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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 Vac, 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 Vac 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 Vac mummies.

The Mycobacterium tuberculosis complex (MTC) has affected South American populations since ca. 200 years BCE. In Argentina, possible cases date from ca. 1000-1400 Common Era (CE). This paper describes the oldest (905-1030 CE) confirmed case of tuberculosis (TB) in a young adult male from Lomitas de Saujil (Tinogasta, Catamarca, Argentina). Osteolytic lesions on the bodies of the lower spine were macroscopically and radiographically identified. Bilateral new bone formation was seen on the visceral vertebral third of several ribs and in long bones, compatible with hypertrophic osteoarthropathy. Representative rib and hand bones gave profiles for MTC-specific C27-C32 mycocerosic acid lipid biomarkers; these were strongest in one heavily-lesioned lower rib, which also had MTC-diagnostic C76-C89 mycolic acids and positive amplification of MTC-typical IS6110 aDNA fragments. During the first millennium CE, the intense social interaction, the spatial circumscription of villages among the pre-Hispanic societies in the mesothermal valleys of Catamarca and the fluid contacts with the Eastern lowlands, valleys and puna, were factors likely to favor disease transmission. It is proposed that TB arrived from northern Chile and dispersed towards the northeast into the Yocavil valley, where several cases of TB infection were macroscopically identified for a later chronology.

Following the report of Mycobacterium tuberculosis proteins found in archaeological bone by Boros-Major et al. (2011), we attempted to identify M. tuberculosis proteins in mummified lung tissues from which ancient DNA success had already been reported. Using a filter-aided sample preparation protocol modified for ancient samples we applied shotgun proteomics to seven samples of mummified lung, chest and pleura tissues. However, we only identified four peptides with unique matches to the M. tuberculosis complex, none of which were unique to M. tuberculosis, although we did identify a range of human proteins and non-mycobacterial bacterial proteins. In light of these results, we question the validity of the peptide mass fingerprint (PMF) approach presented by Boros-Major et al. (2011), especially because the PMF spectrum presented in Boros-Major et al. (2011) has similarities to that of human collagen, the dominant protein in the tissue under investigation. We explore the challenges of using proteomic approaches to detect M. tuberculosis, and propose that, given the contentious outcomes that have plagued ancient protein research in the past, the susceptibility of ancient material to modern contamination, and the degradation inherent in archaeological samples, caution is needed in the acquisition, analysis and reporting of proteomic data from such material.

Mycolic acid methyl esters were extracted from Mycobacterium avium by a mild saponification protocol, designed to preserve labile components. The resulting mixture of α-, keto- and wax ester mycolates was accompanied by some degraded ω-carboxymycolic acid dimethyl esters, whose overall structures were found to support previous studies. Chromatography of the mono-carboxylic mycolates gave an inseparable mixture of keto- and wax ester mycolates and separate α-mycolates. Reduction of the ketomycolate components allowed isolation and characterisation of intact wax ester mycolates for the first time. Minor α- and ω-carboxymycolates were detected in which methyl branches were located on either the proximal or distal sides of trans-alkene groups.

Many tuberculosis and leprosy infections are latent or paucibacillary, suggesting a long time-scale for host and pathogen co-existence. Palaeopathology enables recognition of archaeological cases and PCR detects pathogen ancient DNA (aDNA). Mycobacterium tuberculosis and Mycobacterium leprae cell wall lipids are more stable than aDNA and restrict permeability, thereby possibly aiding long-term persistence of pathogen aDNA. Amplification of aDNA, using specific PCR primers designed for short fragments and linked to fluorescent probes, gives good results, especially when designed to target multi-copy loci. Such studies have confirmed tuberculosis and leprosy, including co-infections. Many tuberculosis cases have non-specific or no visible skeletal pathology, consistent with the natural history of this disease. M. tuberculosis and M. leprae are obligate parasites, closely associated with their human host following recent clonal distribution. Therefore genotyping based on single nucleotide polymorphisms (SNPs) can indicate their origins, spread and phylogeny. Knowledge of extant genetic lineages at particular times in past human populations can be obtained from well-preserved specimens where molecular typing is possible, using deletion analysis, microsatellite analysis and whole genome sequencing. Such studies have identified non-bovine tuberculosis from a Pleistocene bison from 17,500 years BP, human tuberculosis from 9000 years ago and leprosy from over 2000 years ago.

