- Volume 148, Issue 10, 2002

A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.

A polymorphic region was discovered in the genetically uncharacterized opportunistic pathogen Mycobacterium abscessus. The region contains a novel 1·7 kb insertion sequence (IS) named ISMab1. ISMab1 contains two complete ORFs and one partial ORF located in segments with over 80% nucleotide identity to Mycobacterium avium IS1601 and IS999 and to previously unreported IS-like elements from Mycobacterium smegmatis. The marked similarity within this family of elements is supportive of horizontal transfer between environmental mycobacterial species. In clinical isolates, ISMab1 was either present as a single copy or absent. The polymorphic region containing ISMab1 was identified by genomic subtraction between a parental strain and phenotypic variant. The variant has a 14·2 kb genomic deletion and this is flanked in the parental strain by complex arrays of inverted and direct repeats. Clinical isolates of M. abscessus were probed for the deletion and flanking sequences and two were found to be missing more than 20 kb. No regional deletions were found in the type strain, ATCC 19977. Although M. abscessus is a rapidly growing species, comparative sequence analysis of 23 kb from the polymorphic region showed that most local ORFs have greater amino acid identity to proteins encoded by genes from the slowly growing mycobacteria, M. avium and Mycobacterium tuberculosis, than to the rapid-grower M. smegmatis. Several ORFs also have strong similarity to Pseudomonas aeruginosa genes with a potential role in β-oxidation.

mce3 is one of the four mce operons in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence of this bacterium. Upstream of mce3 there is a putative regulatory gene (Rv1963) that harbours a double tetR-family signature. To study the role of this putative regulatory gene in the transcriptional regulation of the mce3 operon, Mycobacterium smegmatis mc2155 and M. tuberculosis H37Rv strains that harboured gene fusions between the mce3 promoter region and the Escherichia coli lacZ gene, either containing or not containing the Rv1963 gene, were used. The presence of the Rv1963 gene in the strains greatly reduced β-galactosidase activity, suggesting that the Rv1963-encoded protein is a transcriptional repressor of the mce3 operon. Expression of mce3 by recombinant M. tuberculosis was increased when it was grown in a macrophage-like cell line (J774), compared to the level of expression seen when the recombinant bacterium was grown under in vitro conditions. However, no lifting of repression was induced. The mce3 promoter was defined by deletion and cloning of the Rv1963–Rv1964 intergenic region in a 200 bp DNA fragment harbouring the region upstream of the Rv1964 start codon. Gel-shift experiments determined that the Rv1963-binding site was located in this region. These results indicate that the mce3 operon is transcriptionally regulated and that under certain, unknown, conditions repression of gene expression could be lifted.

The authors have developed a simple and highly efficient system for generating allelic exchanges in both fast- and slow-growing mycobacteria. In this procedure a gene of interest, disrupted by a selectable marker, is cloned into a conditionally replicating (temperature-sensitive) shuttle phasmid to generate a specialized transducing mycobacteriophage. The temperature-sensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 °C but prevent replication at the non-permissive temperature of 37 °C. Transduction at a non-permissive temperature results in highly efficient delivery of the recombination substrate to virtually all cells in the recipient population. The deletion mutations in the targeted genes are marked with antibiotic-resistance genes that are flanked by γδ-res (resolvase recognition target) sites. The transductants which have undergone a homologous recombination event can be conveniently selected on antibiotic-containing media. To demonstrate the utility of this genetic system seven different targeted gene disruptions were generated in three substrains of Mycobacterium bovis BCG, three strains of Mycobacterium tuberculosis, and Mycobacterium smegmatis. Mutants in the lysA, nadBC, panC, panCD, leuCD, Rv3291c and Rv0867c genes or operons were isolated as antibiotic-resistant (and in some cases auxotrophic) transductants. Using a plasmid encoding the γδ-resolvase (tnpR), the resistance genes could be removed, generating unmarked deletion mutations. It is concluded from the high frequency of allelic exchange events observed in this study that specialized transduction is a very efficient technique for genetic manipulation of mycobacteria and is a method of choice for constructing isogenic strains of M. tuberculosis, BCG or M. smegmatis which differ by defined mutations.

