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.     

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.     

Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery

Mycobacterium tuberculosis encodes mycobactin, a peptide siderophore that is biosynthesized by a nonribosomal peptide synthetase (NRPS) mechanism. Within the mycobactin biosynthetic gene cluster is a gene that encodes a 71-amino-acid protein MbtH. Many other NRPS gene clusters harbor mbtH homologs, and recent genetic, biochemical, and structural studies have begun to shed light on the function(s) of these proteins. In some cases, MbtH-like proteins are required for biosynthesis of their cognate peptides, and non-cognate MbtH-like proteins have been shown to be partially complementary. Biochemical studies revealed that certain MbtH-like proteins participate in tight binding to NRPS proteins containing adenylation (A) domains where they stimulate adenylation reactions. Expression of MbtH-like proteins is important for a number of applications, including optimal production of native and genetically engineered secondary metabolites produced by mechanisms that employ NRPS enzymes. They also may serve as beacons to identify gifted actinomycetes and possibly other bacteria that encode multiple functional NRPS pathways for discovery of novel secondary metabolites by genome mining.     

Production of mycobacterial cell wall glycopeptidolipids requires a member of the MbtH-like protein family

ABSTRACT: BACKGROUND: Glycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production. RESULTS: Deletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility. CONCLUSIONS: Herein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such a gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane.     

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)     

Participation of fad and mbt genes in synthesis of mycobactin in Mycobacterium smegmatis

Colonies of Mycobacterium smegmatis LR222 on iron-limiting (0.1 micro M Fe) minimal medium agar fluoresce under UV light due to the accumulation in the cells of the deferri form of the siderophore mycobactin. Two mutants with little or no fluorescence, designated LUN8 and LUN9, were isolated by screening colonies of transposon (Tn611)-mutagenized M. smegmatis. Ferrimycobactin prepared from iron-restricted cells of the wild type had an R(f) of 0.62 on high-performance thin-layer chromatography (HPTLC) and a characteristic visible absorption spectrum with a peak near 450 nm. Similar extracts from LUN8 cells contained a small amount of ferrimycobactin with an R(f) of 0.58 on HPTLC and an absorption spectrum with the peak shifted to a wavelength lower than that of the wild-type ferrimycobactin. Nuclear magnetic resonance spectroscopy studies suggested that the LUN8 mycobactin may have an altered fatty acid side chain. Mutant strain LUN9 produced no detectable mycobactin. Neither mutant strain produced measurable amounts of excreted mycobactin, although both excreted exochelin (the mycobacterial peptido-hydroxamate siderophore), and both mutants were more sensitive than the wild-type strain to growth inhibition by the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The transposon insertion sites were identified, and sequence analyses of the cloned flanking chromosome regions showed that the mutated gene in LUN9 was an orthologue of the Mycobacterium tuberculosis mycobactin biosynthetic gene mbtE. The mutated gene in LUN8 had homology with M. tuberculosis fadD33 (Rv1345), a gene that may encode an acyl-coenzyme A synthase and which previously was not known to participate in synthesis of mycobactin.     

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.     

Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways

Mycobacteria produce two siderophores, mycobactin and exochelin. Mycobacterium smegmatis mutants defective in the production of exochelin were isolated using agar medium containing chrome azural S for the sensitive detection of siderophores. Cosmids of genomic libraries from M. smegmatis and Mycobacterium bovis BCG were screened for complementation of the mutation. Subcloning of the complementing M. smegmatis cosmid identified a 4.3 kb fragment required for restoring exochelin biosynthesis. Sequencing of the DNA revealed four open reading frames whose genes were named fxuA. fxuB, fxuC, and fxbA. FxuA, FxuB, and FxuC share amino acid sequence homology with the iron permeases FepG, FepC. and FepD from Escherichia coli. respectively. Deletion analysis identified fxbA as the gene required to restore exochelin biosynthesis in our mutant. Although fxbA does not share amino acid sequence homology with any of the published sequences for siderophore biosynthetic genes, it does show limited homology with the phosphoribosyl-glycineamide formyltransferases (GAR enzymes) and methionyl-tRNA formyltransferase over a limited region of the sequence, suggesting that fxbA may code for an enzyme which adds a formyl group in the synthesis of exochelin. A fusion of fxbA with the E. coli lacZ gene demonstrated regulation of gene expression by iron. The ability of M. smegmatis mutants to produce mycobactin in the absence of exochelin supports the hypothesis that exochelin is not a precursor of mycobactin and suggests that the siderophores have independent biosynthetic pathways. In addition, complementation of the M.     

Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in M. tuberculosis

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.     

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.     

Identification of an ABC transporter required for iron acquisition and virulence in Mycobacterium tuberculosis

Iron availability affects the course of tuberculosis infection, and the ability to acquire this metal is known to be essential for replication of Mycobacterium tuberculosis in human macrophages. M. tuberculosis overcomes iron deficiency by producing siderophores. The relevance of siderophore synthesis for iron acquisition by M. tuberculosis has been demonstrated, but the molecules involved in iron uptake are currently unknown. We have identified two genes (irtA and irtB) encoding an ABC transporter similar to the YbtPQ system involved in iron transport in Yersinia pestis. Inactivation of the irtAB system decreases the ability of M. tuberculosis to survive iron-deficient conditions. IrtA and -B do not participate in siderophore synthesis or secretion but are required for efficient utilization of iron from Fe-carboxymycobactin, as well as replication of M. tuberculosis in human macrophages and in mouse lungs. We postulate that IrtAB is a transporter of Fe-carboxymycobactin. The irtAB genes are located in a chromosomal region previously shown to contain genes regulated by iron and the major iron regulator IdeR. Taken together, our results and previous observations made by other groups regarding two other genes in this region indicate that this gene cluster is dedicated to siderophore synthesis and transport in M. tuberculosis.     

