Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin dependent for cultivation in-vitro. The iron dependent regulator (IdeR) is a well-characterized global regulator responsible for maintaining iron homeostasis in Mycobacterium tuberculosis (MTB). We identified an orthologous segment in MAP genome, MAP2827, with >93% amino acid identity to MTB IdeR. Electrophoretic mobility shift assays and DNase protection assays confirmed that MAP2827 protein binds the 19 base pair consensus motif (iron box) on the MAP genome. Resequencing MAP2827 from multiple isolates revealed a non-synonymous change (R91G) exclusive to sheep strains. Reporter gene assays and quantitative real time RT-PCR assays in two diverse MAP strains and in an ideR deletion mutant of M. smegmatis (mc2155) suggested that both sheep MAP IdeR (sIdeR) and cattle MAP IdeR (cIdeR) repressed mbtB transcription at high iron concentration and relieved repression at low iron concentration. On the other hand, bfrA (an iron storage gene) was upregulated by cIdeR when presented with MTB or cattle MAP bfrA promoter and was downregulated by sIdeR in the presence of MTB or sheep or cattle MAP bfrA promoters, at high iron concentration. The differential iron regulatory mechanisms between IdeR regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP.downregulated by sIdeR in the presence of MTB or sheep or cattle MAP bfrA promoters, at high iron concentration. The differential iron regulatory mechanisms between IdeR regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP.

The Mycobacterium tuberculosis PhoPR two-component system is essential for virulence in animal models of tuberculosis. Recent articles have shown that among the reasons for the attenuation of the M. tuberculosis H37Ra strain is a mutation in the phoP gene that prevents the secretion of proteins that are important for virulence. There is a need for new anti-tubercular therapies because of the emergence of multi-drug-resistant M. tuberculosis strains and also the variable efficacy of the currently used bacille Calmette-Guérin vaccine. Because of its major role in M. tuberculosis pathogenicity, PhoP is a potential target candidate. This review summarizes our understanding of PhoPR's role in virulence and discusses areas in which our knowledge is limited.

The PhoP-PhoR two-component signaling system from Mycobacterium tuberculosis is essential for the virulence of the tubercle bacillus. The response regulator, PhoP, regulates expression of over 110 genes. In order to elucidate the regulatory mechanism of PhoP, we determined the crystal structure of its DNA-binding domain (PhoPC). PhoPC exhibits a typical fold of the winged helix-turn-helix subfamily of response regulators. The structure starts with a four-stranded antiparallel beta-sheet, followed by a three-helical bundle of alpha-helices, and then a C-terminal beta-hairpin, which together with a short beta-strand between the first and second helices forms a three-stranded antiparallel beta-sheet. Structural elements are packed through a hydrophobic core, with the first helix providing a scaffold for the rest of the domain to pack. The second and third helices and the long, flexible loop between them form the helix-turn-helix motif, with the third helix being the recognition helix. The C-terminal beta-hairpin turn forms the wing motif. The molecular surfaces around the recognition helix and the wing residues show strong positive electrostatic potential, consistent with their roles in DNA binding and nucleotide sequence recognition. The crystal packing of PhoPC gives a hexamer ring, with neighboring molecules interacting in a head-to-tail fashion. This packing interface suggests that PhoPC could bind DNA in a tandem association. However, this mode of DNA binding is likely to be nonspecific because the recognition helix is partially blocked and would be prevented from inserting into the major groove of DNA. Detailed structural analysis and implications with respect to DNA binding are discussed.

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cationhomeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated as FurB. Since in this paper we show that it represents a Zinc Uptake Regulator, we will refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zur operon, favoring the hypothesis that these genes represent key regulators for zinc homeostasis. In this paper we generated a zur mutant in M. tuberculosis, examined its phenotype and characterized the Zur regulon by using DNA microarray analysis. Thirty two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty four of them belonged to 8 putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zur-regulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to the Early Secretory Antigen Target (ESAT)-6 cluster 3 and three additional proteins belonging to the ESAT-6/Culture Filtrate Protein (CFP)-10 family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.

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 role of iron in mycobacteria as in other bacteria goes beyond the need for this essential cofactor. Limitation of this metal triggers an extensive response aimed at increasing iron acquisition while coping with iron deficiency. In contrast, iron-rich environments prompt these prokaryotes to induce synthesis of iron storage molecules and to increase mechanisms of protection against iron-mediated oxidative damage. The response to changes in iron availability is strictly regulated in order to maintain sufficient but not excessive and potentially toxic levels of iron in the cell. This response is also linked to other important processes such as protection against oxidative stress and virulence. In bacteria, iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. In mycobacteria, this role is fulfilled by the iron-dependent regulator IdeR. IdeR is an essential protein in Mycobacterium tuberculosis, the causative agent of human tuberculosis. It functions as a repressor of iron acquisition genes, but is also an activator of iron storage genes and a positive regulator of oxidative stress responses.

The mycobacterial IdeR protein is a metal-dependent regulator of the DtxR (diphtheria toxin repressor) family. In the presence of iron, it binds to a specific DNA sequence in the promoter regions of the genes that it regulates, thus controlling their transcription. In this study, we provide evidence that ideR is an essential gene in Mycobacterium tuberculosis. ideR cannot normally be disrupted in this mycobacterium in the absence of a second functional copy of the gene. However, a rare ideR mutant was obtained in which the lethal effects of ideR inactivation were alleviated by a second-site suppressor mutation and which exhibited restricted iron assimilation capacity. Studies of this strain and a derivative in which IdeR expression was restored allowed us to identify phenotypic effects resulting from ideR inactivation. Using DNA microarrays, the iron-dependent transcriptional profiles of the wild-type, ideR mutant, and ideR-complemented mutant strains were analyzed, and the genes regulated by iron and IdeR were identified. These genes encode a variety of proteins, including putative transporters, proteins involved in siderophore synthesis and iron storage, members of the PE/PPE family, a membrane protein involved in virulence, transcriptional regulators, and enzymes involved in lipid metabolism.

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis (M. tb) infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a Tet-R like repressor. The fadA5 (Rv3546) gene, annotated as a lipid metabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tb H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tb in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl CoA. This catalytic activity is consistent with a beta-ketoacyl CoA thiolase function in cholesterol beta-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is only essential in the persistent stage of M. tb infection and may be caused by the inability to produce AD/ADD from cholesterol.

Mycobacterium tuberculosis can metabolize cholesterol to both acetate and propionate. The mass of isolated phthiocerol dimycoserate, a methyl-branched fatty acylated polyketide, was used as a reporter for intracellular propionate metabolic flux. When M. tuberculosis is grown using cholesterol as the only source of carbon, a 42 amu increase in average phthiocerol dimycoserate molecular weight is observed, consistent with the cellular pool of propionate and, thus, methylmalonyl CoA increasing upon cholesterol metabolism. In contrast, no shift in phthiocerol dimycoserate molecular weight is observed upon supplementation of medium containing glycerol and glucose with cholesterol. We conclude that cholesterol is a significant source of propionate only in the absence of sugar carbon sources.