A better tuberculosis vaccine is urgently required to control the continuing epidemic. Molecular techniques are now available to produce a better live vaccine than BCG by producing avirulent strains of the Mycobacterium tuberculosis complex with known gene deletions. In this study, 1000 illegitimate recombinants of Mycobacterium bovis were produced by illegitimate recombination with fragments of mycobacterial DNA containing a kanamycin resistance gene. Eight recombinant strains were selected on the basis of their inability to grow when stationary-phase cultures were inoculated into minimal medium. Five of these recombinants were found to be avirulent when inoculated into guinea pigs. Two of the avirulent recombinants produced vaccine efficacy comparable to BCG against an aerosol challenge in guinea pigs with M. bovis. One of these recombinants had an inactivated glnA2 gene encoding a putative glutamine synthetase. Transcriptional analysis showed that inactivation of glnA2 did not affect expression of the downstream glnE gene. The other recombinant had a block of 12 genes deleted, including the sigma factor gene sigG. Two avirulent recombinants with an inactivated pckA gene, encoding phosphoenolpyruvate carboxykinase which catalyses the first step of gluconeogenesis, induced poor protection against tuberculosis. It is clear that live avirulent strains of the M. tuberculosis complex vary widely in their ability as vaccines to protect against tuberculosis. Improved models may be required to more clearly determine the difference in protective effect between BCG and potential new tuberculosis vaccines.

Mycobacterium tuberculosis encodes two-component signal systems. Recently, it was established that the viability of the M. tuberculosis phoP mutant is attenuated in the mouse, suggesting the requirement of the phoP gene for M. tuberculosis intracellular growth. It is now largely acknowledged that M. tuberculosis mannosylated lipoarabinomannans (ManLAM) play a key role in M. tuberculosis intramacrophagic survival by altering the macrophage functions. So ManLAM were extracted and purified from the M. tuberculosis MT103 wild-type strain and from the M. tuberculosis phoP mutant. Their two major functional domains (i) the mannooligosaccharide caps and (ii) the mannosyl phosphatidylinositol anchor were here investigated. Using capillary electrophoresis, it is demonstrated that both mutant and wild-type M. tuberculosis strains share the same capping motifs: mono-, di- and trimannosyl α(1→2) units, with the same relative abundance. Using two-dimensional NMR spectroscopy, the same acyl forms were found to be shared by both strains. However, their relative abundance was quite different. Indeed, in the phoP mutant a decrease of the triacylated ManLAM and an increase of the monoacylated ManLAM were observed. The difference in the proportion of ManLAM acyl forms and the reduced virulence of the M. tuberculosis phoP mutant are discussed.

There has been a general understanding that Mycobacterium smegmatis produces only apolar glycopeptidolipid (GPL), similar in structure to serovar non-specific GPL of Mycobacterium avium. In this study, synthesis of polar GPL in carbon-starved M. smegmatis is reported. Mass spectrometric analysis suggests the polar GPL to be a hyperglycosylated species. The earlier structural studies of polar GPLs from M. avium have invariably shown the presence of an oligosaccharide appendage to D-allo-Thr. However, a further chemical analysis using β-elimination of the newly found polar GPL in M. smegmatis shows that the molecule still contains a monosaccharide at the D-allo-Thr, thus suggesting a new form of polar GPL.

mAb CS-35 is representative of a large group of antibodies with similar binding specificities that were generated against the Mycobacterium leprae lipopolysaccharide, lipoarabinomannan (LAM), and which cross-reacted extensively with LAMs from Mycobacterium tuberculosis and other mycobacteria. That this antibody also cross-reacts with the arabinogalactan (AG) of the mycobacterial cell wall, suggesting that it recognizes a common arabinofuranosyl (Araf)-containing sequence in AG and LAM, is demonstrated. The antibody reacted more avidly with ‘AraLAM’ (LAM with naked Araf termini) compared to ‘ManLAM’ (in which many Araf termini are capped with mannose residues) and mycolylarabinogalactan–peptidoglycan complex (in which the terminal Araf units are substituted with mycolic acids). Neither did the antibody bind to AG from emb knock-out mutants deficient in the branched hexa-Araf termini of AG. These results indicate that the terminal Araf residues of mycobacterial arabinan are essential for binding. Competitive ELISA using synthetic oligosaccharides showed that the branched hexa-Araf methyl glycoside [β-D-Araf-(1→2)-α-D-Araf-(1-)2-(3 and 5)-α-D-Araf-(1→5)-α-D-Araf-OCH3] was the best competitor among those tested. The related linear methyl glycoside, β-D-Araf-(1→2)-α-D-Araf-(1→5)-α-D-Araf-(1→5)-α-D-Araf-OCH3, representing one linear segment of the branched hexa-Araf, was less effective and the other linear tetrasaccharide, β-D-Araf-(1→2)-α-D-Araf-(1→3)-α-D-Araf-(1→5)-α-D-Araf-OCH3, was ineffective. The combined results suggest that the minimal epitope recognized by antibody CS-35 encompasses the β-D-Araf-(1→2)-α-D-Araf-(1→5)-α-D-Araf-(1→5)-α-D-Araf within the branched hexa-Araf motif of mycobacterial arabinans, whether present in LAM or AG.

