Mutational and phylogenetic analyses of the mycobacterial mbt gene cluster

The mycobactin siderophore system is present in many Mycobacterium species, including M. tuberculosis and other clinically relevant mycobacteria. This siderophore system is believed to be utilized by both pathogenic and nonpathogenic mycobacteria for iron acquisition in both in vivo and ex vivo iron-limiting environments, respectively. Several M. tuberculosis genes located in a so-called mbt gene cluster have been predicted to be required for the biosynthesis of the core scaffold of mycobactin based on sequence analysis. A systematic and controlled mutational analysis probing the hypothesized essential nature of each of these genes for mycobactin production has been lacking. The degree of conservation of mbt gene cluster orthologs remains to be investigated as well. In this study, we sought to conclusively establish whether each of nine mbt genes was required for mycobactin production and to examine the conservation of gene clusters orthologous to the M. tuberculosis mbt gene cluster in other bacteria. We report a systematic mutational analysis of the mbt gene cluster ortholog found in Mycobacterium smegmatis. This mutational analysis demonstrates that eight of the nine mbt genes investigated are essential for mycobactin production. Our genome mining and phylogenetic analyses reveal the presence of orthologous mbt gene clusters in several bacterial species. These gene clusters display significant organizational differences originating from an intricate evolutionary path that might have included horizontal gene transfers. Altogether, the findings reported herein advance our understanding of the genetic requirements for the biosynthesis of an important mycobacterial secondary metabolite with relevance to virulence.     

Isolation and characterization of carboxymycobactins as the second extracellular siderophores in Mycobacterium smegmatis

The structure of a chloroform-extractable siderophore from the supernatant of a nonpathogenic mycobacteria, Mycobacterium smegmatis, has been determined and it closely resembles the structure of mycobactin, the intracellular siderophore found in all mycobacteria. The difference in structures is that the extracellular siderophore has a family of short carboxylic chains attached to the mycobactin nucleus instead of a long alkyl chain and hence the name 'carboxymycobactin' is proposed to distinguish it from mycobactin itself as well as from the major siderophore that is produced - exochelin MS. © Rapid Science 1998.     

Characterization of Fe(III) sequestration by an analog of the cytotoxic siderophore brasilibactin A: implications for the iron transport mechanism in mycobacteria

Mycobacteria such as M. tuberculosis represent a significant health concern throughout much of the developing world. In mycobacteria and other pathogenic bacteria, an important virulence factor is the ability of the bacterium to obtain iron from its host. One means of obtaining iron is through the use of siderophores. Brasilibactin A is a membrane bound siderophore produced by Nocardia brasiliensis with structural similarity to the mycobactin class of siderophore in mycobacteria. A characterization of the protonation constants and Fe(III) affinity of a water soluble Brasilibactin A analog (Bbtan) has been performed. Using protonation constants and competition with EDTA, the stability constant of the 1?:?1 Fe(III)-Bbtan complex was found to be log β(110) = 26.96. The pFe of Bbtan is 22.73, somewhat low for a proposed siderophore molecule. The redox potential of the Fe-Bbtan complex was found to be -300 mV vs. NHE, very high for an iron-siderophore complex. The combination of relatively low complex stability and ease of iron reduction may play a crucial role in the mechanism of mycobactin siderophore-mediated iron uptake in mycobacteria and related organisms.     

Catecholates and mixed catecholate hydroxamates as artificial siderophores for mycobacteria

Different mono-, bis- or triscatecholates and mixed mono- or biscatecholate hydroxamates were synthesized as potential siderophores for mycobacteria. Siderophore activity was tested by growth promotion assays using wild type strains and iron transport mutants of mycobacteria as well as Gram-negative bacteria. Some triscatecholates and biscatecholate hydroxamates were active in mutants of Mycobacterium smegmatis deficient in mycobactin and exochelin biosynthesis or exochelin permease, respectively, indicating an uptake route independent of the exochelin/mycobactin pathway. Structure activity relationships were studied. Ampicillin conjugates of some of these compounds were inactive against mycobacteria but active against Gram-negative bacteria.     

