The Cell Wall Composition and Distribution of Free Mycolic Acids in Named Strains of Coryneform Bacteria and in Isolates from Various Natural Sources

The major cell wall amino acids and sugars in 177 strains of coryneform bacteria were determined using a 'rapid'method. Representatives were examined for free mycolic acids and the oxygen requirements of all strains were determined. Included were named strains, most of which were labelled Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium or Microbacterium , and a similar number of unnamed isolates from various natural sources. Strains which contained meso -diaminopimelic acid (DAP) were divided into four groups according to their oxygen requirements, the wall sugars and the occurrence and nature of free mycolic acids. Group 1 strains were mainly facultatively anaerobic and contained arabinose and mycolic acids of the Corynebacterium type. They were considered to be members of Corynebacterium sensu stricto and included Cor. diphtheriae and related animal parasites, Microbact. flavum , and Cor. glutamicum and similar species. Group 2 strains were aerobic, contained arabinose and mycolic acids of the 'rhodochrous'type and were considered members of the ' rhodochrous 'complex. Group 3 strains were aerobic, contained ribose and no mycolic acids. Most were Br. linens strains from cheese but a few, possibly related strains, were from other habitats. Group 4 strains were aerobic and contained neither a pentose sugar nor mycolic acids and were of unknown taxonomic status. Most remaining strains contained lysine or ornithine in the wall and smaller numbers contained L-DAP or diaminobutyric acid; none contained mycolic acids. The chemotaxonomic data are discussed in relation to recent numerical taxonomic studies of coryneform bacteria.     

Structure determination of mycolic acids by using charge remote fragmentation

The collision-induced remote site fragmentation process of closed-shell ions, such as carboxylate anions, is a very potent analytical tool for the structural determination of fatty acids. This leads to an easy location of branch points, double bonds, cyclopropane rings and other functional groups. Although corynomycolic acid mixtures from Corynebacterium diphtheriae can be directly analyzed by negative-ion fast atom bombardment combined with collisionally activated decomposition spectra, mycolic acid mixtures from mycobacteria need a preliminary chemical cleavage. They are oxidized to beta-keto esters and then submitted to a retro-Claisen reaction. The resulting fatty acids were then converted into pentafluorobenzyl derivatives and introduced directly into a high pressure ion source working in the negative ion mode. The resulting gas phase carboxylate anions are activated to decompose by collision with helium atoms. When applied to M3-mycolic acids from Mycobacterium fallax, this method allows for the characterization of a new tri-unsaturated mycolic acid, which has the middle and the remote double bonds separated by two methylene groups.     

The acyl-AMP ligase FadD32 and AccD4-containing acyl-CoA carboxylase are required for the synthesis of mycolic acids and essential for mycobacterial growth: identification of the carboxylation product and determination of the acyl-CoA carboxylase components

Mycolic acids are major and specific long-chain fatty acids of the cell envelope of several important human pathogens such as Mycobacterium tuberculosis, M. leprae, and Corynebacterium diphtheriae. Their biosynthesis is essential for mycobacterial growth and represents an attractive target for developing new antituberculous drugs. We have previously shown that the pks13 gene encodes condensase, the enzyme that performs the final condensation step of mycolic acid biosynthesis and is flanked by two genes, fadD32 and accD4. To determine the functions of the gene products we generated two mutants of C. glutamicum with an insertion/deletion within either fadD32 or accD4. The two mutant strains were deficient in mycolic acid production and exhibited the colony morphology that typifies the mycolate-less mutants of corynebacteria. Application of multiple analytical approaches to the analysis of the mutants demonstrated the accumulation of a tetradecylmalonic acid in the DeltafadD32::km mutant and its absence from the DeltaaccD4::km strain. The parental corynebacterial phenotype was restored upon the transfer of the wild-type fadD32 and accD4 genes in the mutants. These data demonstrated that both FadD32 and AccD4-containing acyl-CoA carboxylase are required for the production of mycolic acids. They also prove that the proteins catalyze, respectively, the activation of one fatty acid substrate and the carboxylation of the other substrate, solving the long-debated question of the mechanism involved in the condensation reaction. We used comparative genomics and applied a combination of molecular biology and proteomic technologies to the analysis of proteins that co-immunoprecipitated with AccD4. This resulted in the identification of AccA3 and AccD5 as subunits of the acyl-CoA carboxylase. Finally, we used conditionally replicative plasmids to show that both the fadD32 and accD4 genes are essential for the survival of M. smegmatis. Thus, in addition to Pks13, FadD32 and AccD4 are promising targets for the development of new antimicrobial drugs against pathogenic species of mycobacteria and related microorganisms.     

