycobacterium tuberculosis were studied by gas chromatography–mass spectrometry. Mycobacterial cells were subjected to alkaline hydrolysis and then acidified before being extracted using petroleum ether to collect the acids. Methyl ester, trimethylsilyl and pentafluorobenzyl derivatives of the mycocerosic acids were formed. In the electron impact mode, using selected-ion monitoring, the methyl ester derivative of 2,4,6,8-tetramethyloctacosanoic acid (C 32 mycocerosic acid) was detectable down to 20 pg (injected amount), whereas in the negative-ion chemical ionization mode, the pentafluorobenzyl derivative was detectable down to 2 pg, provided that the temperature of the ion source was at least 240°C. The described methods can be used to demonstrate and quantify certain species of Mycobacterium, including M. tuberculosis, in clinical or environmental samples.

Methylmalonyl coenzyme A selectivity of cloned and expressed acyltransferase and beta-ketoacyl synthase domains of mycocerosic acid synthase from Mycobacterium bovis BCG.

Methyl-branched fatty acids and polyketides occur in a variety of living organisms. Previous studies have established that multifunctional enzymes use methylmalonyl coenzyme A (CoA) as the substrate to generate methyl-branched products such as mycocerosic acids and polyketides. However, we do not know which of the component activities show selectivity for methylmalonyl-CoA in any biological system. A comparison of homologies of the domains of the multifunctional synthases that selectively use malonyl-CoA or methylmalonyl-CoA suggested that the acyltransferase (AT) and beta-ketoacyl synthase (KS) domains might be responsible for the substrate selectivity. To test this hypothesis, we expressed the AT and KS domains of the mycocerosic acid synthase (MAS) gene from Mycobacterium bovis BCG in Escherichia coli and examined whether they confer to synthases that normally do not use methylmalonyl-CoA the ability to incorporate methylmalonyl-CoA into fatty acids. Both the AT and the KS domains of MAS showed selectivity for methylmalonyl-CoA over malonyl-CoA. Acyl carrier protein (ACP)-dependent elongation of the n-C12 acyl primer mainly by one methylmalonyl-CoA unit was catalyzed by an E. coli fatty acid synthase preparation only in the presence of the expressed MAS domains. An ACP-dependent elongation of the n-C20 acyl primer by one methylmalonyl-CoA extender unit was catalyzed by fatty acid synthase from Mycobacterium smegmatis only in the presence of the expressed MAS domains. These results show methylmalonyl-CoA selectivity for the AT and KS domains of MAS. These domains may be useful in producing novel polyketides by genetic engineering.

Open reading frame 3, which is adjacent to the mycocerosic acid synthase gene, is expressed as an acyl coenzyme A synthase in Mycobacterium bovis BCG.

The aim of this study was to test for expression of a 900-bp open reading frame (ORF), ORF3, located at the 5' end of the mycocerosic acid synthase gene in Mycobacterium bovis BCG and to determine the nature of the ORF3 protein. ORF3 was expressed as a 61-kDa C-terminal fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal rabbit antiserum, prepared against this fusion protein, cross-reacted with a 65-kDa protein in M. bovis BCG crude extracts. Since this protein was larger than that predicted from the nucleotide sequence (32 kDa), ORF3 was resequenced, revealing an ORF of 1,749 bp that encodes a 64.8-kDa protein containing 583 amino acids. Reverse transcription-PCR revealed that ORF3 is expressed in M. bovis BCG. The ORF3 product has a high degree of similarity to the acyladenylate family of enzymes. Immunoaffinity absorption chromatography was used to isolate the 65-kDa cross-reacting protein from M. bovis BCG. This purified protein catalyzed coenzyme A (CoA) ester synthesis of n-C10 to n-C18 fatty acids but not mycocerosic acids. ORF3 antibodies severely inhibited acyl-CoA synthase activities of the purified protein and extracts of M. bovis BCG, Mycobacterium smegmatis, and E. coli. They also showed immunological cross-reactivity with proteins in these extracts. Both the ORF3 protein and the acyl-CoA synthase activity were located in the cell cytosol or were loosely associated with the cell membrane. These results indicate that ORF3 encodes an acyl-CoA synthase-like protein.

Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides.

A single gene (mas) encodes the multifunctional enzyme that catalyzes the synthesis of very long chain multiple methyl branched fatty acids called mycocerosic acids that are present only in slow-growing pathogenic mycobacteria and are thought to be important for pathogenesis. To achieve a targeted disruption of mas, an internal 2-kb segment of this gene was replaced with approximately the same size hygromycin-resistance gene (hyg), such that hyg was flanked by 4.7- and 1.4-kb segments of mas. Transformation of Mycobacterium bovis BCG with this construct in a plasmid that cannot replicate in mycobacteria yielded hygromycin-resistant transformants. Screening of 38 such transformants by PCR revealed several transformants representing homologous recombination with single crossover and one with double crossover. With primers representing the hyg termini and those representing the mycobacterial genome segments outside that used to make the transformation construct, the double-crossover mutant yielded PCR products expected from either side of hyg. Gene replacement was further confirmed by the absence of the vector and the 2-kb segment of mas replaced by hyg from the genome of the mutant. Thin-layer and radio-gas chromatographic analyses of the lipids derived from [1-14C]propionate showed that the mutant was incapable of synthesizing mycocerosic acids and mycosides. Thus, homologous recombination with double crossover was achieved in a slow-growing mycobacterium with an intron-containing RecA. The resulting mas-disrupted mutant should allow testing of the postulated roles of mycosides in pathogenesis.

Fast atom bombardment mass spectrometry of mycobacterial phenolic glycolipids

Fast atom bombardment mass spectra were successfully recorded for intact glycosylphenolphthiocerol dimycocerosates (phenolic glycolipids, PGLs) from Mycobacterium kansasii, M. leprae, M. tuberculosis, M. marinum, M. bovis and M. haemophilum. Characteristic fragment ions from the loss of the oligosaccharide moiety and one of the long-chain multimethyl-branched mycocerosic acids were observed in most cases. A tandem mass spectrometric experiment was carried out on the PGL from M. tuberculosis, revealing the type of mycocerosic acids esterified to individual homologues. Mass spectra of homologues separated by reversed-phase high-performance liquid chromatography gave information on the substitution pattern in certain cases. The potential of matrix-assisted laser desorption ionization spectroscopy was demonstrated by a successful analysis of the PGL from M. tuberculosis.