Fine discrimination in the recognition of individual species of phosphatidyl-myo-inositol mannosides from Mycobacterium tuberculosis by C-type lectin pattern recognition receptors

The Mycobacterium tuberculosis (M.tb) envelope is highly mannosylated with phosphatidyl-myo-inositol mannosides (PIMs), lipomannan, and mannose-capped lipoarabinomannan (ManLAM). Little is known regarding the interaction between specific PIM types and host cell C-type lectin pattern recognition receptors. The macrophage mannose receptor (MR) and dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells engage ManLAM mannose caps and regulate several host responses. In this study, we analyzed the association of purified PIM families (f, separated by carbohydrate number) and individual PIM species (further separated by fatty acid number) from M.tb H(37)R(v) with human monocyte-derived macrophages (MDMs) and lectin-expressing cell lines using an established bead model. Higher-order PIMs preferentially associated with the MR as demonstrated by their reduced association with MDMs upon MR blockade and increased binding to COS-1-MR. In contrast, the lower-order PIM(2)f associated poorly with MDMs and did not bind to COS-1-MR. Triacylated PIM species were recognized by MDM lectins better than tetra-acylated species and the degree of acylation influenced higher-order PIM association with the MR. Moreover, only higher-order PIMs that bind the MR showed a significant increase in phagosome-lysosome fusion upon MR blockade. In contrast with the MR, the PIM(2)f and lipomannan were recognized by DC-SIGN comparable to higher-order PIMs and ManLAM, and the association was independent of their degree of acylation. Thus, recognition of M.tb PIMs by host cell C-type lectins is dependent on both the nature of the terminal carbohydrates and degree of acylation. Subtle structural differences among the PIMs impact host cell recognition and response and are predicted to influence the intracellular fate of M.tb.     

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.

The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.     

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.     

Identification of the pgmG gene, encoding a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461

The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K(m) values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway.     

Identification of the pgmG Gene, Encoding a Bifunctional Protein with Phosphoglucomutase and Phosphomannomutase Activities, in the Gellan Gum-Producing Strain Sphingomonas paucimobilis ATCC 31461

The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K_m values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway.     

Characterization of a thermostable enzyme with phosphomannomutase/phosphoglucomutase activities from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.     

Cloning and characterization of phosphoglucomutase and phosphomannomutase derived from Sphingomonas chungbukensis DJ77

The enzymes phosphoglucomutase (PGM) and phosphomannomutase (PMM) play an important role in the synthesis of extracellular polysaccharide. By colony hybridization of the fosmid library of Sphingomonas chungbukensis DJ77, an open reading frame (ORF-1) of 1,626 nucleotides, whose predicted product is highly homologous with other PGM proteins from several bacterial species, was identified. An additional open reading frame (ORF-2) of 1,437 nucleotides was identified, and its encoded protein shows a high level of similarity with the PGM/PMM protein family. The two genes were cloned into a bacterial expression vector pET-15b (+) and expressed in Escherichia coli as fusion proteins with (His)(6)-tag. Both recombinant proteins (designated as SP-1 and SP-2 for ORF-1 and ORF-2, respectively) exhibited PGM and PMM activities. The molecular masses of subunits of SP-1 and SP-2 were estimated to be around 58 and 51 kDa from SDS-PAGE, respectively. However, molecular masses of SP-1 and SP-2 in their native condition were determined to be approximately 59.5 and 105.4 kDa, according to non-denaturing PAGE, respectively. The SP-1 protein has a preference for glucose-1-phosphate rather than mannose-1-phosphate, while the preferred substrate of SP-2 is mannose-1-phosphate. Thus, the existence of two proteins with bifunctional PGM/PMM activities was first found S. chungbukensis DJ77.     

Structure of Leishmania mexicana phosphomannomutase highlights similarities with human isoforms

Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.     