Mycobactin and the competition for iron between Mycobacterium neoaurum and M. vaccae

Two closely related species of mycobacteria, Mycobacterium vaccae and M. neoaurum, were grown under conditions of iron-deficiency (0.02-0.05 microgram Fe ml-1) and iron-sufficiency (2-4 micrograms Fe ml-1) in a simple glycerol/asparagine medium. The strain of M. vaccae used was a nonmycobactin producer whereas M. neoaurum synthesized between 6-8% of its cell biomass as the lipid-soluble siderophore when grown under iron-limitation. The role of mycobactin for iron-acquisition was examined using both pure and mixed cultures, with cell viability determined following growth at various iron concentrations. M. neoaurum, the mycobactin producer, outgrew M. vaccae when iron was readily available. When grown under conditions where iron was limiting, M. neoaurum showed a decline in viable cell number compared with its competitor, highlighting its increased requirement for the metal. Some recovery was observed following mycobactin biosynthesis, this being greatly enhanced by the addition of an iron supplement to the growing cells. Mycobactin biosynthesis allowed M. neoaurum to rapidly acquire any additional iron presented to the bacteria when growing under iron-limitation. However, M. vaccae did not synthesize the lipid-soluble siderophore with its iron-requirement satisfied by production of extracellular exochelin.     

Isolation, identification, and structural analysis of the mycobactins of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium paratuberculosis.

Methods were devised to purify the cell-associated, iron-binding compounds known as mycobactins from the closely related species Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (i.e., the MAIS complex of organisms). The mycobactins from these three species showed a structure that is common to the mycobactins from all the mycobacteria examined to date. However, these mycobactins were unique in that they had more than one alkyl chain. The M. scrofulaceum mycobactins differed from other MAIS mycobactins by a shift in the position of the double bond in the R1 alkyl chain. Traces of other mycobactin types were observed in ethanol extracts of the three species, and examination of the chromatographic properties of these mycobactins showed that each species produced five mycobactin types. Each mycobactin could be subdivided further by the length of its R1 alkyl chain. No differences in the production of these novel mycobactin were observed among species. Mycobactins from three strains of Mycobacterium paratuberculosis and two wood pigeon strains of Mycobacterium avium which had lost their original growth requirements for mycobactin after repeated subculturing in laboratory growth media were examined by thin-layer chromatography and high-pressure liquid chromatography. Each organism produced a mycobactin with similar chromatographic properties to those synthesized by MAIS organisms. M. paratuberculosis NADC 18 produced at least two components in our laboratory, and nuclear magnetic resonance analysis of the major component showed this mycobactin to be identical to that produced by M. intracellulare M12. However, a sample of mycobactin J isolated by Merkal and McCullough (Curr. Microbiol. 7:333-335, 1982) from M. paratuberculosis NADC 18 was different from our isolates and appeared to correspond to a minor mycobactin component we had seen by thin-layer chromatography. No reason for this difference could be evinced. Our findings indicate that there is a close taxonomic relationship between M. paratuberculosis and the MAIS complex.     

Chemical and biological properties of mycobactins isolated from various mycobacteria

Nine different strains of mycobacteria grown on media deficient in iron all produced mycobactins. Most strains produced one mycobactin in great preponderance. Mycobacteria from clearly distinct taxonomic groups gave mycobactins differing in the structure of their nuclei. One group of taxonomically related mycobacteria produced mycobactins having the same nucleus but with different distributions of side chains within the homologous mixtures. Simple methods are described for identifying mycobactins on a small scale; these may be of value in classifying mycobacteria. Structures are proposed for mycobactin A from Mycobacterium aurum, mycobactin R from M. terrae, mycobactin F, produced together with mycobactin H by M. fortuitum, and mycobactins M and N from M. marinum. The first three of these differ from known mycobactins in details of substitution and configuration of asymmetric centres in the nucleus. Mycobactins M and N are substantially different, having only small acyl groups (acetyl and propionyl respectively) at the hydroxamic acid centre of the mycobactic acid moiety. Both are homologous mixtures having long-chain saturated 3-hydroxy-2-methyl acid fragments in the cobactin moiety. All mycobactins so far isolated promote almost maximal growth of M. johnei at 30ng./ml. in liquid medium. The activity of some mycobactins extends to much lower concentrations, mycobactin S showing significant growth promotion at 0.3ng./ml. Mycobactin M or N in combination with mycobactins having a long side chain in the mycobactic acid moiety exerts a mutually antagonistic effect on the growth of M. johnei, the mixture giving less growth than either mycobactin separately. Mycobactin M also decreases the growth of M. kansasii and M. tuberculosis on liquid media. These antagonistic effects are probably caused by a lengthening of the lag phase.     