Mass spectrometry as a tool for identifying group D2 corynebacteria by their fatty acid profiles

Corynebacterium group D2 (CGD2) are lipophilic antibiotic-multiresistant bacteria involved in some infections of immunocompromised patients. The fatty acid composition and structure of different strains was established by several mass spectrometric methods, particularly negative ion tandem mass spectrometry coupled with capillary gas chromatography. Non-hydroxylated fatty acid profiles of three strains of CGD2 (ATCC 43042, ATCC 43043, ATCC 43044) were almost identical and revealed the presence of several straight chain unsaturated fatty acids from the omega-9 series, with even carbon numbers ranging from 14 to 24. Branched saturated fatty acids were mainly anteiso-heptadecanoic acid and tuberculostearic acid. Surprisingly, a relatively large quantity of 10-methylene octadecanoic acid was found. The non-hydroxylated fatty acid profile of one rare beta-lactam susceptible strain (SC1) was different; 10-methylene octadecanoic acid was lacking whereas tuberculostearic acid was much more abundant. In contrast, the four CGD2 strains displayed highly similar mycolic acid patterns. The major mycolic acid species corresponded to C32, C30 and C28 bis-unsaturated with a double bond on each branch at the omega-9 position. The comparison of the mycolic acid composition and structure with those of other medically important corynebacteria strains, revealed a characteristic pattern for CGD2 strains, and CGD2 strains were easily distinguished from Corynebacterium jeikeium (CIP 82.51).     

Analysis of cellular fatty acids by gas chromatography as a tool in the identification of medically important coryneform bacteria

The fatty acid methyl esters of nineteen unidentified pathogenic coryneform bacteria were analysed by gas-liquid chromatography and the resulting profiles were compared with those of type or reference strains of possibly related species, namely Caseobacter polymorphus, Corynebacterium bovis, C. diphtheriae, C. xerosis and Rhodococcus equi. All of the strains had distinct fatty acid profiles but most of them conformed to a general pattern, with high levels of octadecanoic acids and only trace amounts of 10-methyl octadecanoic acid (tuberculostearic acid). These profiles were very similar to those from C. diphtheriae and C. xerosis but could be differentiated from C. bovis, Cas. polymorphus, R. equi and two unidentified pathogenic strains which had significantly higher levels of tuberculostearic acid.     

Analysis of cellular fatty acids by gas chromatography as a tool in the identification of medically important coryneform bacteria

The fatty acid methyl esters of nineteen unidentified pathogenic coryneform bacteria were analysed by gas-liquid chromatography and the resulting profiles were compared with those of type or reference strains of possibly related species, namely Caseobacter polymorphus, Corynebacterium bovis, C. diphtheriae, C. xerosis and Rhodococcus equi. All of the strains had distinct fatty acid profiles but most of them conformed to a general pattern, with high levels of octadecanoic acids and only trace amounts of 10-methyl octadecanoic acid (tuberculostearic acid). These profiles were very similar to those from C. diphtheriae and C. xerosis but could be differentiated from C. bovis, Cas. polymorphus, R. equi and two unidentified pathogenic strains which had significantly higher levels of tuberculostearic acid.     

The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis

The suborder Corynebacterianeae comprises bacteria like Mycobacterium tuberculosis and Corynebacterium glutamicum, and these bacteria contain in addition to the linear fatty acids, unique alpha-branched beta-hydroxy fatty acids, called mycolic acids. Whereas acetyl-coenzyme A (CoA) carboxylase activity is required to provide malonyl-CoA for fatty acid synthesis, a new type of carboxylase is apparently additionally present in these bacteria. It activates the alpha-carbon of a linear fatty acid by carboxylation, thus enabling its decarboxylative condensation with a second fatty acid to afford mycolic acid synthesis. We now show that the acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated alpha-subunit AccBC, the beta-subunit AccD1, and the small peptide AccE of 8.9 kDa, forming an active complex of approximately 812,000 Da. The carboxylase involved in mycolic acid synthesis is made up of the two highly similar beta-subunits AccD2 and AccD3 and of AccBC and AccE, the latter two identical to the subunits of the acetyl-CoA carboxylase complex. Since AccD2 and AccD3 orthologues are present in all Corynebacterianeae, these polypeptides are vital for mycolic acid synthesis forming the unique hydrophobic outer layer of these bacteria, and we speculate that the two beta-subunits present serve to lend specificity to this unique large multienzyme complex.     