Pathogenic Mycobacterium bovis strains differ in their ability to modulate the proinflammatory activation phenotype of macrophages

ABSTRACT: BACKGROUND: Tuberculosis, caused by Mycobacterium tuberculosis or Mycobacterium bovis, remains one of the leading infectious diseases worldwide. The ability of mycobacteria to rapidly grow in host macrophages is a factor contributing to enhanced virulence of the bacteria and disease progression. Bactericidal functions of phagocytes are strictly dependent on activation status of these cells, regulated by the infecting agent and cytokines. Pathogenic mycobacteria can survive the hostile environment of the phagosome through interference with activation of bactericidal responses. To study the mechanisms employed by highly virulent mycobacteria to promote their intracellular survival, we investigated modulating effects of two pathogenic M. bovis isolates and a reference M. tuberculosis H37Rv strain, differing in their ability to multiply in macrophages, on activation phenotypes of the cells primed with major cytokines regulating proinflammatory macrophage activity. RESULTS: Bone marrow- derived macrophages obtained from C57BL/6 mice were infected by mycobacteria after a period of cell incubation with or without treatment with IFN-gamma, inducing proinflammatory type-1 macrophages (M1), or IL-10, inducing anti-inflammatory type-2 cells (M2). Phenotypic profiling of M1 and M2 was then evaluated. The M. bovis strain MP287/03 was able to grow more efficiently in the untreated macrophages, compared with the strains B2 or H37Rv. This strain induced weaker secretion of proinflammatory cytokines, coinciding with higher expression of M2 cell markers, mannose receptor (MR) and arginase-1 (Arg-1). Treatment of macrophages with IFN-gamma and infection by the strains B2 and H37Rv synergistically induced M1 polarization, leading to high levels of inducible nitric oxide synthase (iNOS) expression, and reduced expression of the Arg-1. In contrast, the cells infected with the strain MP287/03 expressed high levels of Arg-1 which competed with iNOS for the common substrate arginine, leading to lower levels of NO production. CONCLUSIONS: The data obtained demonstrated that the strain, characterized by increased growth in macrophages, down- modulated classical macrophage activation, through induction of an atypical mixed M1/M2 phenotype.     

Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana

Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.     

Divergent effects of mycobacterial cell wall glycolipids on maturation and function of human monocyte-derived dendritic cells

Background

Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system.

Principal Findings

We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect.

Conclusions

These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.     

Structural basis of diverse substrate recognition by the enzyme PMM/PGM from P. aeruginosa

Enzyme-substrate complexes of phosphomannomutase/phosphoglucomutase (PMM/PGM) reveal the structural basis of the enzyme's ability to use four different substrates in catalysis. High-resolution structures with glucose 1-phosphate, glucose 6-phosphate, mannose 1-phosphate, and mannose 6-phosphate show that the position of the phosphate group of each substrate is held constant by a conserved network of hydrogen bonds. This produces two distinct, and mutually exclusive, binding orientations for the sugar rings of the 1-phospho and 6-phospho sugars. Specific binding of both orientations is accomplished by key contacts with the O3 and O4 hydroxyls of the sugar, which must occupy equatorial positions. Dual recognition of glucose and mannose phosphosugars uses a combination of specific protein contacts and nonspecific solvent contacts. The ability of PMM/PGM to accommodate these four diverse substrates in a single active site is consistent with its highly reversible phosphoryl transfer reaction and allows it to function in multiple biosynthetic pathways in P. aeruginosa.     

The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan

Interactions between dendritic cells (DCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, most likely play a key role in anti-mycobacterial immunity. We have recently shown that M. tuberculosis binds to and infects DCs through ligation of the DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and that M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) inhibits binding of the bacilli to the lectin, suggesting that ManLAM might be a key DC-SIGN ligand. In the present study, we investigated the molecular basis of DC-SIGN ligation by LAM. Contrary to what was found for slow growing mycobacteria, such as M. tuberculosis and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin, our data demonstrate that the fast growing saprophytic species Mycobacterium smegmatis hardly binds to DC-SIGN. Consistent with the former finding, we show that M. smegmatis-derived lipoarabinomannan, which is capped by phosphoinositide residues (PILAM), exhibits a limited ability to inhibit M. tuberculosis binding to DC-SIGN. Moreover, using enzymatically demannosylated and chemically deacylated ManLAM molecules, we demonstrate that both the acyl chains on the ManLAM mannosylphosphatidylinositol anchor and the mannooligosaccharide caps play a critical role in DC-SIGN-ManLAM interaction. Finally, we report that DC-SIGN binds poorly to the PILAM and uncapped AraLAM-containing species Mycobacterium fortuitum and Mycobacterium chelonae, respectively. Interestingly, smooth colony-forming Mycobacterium avium, in which ManLAM is capped with single mannose residues, was also poorly recognized by the lectin. Altogether, our results provide molecular insight into the mechanisms of mycobacteria-DC-SIGN interaction, and suggest that DC-SIGN may act as a pattern recognition receptor and discriminate between Mycobacterium species through selective recognition of the mannose caps on LAM molecules.     