Mycobactins in the classification and identification of armadillo-derived mycobacteria

Abstract Seven strains of armadillo-derived mycobacteria (ADMs) were encouraged to produce the lipid-soluble siderophore mycobactin when grown under conditions of iron limitation. These compounds have recently been shown to be excellent chemotaxonomic markers for the mycobacteria being both species-specific and highly conserved. Characterization of the mycobactins was carried out by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Examination of the mycobactins isolated from the ADM strains showed them to be heterogeneous. Four strains synthesized mycobactins closely resembling those of the M. avium-intracellularescrofulaceum (MAIS) complex, whereas the remaining three strains formed mycobactins which differed in structure to those of any other mycobacterium previously examined.     

Cultural and Biochemical Characteristics of Mycobacterium Paratuberculosis Isolated from Goats in Norway

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant. The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria. Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

Iron uptake processes in Mycobacterium vaccae R877R, a mycobacterium lacking mycobactin

The production of exochelins (MV) was established in Mycobacterium vaccae R877R under iron-deficient conditions in concentrations about five times greater than in Mycobacterium smegmatis. M. vaccae does not produce mycobactin nor is salicylic acid secreted into the medium. A simple method is described using 55Fe-labelled culture filtrates for assessing exochelin production and which would be applicable to other mycobacteria. One of the exochelins produced (MV3) is part of an active iron uptake system and another (MV1) is responsible for a passive uptake system. MV3 exochelin has similar chromatographic properties and biological activity to the major exochelin produced by M. smegmatis: iron uptake from MV3 exochelin was inhibited by dinitrophenol, NaN3 and HgCl2, and was judged to be an active transport process. This process was not inhibited by equimolar amounts of ferri-salicylate or ferri-citrate both of which could be used separately as sources of iron for the organism. Uptake from these latter sources was insensitive to metabolic inhibitors and uncouplers. The multiplicity of pathways for iron uptake in a single organism is discussed.     

The structure of MbtI from Mycobacterium tuberculosis, the first enzyme in the biosynthesis of the siderophore mycobactin, reveals it to be a salicylate synthase

The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 A, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg2+ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis.     

The separation of the mycobactins from Mycobacterium smegmatis by using high-pressure liquid chromatography.

The family of mycobactins from Mycobacterium smegmatis were resolved into seven fractions by high-pressure liquid chromatography. This separation was by virtue of the differences in length and character of the long acyl substituents as shown by g.l.c. of the methyl esters of the isolated fatty acids from the fractions. As t.l.c. could also resolve the individual mycobactin fractions, it too must rely on the same differences to effect separation. As the lengths of the acyl chains were modulated by the growth conditions, a specific range of acyl groups may not be needed for mycobactin to function. This technique provides a simple means of rapidly characterizing crude mycobactins from all mycobacteria.     

Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5′ region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.     