High molecular weight long-chain mycolic acids are key structural components of the cell envelope of Mycobacterium tuberculosis and they are established as biomarkers for the identification of both ancient and modern tuberculosis. Mycolic acids from M. tuberculosis have a characteristic profile, reflecting contributions from five major distinct homologous series of mycolate structural types. Diagnosis of tuberculosis in archaeological material, using mycolic acid biomarkers, depends on objective recognition of the key characteristic mycolic acid components. A recent article in this journal claimed that tuberculosis could be confirmed in ancient bones by high throughput mass spectrometric analysis of mycolic acids. Scrutiny of the data presented reveals no convincing evidence for the presence of mycolic acids, characteristic of the M. tuberculosis complex, in the skeletal remains examined. This communication reviews the essential criteria necessary for positive tuberculosis diagnosis, using mycolic acids.

It is sixteen years since the first detection of Mycobacterium tuberculosis DNA in archaeological specimens, yet the validity of findings continues to be questioned. Rigorous scientific scrutiny and debate is valuable and has led to a coalescence of procedures and precautions amongst those actively engaged in this work. It is disappointing that these good practices are not recognised by certain scientists whose primary expertise is in the related fields of archaeology, palaeopathology, and eukaryote ancient DNA. There is a danger that by constant repetition, disputable and inadequately justified concerns will assume the status of self-perpetuating myths and misunderstandings. We discuss these issues with reference to a recent article in this journal, in which clear peer-reviewed scientific data were specifically targeted as part of a general critique of the field of the palaeomicrobiology of tuberculosis. We believe we have given sufficient evidence and cogent argument to persuade the unbiased reader that the views in the critique by Wilbur et al. are unjustified. (C) 2009 Elsevier Ltd. All rights reserved.

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.

Mycolic acids are essential components of the mycobacterial cell wall. In this study, we show that a gene encoding a reductase involved in the final step of mycolic acid biosynthesis can be deleted in Mycobacterium smegmatis without affecting cell viability. Deletion of MSMEG4722 (ortholog of Mycobacterium tuberculosis Rv2509) altered culture characteristics and antibiotic sensitivity. The DeltaMSMEG4722 strain synthesized alpha-alkyl, beta-oxo intermediates of mycolic acids, which were found esterified to cell wall arabinogalactan. While the precursors could not be isolated directly due to their inherent instability during base treatment, their presence was established by prior reduction of the beta-oxo group by sodium borohydride. Interestingly, the mutant also accumulated unsaturated ketones, similar to tuberculenone from M. tuberculosis, which were shunt products derived from spontaneous decarboxylation of alpha-alkyl, beta-oxo fatty acid precursors of mycolic acids.

Phase diagrams of Langmuir monolayers of oxygenated mycolic acids, i.e. methoxy mycolic acid (MeO-MA), ketomycolic acid (Keto-MA), and artificially obtained deoxo-mycolic acid (deoxo-MA) from Mycobacterium bovis BCG were obtained by thermodynamic analysis of the surface pressure (pi) vs. average molecular area (A) isotherms. At lower temperatures and lower surface pressures, both Keto- and MeO-MAs formed rigid condensed monolayers where each MA molecule was considered to be in a 4-chain form, in which the three carbon chain segments due to bending of the 3-hydroxy aliphatic carboxylate chain and the 2-side chain were in compact parallel arrangement. At higher temperatures and surface pressures, MeO-MA and deoxo-MA tended to take stretched-out conformations in which the 3-hydroxy aliphatic carboxylate chain was more or less in an extended form, but Keto-MA retained the original 4-chain structure. The thickness measurement of the monolayers in situ by ellipsometry at different pi values and temperatures supported the above conclusions derived from the phase diagrams. The enthalpy changes associated with the phase transitions of MeO-MA and deoxo-MA implied that the MeO-MA needed larger energy to change from a compact conformation to an extended one, possibly and partly due to the dehydration of the methoxy group from water surface involved. Molecular dynamics studies of MA models derived from Monte Carlo calculations were also performed, which confirmed the conformational behavior of MAs suggested by the thermodynamic studies on the Langmuir monolayers. (c) 2007 Elsevier B.V. All rights reserved., The corrected Table 2 appears here. : BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Volume: 1768 Issue: 9 Pages: 2343-2343 Published: SEP 2007

Three stereoisomers of a wax ester meromycolate have been prepared starting from mannitol. A detailed comparison of their NMR spectra with those reported for a homologous series of natural wax esters allows the relative configurations of the α-methyl group and adjacent trans-cyclopropane to be determined.