Identification of free mannose and partial characterization of a mannose-6-phosphate isomerase from Lilium longiflorum bulbs

The existence of free mannose in storage bulbs of Lilium longiflorum Thunb, was established using preparative high performance liquid chromatography, gas chromatography and gas chromatography-mass spectroscopy. Free mannose was not detected in developing (importing) bulb tissues. Mannose, a relatively rare hexose in plant tissue, probably arises from the hydrolysis of glucomannan, a hemicellulosic carbohydrate polymer known to be present in Lilium storage tissues. A calculation of total mannose residues per bulb (prior to versus after reserve hydrolysis and export) indicated that mannose is metabolized, probably in sucrose biosynthesis. A mannose-6-phosphate isomerase (EC 5.3.1.8) was isolated from Lilium bulbs and purified 155-fold with 29% yield. The molecular weight of the enzyme was estimated by gel filtration to be 64 kDa, and the K_m for mannose-6-phosphate was 0.42 m M . It is concluded that glucomannan is functioning as a reserve carbohydrate in Lilium storage tissues and that the mannose-6-phosphate isomerase is responsible for the entry of mannose into the sucrose biosynthetic pathway.     

A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency

Congenital disorder of glycosylation (PMM2-CDG) results from mutations in pmm2, which encodes the phosphomannomutase (Pmm) that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Although it has been widely proposed that Pmm2 deficiency depletes M1P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. Here we report a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. Although M1P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of M6P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly twofold. Suppression of the M6P-synthesizing enzyme mannose phosphate isomerase within the pmm2 background normalized M6P levels and certain aspects of the craniofacial phenotype and abrogated pmm2-dependent LLO cleavage. In summary, we report the first zebrafish model of PMM2-CDG and uncover novel cellular insights not possible with other systems, including an M6P accumulation mechanism for underglycosylation.     

Differential immune response of adipocytes to virulent and attenuated Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tb) takes advantage of various cell types, allowing it to remain in the host for long periods. Because adipocytes have been proposed as niches for dormant M. tb in the latent state, understanding the interaction of virulent M. tb with adipocytes is important. We compared changes in cytokine secretion from 3T3-L1 murine adipocytes infected with virulent M. tb H37Rv (V-M. tb) and attenuated M. tb H37Ra (A-M. tb) strains. Both strains maintained non-replicating states within adipocytes until 10 days post-infection. Adipocytes infected with V-M. tb secreted lower levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12p40, IL-6, and IL-17, and lower levels of nitric oxide than those infected with A-M. tb. In contrast, the anti-inflammatory cytokines, IL-10 and IL-4, were markedly induced in V-M. tb-infected adipocytes versus those infected with A-M. tb at an early time point. Heat-killed or formalin-fixed bacteria induced lower levels of cytokines and no difference was observed between strains. Moreover, V-M. tb induced a high level of necrosis versus A-M. tb in conjunction with increased levels of LHD. These results suggest that V-M. tb regulates cytokine expression in its favor, increasing cytokines necessary for immune evasion and decreasing those required for protective immunity.     

Comparative analysis of iron regulated genes in mycobacteria

Iron dependent regulator, IdeR, regulates the expression of genes in response to intracellular iron levels in M. tuberculosis. Orthologs of IdeR are present in all the sequenced genomes of mycobacteria. We have used a computational approach to identify conserved IdeR regulated genes across the mycobacteria and the genes that are specific to each of the mycobacteria. Novel iron regulated genes that code for a predicted 4-hydroxy benzoyl coA hydrolase (Rv1847) and a protease dependent antibiotic regulatory system (Rv1846c, Rv0185c) are conserved across the mycobacteria. Although Mycobacterium natural-resistance-associated macrophage protein (Mramp) is present in all mycobacteria, it is, as predicted, an iron-regulated gene in only one species, M. avium subsp. paratuberculosis. We also observed an additional iron-regulated exochelin biosynthetic operon, which is present only in non-pathogenic Mycobacterium, M. smegmatis.