Ultrafast pyrosequencing of Corynebacterium kroppenstedtii DSM44385 revealed insights into the physiology of a lipophilic corynebacterium that lacks mycolic acids

Corynebacterium kroppenstedtii is a lipophilic corynebacterial species that lacks in the cell envelope the characteristic alpha-alkyl-beta-hydroxy long-chain fatty acids, designated mycolic acids. We report here the bioinformatic analysis of genome data obtained by pyrosequencing of the type strain C. kroppenstedtii DSM44385 that was initially isolated from human sputum. A single run with the Genome Sequencer FLX system revealed 560,248 shotgun reads with 110,018,974 detected bases that were assembled into a contiguous genomic sequence with a total size of 2,446,804bp. Automatic annotation of the complete genome sequence resulted in the prediction of 2122 coding sequences, of which 29% were considered as specific for C. kroppenstedtii when compared with predicted proteins from hitherto sequenced pathogenic corynebacteria. This comparative content analysis of the genome data revealed a large repertoire of genes involved in sugar uptake and central carbohydrate metabolism and the presence of the mevalonate route for isoprenoid biosynthesis. The lack of mycolic acids and the lipophilic lifestyle of C. kroppenstedtii are apparently caused by gene loss, including a condensase gene cluster, a mycolate reductase gene, and a microbial type I fatty acid synthase gene. A complete beta-oxidation pathway involved in the degradation of fatty acids is present in the genome. Evaluation of the genomic data indicated that lipophilism is the dominant feature involved in pathogenicity of C. kroppenstedtii.     

Changes in composition and content of mycolic acids in glutamate-overproducing Corynebacterium glutamicum

Overproduction of glutamate by Corynebacterium glutamicum is induced by biotin limitation or by the supplementation of specific detergents, sublethal amounts of penicillin, or cerulenin. But, it remains unclear why these different treatments, which have different sites of primary action, produce similar effects.In this study, it was found that the cellular content of mycolic acids--characteristic constituents of Corynebacterineae that are synthesized from fatty acids and form a cell surface layer--decreased under all conditions that induced glutamate overproduction. Furthermore, short mycolic acids increased under conditions of biotin limitation and cerulenin supplementation. These results suggest that different treatments produce the same effect that causes defects in the mycolic acid layer. This is perhaps one of the key factors in overproduction of glutamate by C. glutamicum.     

Current knowledge on mycolic acids in Corynebacterium glutamicum and their relevance for biotechnological processes

Corynebacterium glutamicum is the world’s largest producer of glutamate and lysine. Industrial glutamate overproduction is induced by empirical processes, such as biotin limitation, supplementation with specific surfactants or addition of sublethal concentration of certain antibiotics to the culture media. Although Gram-positive bacteria, C. glutamicum and related bacterial species and genera contain, in addition to the plasma membrane, an outer permeability membrane similar to that of Gram-negative microorganisms. As the amino acids have to cross both membranes, their integrity, composition and fluidity influence the export process. While the precise mechanism of the export of the amino acids by C. glutamicum is not fully understood, the excretion of amino acids through the inner membrane involved at least a major export system mechanosensitive channel MscS family (MscCG) encoded by NCgl1221. As the various industrial treatments have been shown to affect the lipid content of the bacterial cell, it is strongly believed that defects in the hallmark of the outer membrane, 2-alkyl, 3-hydroxylated long-chain fatty acids (mycolic acids), could be key factors in the glutamate overproduction. This review aims at giving an overview of the current knowledge on mycolic acids structure, biosynthesis and transfer in C. glutamicum and their relevance for amino acid biotechnological production.     