Symmetrical and unsymmetrical analogues of the antimycobacterial agent isoxyl have been synthesized and tested against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG, some showing an increased bactericidal effect. In particular, compounds 1-(p-n-butylphenyl)-3-(4-propoxy-phenyl) thiourea (10) and 1-(p-n-butylphenyl)-3-(4-n-butoxy-phenyl) thiourea (11) showed an approximate 10-fold increase in in vitro potency compared to isoxyl, paralleled by increased inhibition of mycolic acid biosynthesis in M. bovis BCG. Interestingly, these isoxyl analogues showed relatively poor inhibition of oleate production, suggesting that the modifications have changed the spectrum of biological activity.

Analogues of the natural antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis β-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-[3-(4-acetyl-phenyl)-prop-2-ynyl]-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited more than an 18-fold increased potency, compared to thiolactomycin, against this key condensing enzyme, involved in M. tuberculosis mycolic acid biosynthesis. Analogues of the antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded activity against cloned mtFabH condensing enzyme.

(Z)-tetracos-5-enoic acid and racemic cis-4-(2-octadecylcyclopropane-1-yl)-butanoic acid have been prepared from 1-eicosene by a new facile route. Periodic acid cleavage of the epoxide of 1-eicosene gave nonadecanal which was condensed with 4-carboxybutyltriphenylphosphonium bromide to give predominately (Z)-tetracos-5-enoic acid. Simmons-Smith type cyclopropanation of (Z)-tetracos-5-enoic acid gave a minor proportion of racemic cis-4-(2-octadecylcyclopropane-1-yl)-butanoic acid accompanied by major amounts of its methyl ester.

(11R,12S)-Lactobacillic acid has been prepared from 2,3-O-isopropylidene-d-glyceraldehyde, in a sequence involving asymmetric cyclopropanation, and from cis-cyclopropane-1,2-dimethanol, using enzymatic desymmetrisation. The key step in the former route was the stereochemically controlled cyclopropanation of (1Z,4′S)-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1-octene via a Simmons–Smith type reaction, using diethylzinc and chloroiodomethane. This product was converted into the key intermediate (1R,2S)-1-formyl-2-hexylcyclopropane, which was also obtained by a known sequence from the (1R,2S)-monobutyrate ester of cis-cyclopropane-1,2-dimethanol. This pivotal aldehyde was converted into (11R,12S)-lactobacillic acid. Using analogous chemistry, the (11S,12R)-enantiomer of lactobacillic acid was prepared from 2,3-O-isopropylidene-d-glyceraldehyde or from the (1S,R)-monobutyrate ester of cis-cyclopropane-1,2-dimethanol.

Mycobacterium tuberculosis continues to be the predominant global infectious agent, annually killing over three million people. Recommended drug regimens have the potential to control tuberculosis, but lack of adherence to such regimens has resulted in the emergence of resistant strains. Mycobacterium tuberculosis has an unusual cell envelope, rich in unique long-chain lipids, that provides a very hydrophobic barrier to antibiotic access. Such lipids, however, can be drug targets, as exemplified by the action of the front-line drug isoniazid on mycolic acid biosynthesis. A number of these lipids are potential key virulence factors and their structures are based on very characteristic methyl-branched long-chain acids and alcohols. This review details the history, structure, and genetic aspects of the biosynthesis of these methyl-branched components, good examples of which are the phthiocerols and the mycocerosic and mycolipenic acids.

Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.

(11 R , 12 S )-Lactobacillic acid ( 1 ) has been prepared either from D-mannitol by asymmetric cyclopropanation or from cis -cyclopropane-1, 2-dimethanol by enzymatic desymmetrisation. The syntheses of (11 S 12 R )-lactobacillic acid ( 2 ) and (1 R , 2 S ) 1-(3′-methoxycarbonylpropyl)-2-octadecylcyclopropane ( 26 ) and related analogues ( 27 and 28 ) have also been achieved.