To study the late events of cell wall assembly in Mycobacterium smegmatis, specific in vivo radiolabelling of exponentially growing liquid cultures over periods of less than one cell generation were carried out. N-Acetyl-[14C]glucosamine was used to label peptidoglycan and [14C]glucose to label arabinogalactan and arabinomannan. Over periods of several generations, radioactive cell wall material was turned over as soluble autolysis products into the culture fluid. However, turnover of newly synthesized and labelled cell wall was delayed for about one cell generation, implying inside-to-outside growth of the wall as observed in Bacillus. Little radioactive wall material was released into the culture fluid during the first generation of labelling in growing cultures, but the addition of amoxicillin plus the β-lactamase inhibitor clavulanic acid, at the minimum inhibitory concentration of amoxicillin, led to the release of radioactive peptidoglycan that could be isolated by gel filtration chromatography and contained nearly 3 mol alanine per glutamic acid residue, indicating that it was linear, un-crosslinked peptidoglycan that had never been substantially cross-linked to the cell wall due to inhibition of transpeptidation by amoxicillin. This peptidoglycan had no covalently attached arabinogalactan. Radioactive arabinogalactan was synthesized and released from the amoxicillin-treated bacteria without attachment to peptidoglycan. The results indicate that during growth, incorporation of arabinogalactan into the cell wall requires its ligation to newly synthesized peptidoglycan and that the peptidoglycan must be undergoing concomitant cross-linking to the inner surface of the cell wall. Inhibition of peptidoglycan transpeptidation prevents ligation of arabinogalactan to peptidoglycan and its consequent incorporation into the wall.

Attempts to construct Mycobacterium tuberculosis strains with a defect in the common aromatic amino acid biosynthesis pathway were made. In other bacteria the genes of this pathway (aro) can be disrupted in the presence of suitable media supplements. The genomic organization of the aro genes in M. tuberculosis reveals that there is one operon (aroCKBQ) and three isolated aro genes (aroE, aroG and aroA). The aroK gene was chosen as a target for disruption; this encodes shikimate kinase, which catalyses the fifth step in chorismate biosynthesis. Attempts to replace the wild-type aroK gene with a disrupted allele (aroKΔ::hyg) by a two-step homologous recombination procedure were unsuccessful in a wild-type strain. When a second functional copy of aroK was integrated into the chromosome, it was possible to isolate a strain carrying the disrupted gene. Excision of the L5-integrated copy of aroK by the L5 excisionase could be not be achieved in the strain carrying the disrupted copy, but was possible in a strain carrying a wild-type copy. These results demonstrate that the chorismate pathway is essential for the viability of M. tuberculosis.

Glycopeptidolipids (GPLs) are a major component of the outer layers of the cell walls of several non-tuberculous mycobacteria. The Mycobacterium smegmatis GPLs consist of a diglycosylated lipopeptide core which is variably modified by acetylation and methylation. Analysis of a region of the M. smegmatis chromosome, upstream of the peptide synthetase gene, mps, revealed a GPL biosynthetic locus containing genes potentially involved in glycosylation, methylation, acetylation and transport of GPLs. Methyltransferases are required to modify rhamnose and the fatty acid of GPLs. Of the four methyltransferases encoded within the locus, one methyltransferase, Mtf2, was unlike sugar methyltransferases from other species. An mtf2 mutant was created and was shown to be unable to methylate the GPL fatty acids. Direct evidence is presented that Mtf2 is a methyltransferase that modifies the GPL fatty acid.