Iron as a candidate in virulence and pathogenesis in mycobacteria and other microorganisms

Iron acquisition is one of the important virulence characteristics of a pathogen. Microorganisms elaborate different ways of acquiring iron. Siderophore-mediated iron acquisition is common in many microorganisms, while some like Neisseriae directly acquire iron from the host proteins. Microorganisms also elaborate systems for iron acquisition from ferric citrate and xenosiderophores. Such modes of uptake are also seen in mycobacteria. The siderophores produced by mycobacteria are well characterised. Iron-regulated envelope proteins (IREPs) are expressed in both saprophytic and pathogenic mycobacteria. In many bacterial systems, like that of Corynebacterium diphtheriae, low iron conditions favour the expression of virulence factors. The siderophore mycobactin has been recently implicated as a virulence factor in Mycobacterium tuberculosis. The role of the IdeR repressor in controlling the expression of the iron acquisition machinery was studied with the generation of the IdeR mutant. Development of other such mutants will facilitate the study of these mechanisms in greater detail and help to develop new drugs for combating tuberculosis and leprosy. This revised version was published online in November 2006 with corrections to the Cover Date.     

Mycobactins as chemotaxonomic characters for some rapidly growing mycobacteria

Thirty-nine strains of rapidly growing mycobacteria were examined for the production of mycobactins (lipid-soluble, iron-binding compounds) when grown under conditions of iron-limitation on solidified medium. Different growth conditions had little effect on the structure of individual mycobactins, indicating them to be strongly conserved molecules showing intra-species consistency and thus suitable for use as chemotaxonomic characters of high discriminatory power. Strains of Mycobacterium aurum, M. chitae, M. chelonae subsp. abscessus, 'M. diernhoferi', M. duvalii, M. flavescens, M. fortuitum, M. gadium, 'M. gallinarum', M. neoaurum, M. parafortuitum, 'M. peregrinum', M. phlei, M. smegmatis, M. thermoresistible and M. vaccae formed mycobactins which were readily isolated and characterized by a combination of thin-layer and high-performance liquid chromatography. All strains of M. komossense and 'M. kanazawa' failed to produce a mycobactin; some strains of M. aurum, M. chelonae, M. parafortuitum, M. thermoresistible and M. vaccae were similarly negative. Mycobacteria of the M. fortuitum complex (M. fortuitum, M. chelonae and 'M. peregrinum') formed distinctive mycobactins, as did those in the M. parafortuitum complex (M. aurum, M. neoaurum, 'M. diernhoferi', M. vaccae and M. parafortuitum). The mycobactin from 'M. gallinarum' was different from those of the related species M. flavescens, for which four distinct mycobactin patterns were recorded. For routine examination of mycobactins in a diagnostic laboratory with limited resources, thin-layer chromatography used alone offers a simple but adequate means of characterization and final identification of the producing mycobacterium. High-performance liquid chromatography is only needed in those few instances where a high degree of discrimination is required.     

Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov

We performed a numerical taxonomy analysis of 38 Mycobacterium paratuberculosis and related mycobacterial strains, including wood pigeon mycobacteria; this analysis was based on 22 tests, which were selected for their potential discriminative value from a total of 51 tests studied and produced four well-defined clusters. Cluster 1 contained the M. paratuberculosis strains, including two strains isolated from Crohn's disease patients; cluster 2 contained Mycobacterium avium and Mycobacterium intracellulare reference strains; cluster 3 consisted of the wood pigeon mycobacteria; and the only strain in cluster 4 was M. paratuberculosis 316F, which is used for antigen and vaccine production. Strains in cluster 1 were mycobactin dependent even when they were subcultured, whereas strains in cluster 3 were unable to grow on egg medium and their growth was stimulated by pH 5.5. Growth stimulation by pyruvate, resistance to D-cycloserine (50 micrograms/ml), and alkaline phosphatase activity also were characteristics that were useful for discriminating between clusters 1 and 3. The results of previous DNA-DNA hybridization studies have demonstrated that M. avium Chester 1901, M. paratuberculosis Bergey et al. 1923, and the wood pigeon mycobacteria belong to a single genomic species, and we propose that the name of this species should be M. avium. On the basis of the results of previous genomic analyses based on restriction fragment length, the results of polymorphism studies, and DNA patterns determined by field inversion gel electrophoresis as well as the results of our phenotypic study, we propose that the species should be divided into subspecies which correspond to pathogenicity and host range characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial growth promotion studies of exochelin MN and analogues thereof

The ability of exochelin MN and three synthetic analogues to promote the growth of various strains of mycobacteria and Gram-negative bacteria was investigated. The results indicated that growth promotion ability of these compounds depends either on ligand exchange with mycobactin or on the exochelin permease. Despite stronger iron complexing capacity, the structural analogues showed weaker growth promotion ability than exochelin MN, which further supported our hypothesis of pH-dependent iron(III)-release of exochelin MN.     