Racemic 2,4-dimethyldocosanal, available from previous studies, was oxidised to the aldehyde using pyridinium chlorochromate. Aldol reaction of 2,4-dimethyldocosanal with methyl propionate afforded racemic methyl 3-hydroxy-2,4,6-trimethyltetracosanoic acid. The hydroxy function was protected as a tert -butyldimethylsilyl ether and the ester functionality hydrolysed to the free acid. Wittig reaction of 2,4-dimethyldocosanal using 1-carboethoxyethylidenetriphenylphosphorane afforded the ( E )-stereoisomer following removal of the small quantities of ( Z )-isomer by column chromatography. The ethyl ester was hydrolysed to afford racemic 2,4,6-trimethyltetracos-2-enoic acid. These unusual fatty acids are characteristic of Mycobacterium tuberculosis .

A known intermediate, (3 R, S; 5 R,S)-3,5-dimethyl-6- triphenylmethyloxyhexanal gave, in a Wittig reaction with 1-octyltriphenylphosphonium bromide and n-butyl lithium in anhydrous tetrahydrofuran, a mixture of olefininic compounds. Trityl deprotection and hydrogenation of the double bond afforded the (2 R, S; 4 R,S)-2,4- dimethyltetradecanols. Oxidation of the alcohol functionality using pyridinium dichromate in anhydrous N,N′-dimethylformamide gave racemic, (2 R, S; 4 R,S)-2,4-dimethyltetradecanoic acids, which were converted to their methyl esters. 2,4-Dimethyltetradecanoic acids are constituents of glycolipid antigens from Mycobacterium kansasii.

The absolute stereochemistry of 2,4-dimethyleicos-2-enoic acid, isolated from the pyruvylated glycolipid of Mycobacterium smegmatis, has been determined. The two enantiomers of methyl 2,4-dimethyleicos-2-enoate were synthesised for the first time but could not be separated by gas chromatography on cyclodextrin phases. (E)-2-Methyloctadec-2-enoate, an intermediate in the synthesis, is a characteristic component of acyl trehalose glycolipids from Mycobacterium fortuitum. Ozonolysis of the fatty acid ester mixture, obtained from the pyruvylated glycolipid produced 2-methyloctadecanoate. It was identified as the (S)-enantiomer by comparison with (2R) and (2S)-2-methyloctadecanoic acid, intermediates in the synthesis of (4R)- and (4S)-2,4,-dimethyleicos-2-enoic acid, using enantioselective gas chromatography of the methyl esters with heptakis(2,6-di-O-methyl-3-O-pentyl)-beta-cyclodextrin as a chiral stationary phase. The natural acid was therefore determined to be 2E-(4S)-2,4-dimethyleicos-2-enoic acid.

The difference in relative stereochemistry of the 1.3-diol unit of mycobacterial phthiocerols can be determined by simple thin-layer chromatographic analysis of pentafluorobenzylidene acetal derivatives. The threo phthiocerol acetals from Mycobacterium tuberculosis are composed of equal amounts of two axial-equatorial stereoisomers but the erythro phthiocerols from Mycobacterium marinum form only one acetal with diequatorial substituents. The two acetals formed from a threo phthiocerol can be separated by thin-layer chromatography and mass spectra of the two stereoisomers allowed assignment of their structures.