The crystal structure of Rv1347c, a putative antibiotic resistance protein from Mycobacterium tuberculosis, reveals a GCN5-related fold and suggests an alternative function in siderophore biosynthesis

Mycobacterium tuberculosis, the cause of tuberculosis, is a devastating human pathogen. The emergence of multidrug resistance in recent years has prompted a search for new drug targets and for a better understanding of mechanisms of resistance. Here we focus on the gene product of an open reading frame from M. tuberculosis, Rv1347c, which is annotated as a putative aminoglycoside N-acetyltransferase. The Rv1347c protein does not show this activity, however, and we show from its crystal structure, coupled with functional and bioinformatic data, that its most likely role is in the biosynthesis of mycobactin, the M. tuberculosis siderophore. The crystal structure of Rv1347c was determined by multiwavelength anomalous diffraction phasing from selenomethionine-substituted protein and refined at 2.2 angstrom resolution (r = 0.227, R(free) = 0.257). The protein is monomeric, with a fold that places it in the GCN5-related N-acetyltransferase (GNAT) family of acyltransferases. Features of the structure are an acyl-CoA binding site that is shared with other GNAT family members and an adjacent hydrophobic channel leading to the surface that could accommodate long-chain acyl groups. Modeling the postulated substrate, the N(epsilon)-hydroxylysine side chain of mycobactin, into the acceptor substrate binding groove identifies two residues at the active site, His130 and Asp168, that have putative roles in substrate binding and catalysis.     

Identification of nocobactin NA biosynthetic gene clusters in Nocardia farcinica

We identified the biosynthetic gene clusters of the siderophore nocobactin NA. The nbt clusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing of Nocardia farcinica IFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of the nbt clusters and the predicted functions of the nbt genes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structure of nocobactin NA. Disruptions of the nbtA and nbtE genes, respectively, reduced and abolished the productivity of nocobactin NA. The heterologous expression of the nbtS gene revealed that this gene encoded a salicylate synthase. These results indicate that the nbt clusters are responsible for the biosynthesis of nocobactin NA. We also found putative IdeR-binding sequences upstream of the nbtA, -G, -H, -S, and -T genes, whose expression was more than 10-fold higher in the low-iron condition than in the high-iron condition. These results suggest that nbt genes are regulated coordinately by IdeR protein in an iron-dependent manner. The ΔnbtE mutant was found to be impaired in cytotoxicity against J774A.1 cells, suggesting that nocobactin NA production is required for virulence of N. farcinica.     

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co-regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.     

MKAN27435 Is Required for the Biosynthesis of Higher Subclasses of Lipooligosaccharides in Mycobacterium kansasii

Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate null mutants in other mycobacteria, we generated a MKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.     

The mycobacterial glycopeptidolipids: structure, function, and their role in pathogenesis

Glycopeptidolipids (GPLs) are a class of glycolipids produced by several nontuberculosis-causing members of the Mycobacterium genus including pathogenic and nonpathogenic species. GPLs are expressed in different forms with production of highly antigenic, typeable serovar-specific GPLs in members of the Mycobacterium avium complex (MAC). M. avium and M. intracellulare, which comprise this complex, are slow-growing mycobacteria noted for producing disseminated infections in AIDS patients and pulmonary infections in non-AIDS patients. Previous studies have defined the gene cluster responsible for GPL biosynthesis and more recent work has characterized the function of the individual genes. Current research has also focused on the GPL's role in colony morphology, sliding motility, biofilm formation, immune modulation and virulence. These topics, along with new information on the enzymes involved in GPL biosynthesis, are the subject of this review.     

Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes

Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2- L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.     

Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.     

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.     

Analysis of the Exochelin Locus in Mycobacterium smegmatis: Biosynthesis Genes Have Homology with Genes of the Peptide Synthetase Family

Mycobacteria secrete the siderophore exochelin when grown under iron-limiting conditions. In order to understand iron uptake mechanisms in mycobacteria, we have taken a genetic approach to identify those genes involved in exochelin biosynthesis and transport in Mycobacterium smegmatis. Of the 6,000 chemically mutagenized clones of M. smegmatis mc²155 screened on agar plates containing chrome azural S, 19 mutants that had lost the ability to produce or secrete exochelin were identified. Thirteen of these mutants were complemented by a single M. smegmatis cosmid. Sequence analysis of this cosmid revealed nine open reading frames, three of which are homologous to genes encoding transporter proteins, which are likely involved in exochelin transport. Complementation and Tn10 mutagenesis analysis identified two new genes, fxbB and fxbC, which are required for exochelin biosynthesis. The fxbB and fxbC genes encode large proteins of 257 and 497 kDa, respectively, which are highly homologous to peptide synthetases, indicating that exochelin biosynthesis occurs by a nonribosomal mechanism.