Purification and structure analysis of mycolic acids in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is widely used for producing amino acids. Mycolic acids, the major components in the cell wall of C. glutamicum might be closely related to the secretion of amino acids. In this study, mycolic acids were extracted from 5 strains of C. glutamicum, including ATCC 13032, ATCC 13869, ATCC 14067, L-isoleucine producing strain IWJ-1, and L-valine producing strain VWJ-1. Structures of these mycolic acids were analyzed using thin layer chromatography and electrospray ionization mass spectrometry. More than twenty molecular species of mycolic acid were observed in all 5 strains. They differ in the length (20–40 carbons) and saturation (0–3 double bonds) of their constituent fatty acids. The dominant species of mycolic acid in every strain was different, but their two hydrocarbon chains were similar in length (14–18 carbons), and the meromycolate chain usually contained double bonds. As the growth temperature of cells increased from 30°C to 34°C, the proportion of mycolic acid species containing unsaturated and shorter hydrocarbon chains increased. These results provide new information on mycolic acids in C. glutamicum, and could be useful for modifying the cell wall to increase the production of amino acids.     

Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice

Members of the Mycobacterium tuberculosis group synthesize a family of long-chain fatty acids, mycolic acids, which are located in the cell envelope. These include the non-oxygenated alpha-mycolic acid and the oxygenated keto- and methoxymycolic acids. The function in bacterial virulence, if any, of these various types of mycolic acids is unknown. We have constructed a mutant strain of M. tuberculosis with an inactivated hma (cmaA, mma4) gene; this mutant strain no longer synthesizes oxygenated mycolic acids, has profound alterations in its envelope permeability and is attenuated in mice.     

Quantitative comparison of the mycolic and fatty acid compositions of Mycobacterium leprae and Mycobacterium gordonae

The mycolic and fatty acids of three samples each of Mycobacterium leprae and Mycobacterium gordonae were compared. Acids released by whole-organism alkaline hydrolysis were converted to 4-nitrobenzyl esters and mycolic acids were further derivatized to t-butyldimethylsilyl ethers. Thin-layer chromatography of the derivatized long-chain extracts showed that all three M. leprae preparations contained so-called alpha-mycolates and ketomycolates but that the M. gordonae samples had a methoxymycolate in addition to the above types. Silica gel normal-phase high-performance liquid chromatography of the total mycolic acid derivatives confirmed the lack of detectable amounts of methoxymycolates in M. leprae and reverse-phase chromatography of the individual mycolate types demonstrated the homogeneity of the chain lengths of the mycolic acids in each species. Non-hydroxylated fatty acid 4-nitrobenzyl esters were transformed to methyl esters and examined by gas chromatography. Tuberculostearic (10-methyloctadecanoic) acid was a major component of the lipids of all three M. leprae preparations but it was absent in one M. gordonae strain and a very minor component in the other representatives of this latter species. On the basis of fatty and mycolic acid compositions, therefore, a previously suggested close relationship between M. leprae and M. gordonae was not supported.     

Trafficking pathways of mycolic acids: structures, origin, mechanism of formation, and storage form of mycobacteric acids

Mycolic acids, the hallmark of mycobacteria and related bacteria, are major and specific components of their cell envelope and essential for the mycobacterial survival. Mycobacteria contain structurally related long-chain lipids, but the metabolic relationships between these various classes of compounds remain obscure. To address this question a series of C(35) to C(54) nonhydroxylated fatty acids (mycobacteric acids), ketones, and alcohols structurally related to the C(70-80) dicyclopropanated or diethylenic mycolic acids were characterized in three mycobacterial strains and their structures compared. The relationships between these long-chain acids and mycolic acids were established by following the in vivo traffic of (14)C labeled alpha-mycolic acids purified from the same mycobacterial species. The labeling was exclusively found in mycobacteric acids. The mechanism of this degradation was established by incorporation of (18)O(2) into long-chain lipids and shown to consist in the rupture of mycolic acids between carbon 3 and 4 by a Baeyer-Villiger-like reaction. We also demonstrated that mycobacteric acids occur exclusively in the triacylglycerol (TAG) fraction where one molecule of these acids esterifies one of the three hydroxyl groups of glycerol. Altogether, these data suggest that these compounds represent a pathway of metabolic energy that would be used by mycobacteria in particular circumstances.     