The aim was to 'triprotect' trehalose by placing various acetals, or related protecting groups, across the 4,6-, 2',3'-, and 4',6'-positions, leaving the 2,3-positions free for subsequent acylation. Isopropylidene and ethylidene acetals were studied, with the formation of a small amount of 4,6:2',3':4',6'-tri-O-isopropylidene-alpha,alpha'-trehalose. 4,6:4',6'-Di-O-benzylidene-2',3'-O-(tetraphenyldisiloxane-1,3-d iyl)-alpha, alpha'-trehalose 2,3-diacetate was prepared in low yield. 1,1-Dimethoxycyclohexane reacted with methyl alpha-D-glucopyranoside to afford the 4,6-O-cyclohexylidene derivative, isolated as the diacetate; mild acid cleavage of the acetal gave the 2,3-diacetate. 4,6:2',3':4',6'-Tri-O-cyclohexylidene-alpha,alpha'-trehalose is the major product of the reaction between alpha,alpha'-trehalose and 1,1-dimethoxycyclohexane. 2,3:4,6:2',3':4',6'-Tetra-O-cyclohexylidene-, 4,6:4',6'-di-O-cyclohexylidene-, and 4,6-O-cyclohexylidene-alpha,alpha'-trehaloses were also isolated in lower yields, all acetals being characterised as their peracetates. The proportions of the different trehalose acetals were dependent upon the molar ratio of 1,1-dimethoxycyclohexane and particularly on the reaction temperature. The triprotected trehalose acetal was acylated with palmitic acid, with excellent regioselectivity, affording the 2-O-palmitoyl ester. This 2-monoacylated, triprotected trehalose is a key intermediate for the synthesis of 2,3-di-O-acyl-alpha,alpha'-trehalose glycolipid antigens, isolated from Mycobacterium fortuitum and Mycobacterium tuberculosis.

2,4-Dimethyldocosanoic acid is a major acyl component of 2,3-di- O -acyl- α , α -trehalose glycolipid antigens isolated from Mycobacterium tuberculosis . Racemic 2,4-dimethyldocosanoic acid has been synthesised and the chiral centre at C-2 resolved as an (R)-(−)-naphthylethylamide. An isomeric mixture of 3,5-dimethylcyclohexan-l-ols was oxidised to isomeric 3,5-dimethylcyclohexan-l-ones. This was subjected to Baeyer-Villiger oxidation to give isomeric 3,5-dimethylcaprolactones. Ring opening under alkaline conditions followed by phase-transfer catalysed esterification gave isomeric methyl 3,5-dimethyl-6-hydroxyhexanoates. Protection of the alcohol with triphenylmethyl chloride followed by lithium aluminium hydride reduction and pyridinium chlorochromate oxidation gave isomeric 3,5-dimethyl-6-triphenylmethyloxyhexanals. Coupling with hexadecyltriphenylphosphonium bromide, followed by trityl deprotection and hydrogenation of the remaining alkene, yielded isomeric 2,4-dimethyldocosanols. Oxidation with pyridinium dichromate in N , N ′-dimethylformamide produced isomeric 2,4-dimethyldocosanoic acids, which were resolved at C-2 as diastereoisomeric (R)-(−)-naphthylethylamides.

The high molecular weight 2-alkyl-3-hydroxy mycolic acids are key structural components of the cell envelope of pathogenic mycobacteria, such as Mycobacterium tuberculosis. A prime target for drug action would be the initial stages where the biosynthetic pathways diverge from those of ordinary fatty acids. It has been postulated that the pathway for the α-mycolates, without oxygen functions in addition to the hydroxy-acid unit, appears to diverge from (Z)-tetracos-5-enoic acid. The biosynthesis of oxygenated mycolic acids is considered to possibly diverge from (E)-6-(R)-methyltetracos-4-enoic and (E)-6-(S)-methyltetracos-4-enoic acids. This communication describes the synthesis of esters of these acids in order to test their potential role in the biosynthesis of mycolic acids.

The high molecular weight 2-alkyl-3-hydroxy mycolic acids are key structural components of the cell envelope of pathogenic mycobacteria such as Mycobacterium tuberculosis. It has been shown recently that (Z)-tetracos-5-enoic acid is a key initial intermediate in mycolic acid biosynthesis in extracts of Mycobacterium smegmatis. This acid is presumably formed by desaturation of tetracosanoate, and previous studies on oleic acid biosynthesis suggest that a cyclopropene analogue may be a potential inhibitor. This communication describes the synthesis of methyl 4-(2-octadecylcyclopropen-l-yl)butanoate, which is shown elsewhere to be an inhibitor of the initial stages of mycolic acid synthesis.

