We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.

Mycobacterium tuberculosis is a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyte Mycobacterium smegmatis. In this work we investigated the role of sigH in the M. tuberculosis response to heat and oxidative stress. We determined that a M. tuberculosis sigH mutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genes trxB2/trxC and a gene of unknown function, Rv2466c, is regulated by sigH via expression from promoters directly recognized by SigH. We also determined that the sigH mutant is more susceptible to heat stress and that inducible expression of the heat shock genes dnaK and clpB is positively regulated by sigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role of sigH in the M. tuberculosis stress response, we investigated the regulation of the stress-responsive sigma factor genes sigE and sigB. We determined that inducible expression of sigE is regulated by sigH and that basal and inducible expression of sigB is dependent on sigE and sigH. These data indicate that sigH plays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important in M. tuberculosis pathogenesis.Tuberculosis remains a major cause of human suffering, exacting an enormous toll of morbidity and mortality in much of the world (11). The cause of tuberculosis, the obligate pathogen Mycobacterium tuberculosis, is highly adapted for survival in the host organism. Following infection M. tuberculosis is ingested by macrophages and must persist in this environment in order to survive, either in a quiescent state or through active replication that results in tissue destruction and the disease of tuberculosis. In adapting to this intracellular environment, this bacterium must regulate its physiology to survive a variety of stresses produced by the macrophage, including reactive oxygen and reactive nitrogen species produced by these cells (1, 4, 28). In addition M. tuberculosis has been shown to alter the physiology of the macrophage to modulate host defenses (51). Although the sequencing of the M. tuberculosis genome and recent insights are beginning to shed light on pathogenic mechanisms of this organism (7,8,27,30), the means by which M. tuberculosis adapts to survive and replicate in the host remain poorly understood.Data from a number of laboratories have implicated several alternative sigma factors of mycobacteria, including SigB, SigE, SigF, and SigH, in the adaptation of the pathogen M. tuberculosis and the saprophyte Mycobacterium smegmatis to several stresses (5,9,12,20,29,53). M. smegmatis SigH has been shown ...

Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rv1139c-Rv1138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the ؊35 region and CGTgtc in the ؊10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE_PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis.The Mycobacterium tuberculosis genome encodes 13 sigma factors, of which 10 fall into the extracytoplasmic function (ECF) subfamily. Three of these sigma factor genes are located 5Ј of genes that encode proteins containing an HXXXCXXC motif, which is found in several anti-sigma factors, including Streptomyces coelicolor RsrA (2,15,17,25), Rhodobacter sphaeroides ChrR (23), and M. tuberculosis RshA (35). For both S. coelicolor SigR-RsrA and M. tuberculosis SigH-RshA, it has been demonstrated that this motif in the anti-sigma protein is a key element of a redox switch that affects the interaction between sigma and anti-sigma and thus regulates sigma factor activity in response to intracellular oxidative stress (24, 35). In the case of RsrA and ChrR, this cysteine-rich motif has been shown to bind zinc, and it has been suggested that this is a general property of this motif, leading to the term zinc-associated anti-sigma factor (ZAS) (17,23,24).In addition to sigH, the other M. tuberculosis sigma factor genes linked to these putative ZAS protein genes are sigE and sigL. SigE, like SigH, has been shown to be a key regulator of the mycobacterial response to oxidative and heat stresses (21, 37). In addition, M. tuberculosis SigB, though it is not linked to a similar putative anti-sigma factor gene, is regulated by SigE and SigH and has be...