Glycopeptidolipids (GPLs) are a class of species- or type-specific mycobacterial lipids and major constituents of the cell envelopes of many non-tuberculous mycobacteria. To determine the function of GPLs in the physiology of these bacteria, a mutant of Mycobacterium smegmatis in which the gene encoding a mycobacterial nonribosomal peptide synthetase has been inactivated by transposon mutagenesis was analysed. Labelling experiments indicated that half of the bacterial GPLs were located on the cell surface and represented 85% of the surface-exposed lipids of the parent strain whereas the mutant was defective in the production of the GPLs. Compared to the parent smooth morphotype strain, the GPL-deficient mutant strain exhibited a rough colony morphology, an increase of the cell hydrophobicity and formed huge aggregates. As a consequence, the mutant cells were no longer able to bind ruthenium red, as observed by transmission electron microscopy. The altered surface properties of the mutant cells also affected the phagocytosis of individual bacilli by human monocyte-derived macrophages since mutant cells were internalized more rapidly than cells from the parent strain. Nevertheless, no specific release of surface constituents into the culture broth of the mutant was observed, indicating that the cell surface is composed of substances other than GPLs and that these are essential for maintaining the architecture of the outermost layer of the cell envelope. Importantly, the absence of these major extractable lipids of M. smegmatis from the mutant strain has a profound effect on the uptake of the hydrophobic chenodeoxycholate by cells, indicating that GPLs are involved in the cell wall permeability barrier of M. smegmatis. Altogether, these data showed that, in addition to being distinctive markers of numerous mycobacterial species, GPLs play a role in the bacterial phenotype, surface properties and cell wall permeability.

Analogues of the antibiotic thiolactomycin (TLM) have been synthesized and have been shown to have enhanced activity against whole cells of Mycobacterium tuberculosis H37Rv and against mycolic acid biosynthesis in cell extracts of Mycobacterium smegmatis. TLM has a methyl-branched butadienyl side chain attached at position 5 on a ‘thiolactone’ ring, namely 4-hydroxy-3,5-dimethyl-5H-thiophen-2-one. Various combinations of strong bases were explored to create a reactive anion at position 5 on the thiolactone ring which could react with halides to produce 5-substituted derivatives; the best reagent was two equivalents of lithium-bis-(trimethylsilyl)amide in tetrahydrofuran. The analogue with a 5-tetrahydrogeranyl substituent showed the best biological activity with an MIC90 for M. tuberculosis of 29 μM and 92% mycolate inhibition in extracts of M. smegmatis, as compared to 125 μM and 54%, respectively, for TLM; other related C10 and C15 isoprenoid derivatives had similar biological activity. These isoprenoid-based derivatives did not inhibit type II fatty acid synthase from M. smegmatis, but compounds with iso-butyl and iso-butenyl side chains did show some inhibitory activity against this enzyme. These short-chain derivatives did not inhibit mycolate synthesis or have significant antibiotic activity. Treatment of the thiolactone with a weaker base, sodium hydride in tetrahydrofuran, gave 3-alkyl-3,5-dimethyl-thiophene-2,4-dione analogues, which had no effect on fatty acid or mycolate synthesis. However, the geranyl derivative had an MIC99 of 60 μM for M. tuberculosis, one quarter that (240 μM) of TLM, demonstrating its excellent antibiotic potential against an unknown cellular target.

Annually, Mycobacterium tuberculosis is the cause of approximately three million deaths worldwide. It would appear that currently available therapies for this disease are inadequate. The identification of genes involved in mycobacterial virulence will facilitate the design of new prophylactic and therapeutic interventions. A method for high-resolution comparison of bacterial genomes has been developed to facilitate the identification of genes possibly involved in the virulence of clinically relevant mycobacteria. This ‘two-dimensional bacterial genome display’ (2DBGD) method utilizes two-dimensional DNA electrophoresis to separate, on the basis of size and G+C content, genomic fragments generated with different restriction endonucleases. The use of this method to identify genomic differences between species, strains and, most importantly, isogenic mutants of mycobacteria is reported. That 2DBGD can be used to identify differences resulting from either insertional mutagenesis using a gentamicin-resistance gene or from a frameshift mutation is demonstrated.