Seventy-nine representative strains of Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum and Mycobacterium senegalense were analysed by thin-layer chromatography for diagnostic glycolipid and mycolic acid patterns. On the basis of glycolipid patterns the 16 M. senegalense strains were assigned to four groups (I, II, III and IV); one strain received as M. farcinogenes had glycolipids that corresponded to those given by the M. senegalense group III strain. Representatives of M. fortuitum produced alkalilabile glycolipids whereas the M. peregrinum strains produced alkali-stable glycopeptidolipids related to those of M. senegalense group I. Members of M. senegalense groups I, III and IV and the M. farcinogenes strains gave the characteristic pattern consisting of alpha- and epoxymycolates on thin-layer chromatographic analysis of the products of alkaline hydrolysis with 5% aqueous tetrabutylammonium hydroxide. Extraction of mycolates from the same strains using acid methanolysis revealed alpha-mycolates and characteristic more polar long-chain components originating from epoxymycolates. In contrast, the M. senegalense group II strains contained an additional more polar omega-1 methoxymycolate that was detected from both acid and alkaline hydrolysates; this spot was also seen in some M. fortuitum and M. peregrinum strains. Preliminary investigations, using a combination of thin-layer chromatography and immunostaining, showed that glycopeptidolipids from M. peregrinum and M. senegalense group I strains were antigenic, a cross-reaction between members of these taxa was shown. In contrast, no reaction was detected between glycopeptidolipids extracted from M. peregrinum and M. senegalense group I strains with antisera from either M. farcinogenes or M. fortuitum.

Mycolic acids are high molecular weight hydroxy fatty acids which are a covalently linked part of the cell wall structure of all mycobacteria and their biosynthetic pathways offer potential drug targets. Three good candidates, cis -tetracos-5-enoic acid and R or S trans -6-methyl-tetracos-4-enoic acids, for the key initial intermediates where mycolic acid biosynthesis might diverge from other metabolic pathways, were tested as possible substrates. A cell-wall preparation from Mycobacterium smegmatis , capable of mycolic acid synthesis, was developed to investigate the possible incorporation of these, and other 16 to 24 carbon acids into mycolic acids. The wall preparations were extracted with hexane and suspended in hexane/water (7:1, v/v), and in this low-water assay, only one of these acids, cis -tetracos-5-enoic acid, stimulated the incorporation of radioactive label from [1- 14 C]acetate into α- and α'-mycolic acids. The extraction method used did, however, abolish some enzyme activity and mycolic acid biosynthesis was not completely restored by cis -tetracos-5-enoate. The two methyl-branched acids did not enhance the amount of label in epoxymycolic acids. An initial key intermediate in the synthesis of α- and α'-mycolic acids has therefore been positively identified for the first time; intermediates in the initial stages of the biosynthesis of oxygenated mycolic acids such as epoxymycolates remain to be defined.

The phenolic glycolipids from two strains of Mycobacterium marinum have been isolated and characterised. The glycolipids from M. marinum MNC 170 were principally glycosides of diacyl C37, C39 and C41, phenolphthiocerols A, but in M. marinum MNC 842, these lipids were accompanied by glycosides of diacyl phenolphthiodiolones A and novel phthiotriols A with the same overall chain-lengths. The main acyl components of the phenolic glycolipids from M. marinum MNC 170 were C26 dimethyl and C27 and C29 trimethyl-branched fatty acids, but in the lipids of M. marinum MNC 842, the C27 trimethyl acid was the only principal component. The sugar composition of all these glycolipids had been previously shown to correspond to 3-O-methylrhamnose.

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.

Seven strains of Rothia dentocariosa were degraded by acid methanolysis and the nonhydroxylated fatty acid methyl esters released were examined by thin-layer and gas chromatography. The fatty acid profiles were composed of iso-, anteiso- and straight chain saturated fatty acids with 12-methyltetradecanoic (anteiso-C15), 14-methylpentadecanoic (iso-C16), 14-methylhexadecanoic (anteiso-C17) and hexadecanoic acid (C16) as major components. A small scale integrated procedure was used for the sequential extraction of isoprenoid quinones and polar lipids. The latter were examined by two-dimensional thin-layer chromatography and all of the test strains contained diphosphatidylglycerol, phosphatidylglycerol and two uncharacterised glycolipids. In all cases the major isoprenoid quinones were unsaturated menaquinones with seven isoprene units. Analyses of the cell wall amino acid composition using gas chromatography showed that the strains contained 2.5 to 5 moles of alanine and 1 mole each of glutamic acid and lysine. The chemical data support the integrity of Rothia dentocariosa and can be used to separate it from all other actinomycetes especially those which contain lysine in the wall peptidoglycan.