Extraction of mycobacterial mycolic acids and other long-chain compounds by an alkaline methanolysis procedure

Treatment of whole organisms with methanolic tetramethylammonium hydroxide and toluene, followed by addition of iodomethane in dimethylformamide, released long-chain compounds and fatty acids, as their methyl esters, from representative strains of Mycobacterium. Two-dimensional thin-layer chromatography was used to analyze methanolysates for the presence of the methyl esters of mycolic acids which are characteristic high molecular weight 3-hydroxy-2-alkyl fatty acids.     

Effect of diets rich in medium-chain and long-chain triglycerides on lipogenic-enzyme gene expression in liver and adipose tissue of the weaned rat.

The activity and mRNA concentrations of two lipogenic enzymes, fatty-acid synthase and acetyl-CoA carboxylase were measured in the liver and white adipose tissue of rats weaned to a carbohydrate-rich diet containing either long-chain or medium-chain fatty acids, and compared to those of rats weaned on a diet containing less than 1% (total energy) fat (high-carbohydrate diet). In the liver, the diet containing long-chain fatty acids inhibited the increase of both lipogenic-enzyme mRNA concentrations and activities seen at weaning on the high-carbohydrate diet but did not prevent the decrease in phosphoenolpyruvate carboxykinase mRNA and activity. In contrast, the diet containing medium-chain fatty acids induced a slower but finally similar increase in lipogenic-enzyme mRNA concentrations and activities. In adipose tissue, a similar trend was observed, although the inhibitory effect of the diet containing long-chain fatty acids was considerably less marked than in liver. It is concluded that medium-chain and long-chain fatty acids have not the same inhibitory potency of the gene expression of lipogenic enzymes, and that long-chain fatty acids have a more marked effect in the liver.     

Cell envelope composition and organisation in the genus Rhodococcus

A knowledge of the organisation of the rhodococcal cell envelope is of fundamental importance if the environmental and biotechnological significance of these bacteria are to be understood and succesfully exploited. The genus Rhodococcus belongs to a distinctive suprageneric taxon, the mycolata, which includes among others the genera Corynebacterium, Mycobacterium and Nocardia. Members of this taxon exhibit an unusual complexity in their cell envelope composition and organisation compared to other Gram-positive bacteria. Models that describe the architecture of the mycobacterial cell envelope are extrapolated here to provide a model of the rhodococcal cell envelope. The rhodococcal cell envelope is dominated by the presence of an arabinogalactan cell wall polysaccharide and large 2-alkyl 3-hydroxy branched-chain fatty acids, the mycolic acids, which are covalently assembled into a peptidoglycan–arabinogalactan–mycolic acid matrix. This review further emphasises that the mycolic acids in this complex form the basis of an outer lipid permeability barrier. The localisation and roles of other cell envelope components, notably complex free lipids, lipoglycans, proteins and lipoproteins are also considered.     

Biosynthesis of C30 to C56 fatty acids by an extract of Mycobacterium tuberculosis H37Ra.

A 10,800 X g supernatant fraction from disrupted cells of Mycobacterium tuberculosis H37Ra was incubated with [2-14C]malonate to produce labeled long-chain fatty acids upon saponification. These acids were derivatized to the p-bromophenacyl ester and separated into the nonmycolic saturated, monounsaturated, and multiunsaturated esters by argentation thin-layer chromatography. Each of these fractions was then analyzed by high-performance liquid chromatography by using a C18-bonded silica cartridge and a mobile phase of a linear gradient of 0 to 70% p-dioxane in acetonitrile. The results showed that the cell-free system is able to synthesize both saturated and monounsaturated fatty acids of the sizes C30 to C40 and C48 to C56. This latter series was strikingly similar to meromycolic acid, a putative precursor of mycolic acid. When acetate or oleate was used as the labeled substrate, the major products were no longer than C32. When palmitate was used as the labeled substrate, the saturated acids ranged in size from C18 to C32, whereas the monounsaturated products contained C18, C26 to C30 and C40 fatty acids. Fatty acids greater than C40 were also detected. When methyl-labeled S-adenosyl-L-methionine was used as the substrate, the methyl group was incorporated into short-chain and C48 to C56 fatty acids. Unlabeled malonyl-coenzyme A was included in all of these reactions. This cell-free system was not able to synthesize mycolic acid (final product) or its keto derivative (intermediate product). However, since the meromycolate-like C48 to C56 fatty acids were synthesized, we suggest that the present system is able to take the synthesis to a point before the alpha-alkyl condensation reaction.