The mycolic acids of several strains of Mycobacterium gordonae were examined by chromatographic and spectroscopic techniques. Both HPLC and TLC revealed two patterns of mycolates among the M. gordonae strains studied. As determined by TLC, one pattern was composed of α-, methoxy- and keto-mycolates; the other was composed of these mycolates plus an additional component, which was identified as dicarboxy-mycolates. The dicarboxy-mycolates were only found in those M. gordonae strains that displayed a so-called HPLC-double-cluster pattern. Detailed structural analyses of the dicarboxy-mycolates indicated that these compounds contained predominantly 61–65 carbon atoms (C63 was the major component) and a trans-1,2-disubstituted cyclopropane ring. Thus, the dicarboxy-mycolate content of strains of M. gordonae determines their HPLC pattern. In spite of the differences in their HPLC patterns, and although they belonged to different PCR-restriction length polymorphism clusters, all of the M. gordonae strains examined in this study were closely related on the basis of the structural features of their α-, keto- and methoxy-mycolates; the predominant α-mycolates contained two cis-1,2-disubstituted cyclopropane rings, the major keto-mycolates contained a trans-1,2-disubstituted cyclopropane ring and the methoxy-mycolates contained one cis- or one trans-1,2-disubstituted cyclopropane ring. It is noteworthy that the strains containing dicarboxy-mycolates also displayed significant amounts of α-mycolates that contained one cis-1,2-disubstituted cyclopropane ring and one cis double bond. The results obtained in this study demonstrate heterogeneity among M. gordonae strains.

Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass α-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heat-shock response and is related to a major antigen induced in response to anaerobic stress.

The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc2155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Kmr and M. smegmatis ahpC::Kmr showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Kmr also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNγ activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.

The mycobacterial cell wall consists of a core composed of peptidoglycan linked to the heteropolysaccharide arabinogalactan, which in turn is attached to mycolic acids. A variety of free lipids complements the mycolyl residues, whereas phosphatidylinositol mannosides (PIMs), lipoarabinomannan and proteins are interspersed in this framework. As a consequence, the cell envelope is extremely rich in lipids and early work has shown that the lipid content may vary with environmental conditions. To extend these studies, the influence of growth temperature on cell envelope components in Mycobacterium thermoresistibile, a temperature-resistant mycobacterial species, was investigated. Mycolic acid synthesis was reduced at 55 °C compared to 37 °C and the production of fatty acids, presumably precursors of mycolic acids, was increased. Since fatty acids are elongated by the type II fatty acid synthase complex and consequently by a mycobacterial β-ketoacyl acyl carrier protein synthase (KasA), leading to mycolic acids, the expression level of KasA was analysed by Western blotting. KasA expression was significantly decreased at 55 °C over 37 °C. Important changes in the mycolic acid composition were observed and characterized by reduced levels of cyclopropanation and the concomitant accumulation of the cis-olefin derivatives. In addition, striking differences involved in complex lipid composition, including acylated trehaloses and trehalose dimycolate (TDM) were also observed. At 55 °C, M. thermoresistibile produced less TDM than at 37 °C, which could be explained by the down-regulation of antigen 85 (Ag85) expression as shown by Western blotting. The Ag85 complex represents a family of proteins known to catalyse the transfer of mycolates to trehalose, thereby generating TDM. Furthermore, at 55 °C the level of phosphatidyl-inositol hexamannoside (PIM6) synthesis, but not that of other PIM species, was dramatically reduced. This observation could be correlated to a decrease of mannosyltransferase activity associated with membranes prepared from cells grown at 55 °C as compared to 37 °C. Altogether, this study suggests that mycobacteria are capable of inducing important cell-wall changes in response to temperature variations, which may represent a strategy developed by the bacteria to adapt to environmental changes.

Restriction of the growth of Mycobacterium avium was studied in wild-type and p47phox-deficient macrophages. The ability of gamma interferon and tumour necrosis factor alpha to induce antimycobacterial activity in bone-marrow-derived macrophages or the expression of the NRAMP1-mediated resistance to M. avium were not affected by the deficiency in p47phox. The addition of exogenous iron increased mycobacterial growth in macrophages expressing a functional NRAMP1 protein or a mutant NRAMP1 protein. Reactive oxygen species are therefore not involved in the constitutive or induced anti-M. avium activities of the mouse macrophage.