A simple small-scale procedure for the sequential extraction of isoprenoid quinones and polar lipids from bacterial cells was developed. Extraction with a biphasic mixture of petroleum ether (b.p. 60–80°C) and methanolic saline gave an upper phase containing isoprenoid quinones. The lower phase, containing the partially extracted organisms, was processed according to the Bligh and Dyer extraction method to give a polar lipid extract. As examples of the procedure, the isoprenoid quinones and polar lipids of Bacillus subtilis, Mycobacterium avium, Pseudomonas diminuta and Streptomyces griseus were extracted and analyzed.

Whole-organism methanolysates of representative bacteria containing mycolic acids (long-chain 3-hydroxy 2-branched acids) were prepared by treating dry organisms with methanol-toluene-sulphuric acid (30:15:1) at 75° overnight. The mycolic acid esters from mycobacteria were not resolved satisfactorily by single dimensional thin-layer chromatographic analysis. Three distinct patterns of mycobacterial mycolates were produced by two-dimensional thin-layer chromatography, good examples of each pattern being the mycolates from Mycobacterium tuberculosis , Mycobacterium avium and Mycobacterium fortuitum .

Three characteristic waxes, dimycocerosates of the phthiocerol family, have been detected in the free lipids and also acid methanolysates of Mycobacterium tuberculosis by use of a special two-dimensional thin-layer Chromatographic system. Reductive degradation of the least, intermediate and most polar waxes gave mycocerosic alcohols in all cases and phthiocerol A, phthiocerol B and phthiotriol A, respectively. The most polar wax was selectively reduced by sodium borohydride, showing that the diol constituent was a keto component, phthiodiolone A, rather than the reduced compound, phthiotriol A. Gas chromatography and mass spectrometry of all the wax reduction products showed that the myococerosates were the expected tri- and tetramethyl branched acids, with a C 32 main component, and the phthiocerols A, B and phthiodiolone A components were similar to the C 33 to C 36 methoxy or keto 1,3-diols isolated previously from M. tuberculosis . Dimycocerosates of phthiocerol A have been described previously; the dimycocerosates of phthiocerol B and phthiodiolone A have, in this study, been isolated for the first time.

Previous studies showed that whole-organism acid methanolysates of representatives of Mycobacterium fortuitum, Mycobacterium farcinogenes and Mycobacterium senegalense contained characteristic mixtures of polar oxygenated mycolic acid methyl esters. These polar mycolates were absent in alkaline methanolysates of these organisms, being replaced by a single oxygenated mycolic acid methyl ester. This latter mycolate had the chemical and spectroscopic properties of a long-chain epoxide and was converted, on acid methanolysis, to the characteristic components present in whole-organism acid methanolysates. Spectroscopic analyses, supported by chemical transformations and degradations, showed that the homologous series of epoxymy colic acids present in these bacteria had carbon skeletons (main component C79) similar to those previously found in mycobacterial keto, methoxy and wax-ester mycolates. The characteristic functional group in these epoxymycolic acids is an unusual trans epoxy group with a methyl branch on the adjacent carbon remote from the mycolate 2-alkyl-branched, 3-hydroxy acid unit.

Summary Chemotaxonomic data from investigations of fatty acids, isoprenoid quinones and polar lipids provide further evidence that the genus Stomatococcus is compact and well delineated from both Micrococcus and Staphylococcus .

Comparative immunodiffusion studies were performed on representative strains of “ Mycobacterium album ”, the “ aurantiaca ” taxon and an “ aurantiaca ”-like group of actinomycetes, using reference systems representing 25 taxa assigned to the genera Arthrobacter, Corynebacterium, Kurthia, Mycobacterium, Nocardia, Rhodococcus, Streptomyces and the “ aurantiaca ” taxon. Thin-layer chromatographic analyses of whole-organism methanolysates were carried out on single strains of “ Mycobacterium album ” and the “ aurantiaca ”-like taxon. Both the distribution of precipitinogens and the type of mycolic acids determined showed that the “ Mycobacterium album ” and “ aurantiaca ” strains were closely related to one another, but not to the “ aurantiaca ”-like strains. The aggregate “ aurantiaca ” taxon, containing “ Mycobacterium album ” and the “ aurantiaca ” taxon, can be distinguished from the established mycolic-acid containing taxa, Corynebacterium, Mycobacterium, Nocardia and Rhodococcus , using both serological and lipid data. Further studies on additional strains are needed to determine whether the aggregate “ aurantiaca ” taxon merits generic status.