99mTc-labeling of HYNIC-conjugated cyclic RGDfK dimer and tetramer using EDDA as coligand

In this study, EDDA (ethylenediamine- N, N'-diacetic acid) was used as the coligand for 99mTc-labeling of cyclic RGDfK conjugates: HYNIC-dimer (HYNIC = 6-hydrazinonicotinamide; dimer = E[c(RGDfK)]2) and HYNIC-tetramer (tetramer = E{E[c(RGDfK)]2}2). First, HYNIC-dimer was allowed to react with 99mTcO4 (-) in the presence of excess tricine and stannous chloride to form the intermediate complex [99mTc(HYNIC-dimer)(tricine)2], which was then allowed to react with EDDA to afford [99mTc(HYNIC-dimer)(EDDA)] with high yield (>90%) and high specific activity ( approximately 8.0 Ci/micromol). Under the same radiolabeling conditions, the yield for [99mTc(HYNIC-tetramer)(EDDA)] was always <65%. The results from a mixed-ligand experiment show that there is only one EDDA bonding to the 99mTc-HYNIC core in [99mTc(HYNIC-dimer)(EDDA)]. The athymic nude mice bearing subcutaneous U87MG human glioma xenografts were used to evaluate the impact of EDDA coligand on the biodistribution characteristics and excretion kinetics of the 99mTc-labeled HYNIC-dimer and HYNIC-tetramer. Surprisingly, [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] had almost identical tumor uptake over the 2 h period. The use of EDDA as coligand to replace tricine/TPPTS (TPPTS = trisodium triphenylphosphine-3,3',3'-trisulfonate) did not significantly change the uptake of the 99mTc-labeled HYNIC-dimer in noncancerous organs, such as the liver, kidneys, and lungs; but it did result in a significantly lower kidney uptake for the 99mTc-labeled HYNIC-tetramer due to faster renal excretion. It was also found that the radiotracer tumor uptake decreases in a linear fashion as the tumor size increases. The smaller the tumors are, the higher the tumor uptake is regardless of the identity of radiotracer.     

Role for Mycobacterium tuberculosis Membrane Vesicles in Iron Acquisition

Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host.     

Influence of metal ions on the formation of mycobactin and salicylic acid in Mycobacterium smegmatis grown in static culture

Mycobacterium smegmatis was grown on trace-metal-free medium in static culture. Throughout the growth phase, the concentration of mycobactin increased continuously, reaching a maximum of about 30 to 40 mug of mycobactin/mg of cell dry weight after 6 days; the concentration of salicylic acid remained approximately constant at 1 to 2 mug of salicylic acid/mug of cell dry weight. Fe(2+) (or Fe(3+)), Zn(2+), Mn(2+), and Mg(2+) were all essential to a maximum formation of mycobactin. Optimum concentrations required were: Fe(2+), about 1.8 mum; Mn(2+) and Zn(2+), about 0.5 mum; and Mg(2+), at least 0.17 mm. Higher levels of Fe(2+) (9 to 90 mum) and Zn(2+) (2 to 7 mum) repressed mycobactin to about half the maximum value. No other cation or anion apparently is required for mycobactin biosynthesis. Salicylic acid concentration increased about fourfold when iron was omitted from the medium, but this is not as great as the increase reported previously for this strain of M. smegmatis. Mycobactin formation in another strain of M. smegmatis, NCIB 8548, showed similar dependencies on Fe(2+), Zn(2+), and Mn(2+). Maximum accumulation of mycobactin with this strain was 85 mug of mycobactin/mg of dry cell weight, under iron-deficient (1.8 mum Fe(2+)) conditions.