Epoxides of methyl esters of elaidic and oleic acids were allowed to react with methanol, ethanol, n -propanol, iso -propanol and n -butanol, in the presence of iodine, to give the corresponding alkoxyhydroxy methyl esters. Ethyl elaidate epoxide gave a hydroxymethoxy methyl ester when treated with boron trifluoride in methanol but the ethyl ester group was not attacked with iodine as catalyst. Mass spectra of the alkoxyhydroxy esters contained strong peaks which demonstrated the location in the chain of the original epoxide ring. Iodine also catalysed the addition of water to methyl elaidate or oleate, giving erythro - and threo -9,10-dihydroxyoctadecanoates, respectively. The alkoxyhydroxy esters were quantitatively converted to t -butyldimethylsilyl ethers by reaction with t -butyldimethylchlorosilane/imidazole/dimethylformamide reagent at 100°C but the dihydroxyoctadecanoates were not completely derivatised. Mass spectra of all the t -butyldimethylsilyl ether derivatives contained intense fragments allowing the molecular weights and the positions of the ether functions to be easily determined.

Two strains of Arachnia propionica and two Arachnia-like isolates were degraded by alkaline methanolysis and the non-hydroxylated fatty acid esters released were examined by thin-layer and gas chromatography. The fatty acid profiles obtained were both qualitatively and quantitatively similar and were comprised of iso-, anteiso- and straight chain saturated fatty acids with 13-methyltetradecanoic acid (i-15) and 12-methyltetradecanoic acid (ai-15) as major components. One of the Arachnia-like strains (HIK 288) had, in addition to i-15 and ai-15, major amounts of pentadecanoic acid (15:0). All of the strains gave characteristic polar lipid patterns consisting of diphosphatidylglycerol, phosphatidylglycerol and two incompletely characterised glycolipids. Analyses of wall amino acid preparations by gas and thin-layer chromatography showed that Arachnia propionica strains contain major amounts of alanine, glycine, glutamic acid and LL-diaminopimelic acid. The chemical data support the integrity of Arachnia propionica and provide a valuable means of differentiating it from morphologically and physiologically similar strains of Actinomyces israelii.

Electron-impact mass spectrometric procedures for locating the position of double bonds and cyclopropane rings in long-chain fatty acids are reviewed. Since unsaturation is not located directly by mass spectrometry, the properties of suitable derivatives are summarized. Epoxides are readily prepared from double bonds and on opening of the ring with various reagents useful derivatives are obtained, the most promising to date being hydroxymethoxy esters whose trimethylsilyl ethers give good mass spectra. Trimethylsilyl ethers of vicinal diols, prepared by direct hydroxylation, are recommended for the analysis of polyunsaturated fatty acid esters using combined gas chromatography-mass spectrometry. Oxymercuration-demercuration techniques are very convenient and one particular procedure can specifically locate unsaturation up to five carbons distant from the carboxyl group. An alternative approach enables the location of double bonds and cyclopropane rings in fatty acids by direct mass spectrometry of pyrrolidides. Cyclopropane rings can be positively located in fatty acid esters by mass spectrometry of isomeric ketones or methoxy derivatives prepared by chromium trioxide oxidation on poron trifluoride-catalysed methoxylation, respectively. A variety of other procedures are also considered and some guidelines are given for choosing a method to suit a particular unsaturated acid.

Bacillus subtilis W23 and Bacillus cereus T, grown under conditions of apparent phosphate starvation approaching stationary phase of growth, accumulate diglucosyl diglycerides while the proportion of phosphatidylethanolamine is reduced. The latter organism also produces under these conditions an acidic glycolipid which apparently partially replaces the acidic phospholipids, phosphatidylglycerol and diphosphatidylglycerol.