Serine 83 in DosR,a response regulator from Mycobacterium tuberculosis, promotes its transition from an activated,phosphorylated state to an inactive,unphosphorylated state

A sensor kinase, DosS, and its corresponding response regulator, DosR, constitute a two component system for regulating gene expression under hypoxic conditions in Mycobacterium tuberculosis. Among response regulators in M. tuberculosis, NarL has high sequence similarity to DosR, and autophosphorylated DosS transfers its phosphate group not only to DosR but also to NarL. Phosphorylated DosR is more rapidly dephosphorylated than phosphorylated NarL. DosR and NarL differ with respect to the amino acids at positions T + 1 and T + 2 around the phosphorylation sites in the N-terminal phosphoacceptor domain; NarL has S83 and Y84, whereas DosR has A90 and H91. A DosR S83A mutant shows prolonged phosphorylation. Structural comparison with a histidinol phosphate phosphatase suggests that the hydroxyl group of DosR S83 could play a role in activating the water molecule involved in the triggering of autodephosphorylation.     

Crystal structures of the response regulator DosR from Mycobacterium tuberculosis suggest a helix rearrangement mechanism for phosphorylation activation

The response regulator DosR is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. Here, we report crystal structures of full-length unphosphorylated DosR at 2.2 Å resolution and its C-terminal DNA-binding domain at 1.7 Å resolution. The full-length DosR structure reveals several features never seen before in other response regulators. The N-terminal domain of the full-length DosR structure has an unexpected (βα)₄ topology instead of the canonical (βα)₅ fold observed in other response regulators. The linker region adopts a unique conformation that contains two helices forming a four-helix bundle with two helices from another subunit, resulting in dimer formation. The C-terminal domain in the full-length DosR structure displays a novel location of helix α10, which allows Gln199 to interact with the catalytic Asp54 residue of the N-terminal domain. In contrast, the structure of the DosR C-terminal domain alone displays a remarkable unstructured conformation for helix α10 residues, different from the well-defined helical conformations in all other known structures, indicating considerable flexibility within the C-terminal domain. Our structures suggest a mode of DosR activation by phosphorylation via a helix rearrangement mechanism.     

The Mycobacterium DosR regulon structure and diversity revealed by comparative genomic analysis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), which claims approximately two million people annually, remains a global health concern. The non‐replicating or dormancy like state of this pathogen which is impervious to anti‐tuberculosis drugs is widely recognized as the culprit for this scenario. The dormancy survival regulator (DosR) regulon, composed of 48 co‐regulated genes, is held as essential for Mtb persistence. The DosR regulon is regulated by a two‐component regulatory system consisting of two sensor kinases—DosS (Rv3132c) and DosT (Rv2027c), and a response regulator DosR (Rv3133c). The underlying regulatory mechanism of DosR regulon expression is very complex. Many factors are involved, particularly the oxygen tension. The DosR regulon enables the pathogen to persist during lengthy hypoxia. Comparative genomic analysis demonstrated that the DosR regulon is widely distributed among the mycobacterial genomes, ranging from the pathogenic strains to the environmental strains. In‐depth studies on the DosR response should provide insights into its role in TB latency in vivo and shape new measures to combat this exceeding recalcitrant pathogen. J. Cell. Biochem. 114: 1–6, 2012. © 2012 Wiley Periodicals, Inc.     

The Mycobacterium tuberculosis DosR Regulon Assists in Metabolic Homeostasis and Enables Rapid Recovery from Nonrespiring Dormancy

Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.Mycobacterium tuberculosis, a major human pathogen, infects nearly one-third of the people in the world and causes two million deaths per year (8). Most infections are latent, and a substantial number of new infections are transmitted by individuals in whom latent infections are being reactivated. Latency is a clinical term describing people that are infected with M. tuberculosis but lack symptoms of active disease. Traditionally, it has been thought that bacilli in latently infected individuals reside almost exclusively inside granulomas and mature tubercle lesions. Recent studies indicate that in latently infected individuals M. tuberculosis may also be found outside granulomas in places such as endothelial cells, fibroblasts, and adipose tissue (17, 28). The evidence for M. tuberculosis metabolic activity in vivo is more limited, but two studies by Lillebaek et al. are informative (24, 25). In these studies the researchers used detailed records of tuberculosis epidemiology and strain types in the fairly static population of Denmark. They found that strains isolated from patients thought to have reactivated disease (rather than a primary infection) were nearly identical to strains present 30 years earlier in the same geographic population. The near-identity of the strains and the fact that infections were attributed to reactivation suggest that bacteria in latently infected individuals experience little genetic change during years of latent infection. The researchers concluded that during latency, M. tuberculosis divides infrequently and is likely in a minimal metabolic state.One approach to study the M. tuberculosis metabolic state during latent infection is to use in vitro models that mimic conditions thought to exist in vivo. Such conditions include hypoxia produced in avascular calcified granulomas (40) and nitric oxide (NO) (27) or carbon monoxide (CO) (33) produced by activated immune cells. A widely used model is the “Wayne model” pioneered by Lawrence Wayne. In this model, a low-inoculum culture is sealed in a tube with stirring and allowed to slowly consume oxygen until the culture is anaerobic, resulting in a nonreplicating and apparently dormant state (45, 46). Another model used to look at dormant M. tuberculosis is a constant-hypoxia model that maintains a 0.2% oxygen tension in culture flasks (31).The common theme in these in vitro models used to obtain M. tuberculosis dormancy is inhibition of respiration. The DosR regulon is a set of at least 48 coregulated genes that are induced by three conditions that inhibit aerobic respiration: hypoxia, NO, and CO (42). Induction of the DosR regulon closely mirrors inhibition of respiration, indicating that control of the regulon is linked to the aerobic respiratory state of the bacilli (43). Several studies have shown that the DosR regulon is controlled by a three-component regulatory system composed of two sensor histidine kinases, DosS and DosT, and a response regulator, DosR (42). DosS and DosT both bind the respiration-impairing gases NO and CO (19, 20, 38), further supporting the hypothesis that the DosR regulon responds to, and is important during, conditions that do not allow aerobic respiration. Although the majority of the DosR-regulated genes have not been characterized, the timing of their induction combined with the conditions under which they respond suggests that they may play a role in adaptation of M. tuberculosis to its host environment. Consistent with this notion, DosR regulon genes are induced in the lungs of M. tuberculosis-infected mice (43), as well as in interferon-gamma-activated murine macrophages (34) and guinea pigs (37).Several studies have suggested that the DosR regulon plays a role in latent infection and in persistence in animal models that resemble human infection in some respects. Leyten et al. found that latently infected humans are more likely than humans with active infections to bear T cells specific for DosR regulon antigens (23), suggesting that the regulon is expressed during latency. Two recent studies confirmed that there is an immune response to DosR regulon antigens during latent infection (4, 36). Further evidence for clinical relevance in humans comes from a study showing that M. tuberculosis in sputum expresses the DosR regulon (15). The importance of this regulon for persistence in rabbit and guinea pig models was demonstrated by data showing a 2-log decrease in recovery of a DosR mutant 2 weeks (guinea pig) and 8 weeks (rabbit) after aerosol infection (11). A DosR mutant was also found to be significantly attenuated in guinea pig infection (26), further supporting the notion that the DosR regulon is required for persistence in vivo. It should be noted that in both studies showing the DosR phenotype (11, 26), full complementation and reversion to full virulence were not observed. However, it is now known that regulation of dosR expression is quite complex. Multiple regulatory sequences exist in and upstream of Rv3134c, the gene directly upstream of dosR (8). Failure to include such a regulatory sequence in a complemented strain would likely result in misregulation of dosR and poor complementation. Studies of DosR regulon mutants for murine infection have produced inconsistent findings that vary from hypervirulent (30) to attenuated (11) and not attenuated (3, 31). When animal models are compared, it is important to remember that M. tuberculosis-induced granulomas in primates, rabbits, and guinea pigs develop caseous necrosis and are hypoxic and/or anaerobic, while M. tuberculosis induced-granulomas in mice are neither hypoxic nor anaerobic (2, 21, 41). Furthermore, M. tuberculosis divides regularly in chronic murine infections (16), in contrast to the replication during latent infections, as demonstrated in the studies of Lillebaek et al. (24, 25). Such studies underscore the significant differences between models.A previous study with a DosR mutant in a closely related Mycobacterium bovis BCG strain showed that DosR expression is required for survival in an in vitro Wayne-like model of dormancy (5). Unexpectedly, two similar studies in M. tuberculosis did not show a strong survival defect for a DosR mutant (31, 43). The most recent study showed that there was only a modest survival defect in an H37Rv DosR mutant and concluded that the DosR regulon is a short-term phenomenon and is not responsible for the adaptation necessary to survive under primarily hypoxic conditions in vitro (31, 32).In this study we showed that the DosR regulon is required for M. tuberculosis survival during anaerobic dormancy. We also used a combination of genetic and biochemical approaches to demonstrate that this regulon is necessary to shift away from oxygen consumption, maintain ATP levels, and balance the redox state (NAD/NADH ratio) of the cell as oxygen becomes scarce. Furthermore, we showed that the DosR regulon is necessary for optimal transition of M. tuberculosis back to aerobic growth from an anaerobic or nitric oxide-induced nonrespiring state.     

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.     

Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides

Background

High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the α-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen.

Results

One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation.

Conclusion

Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a “core iron response” that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-794) contains supplementary material, which is available to authorized users.     

Modeling Phenotypic Metabolic Adaptations of Mycobacterium tuberculosis H37Rv under Hypoxia

The ability to adapt to different conditions is key for Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), to successfully infect human hosts. Adaptations allow the organism to evade the host immune responses during acute infections and persist for an extended period of time during the latent infectious stage. In latently infected individuals, estimated to include one-third of the human population, the organism exists in a variety of metabolic states, which impedes the development of a simple strategy for controlling or eradicating this disease. Direct knowledge of the metabolic states of M. tuberculosis in patients would aid in the management of the disease as well as in forming the basis for developing new drugs and designing more efficacious drug cocktails. Here, we propose an in silico approach to create state-specific models based on readily available gene expression data. The coupling of differential gene expression data with a metabolic network model allowed us to characterize the metabolic adaptations of M. tuberculosis H37Rv to hypoxia. Given the microarray data for the alterations in gene expression, our model predicted reduced oxygen uptake, ATP production changes, and a global change from an oxidative to a reductive tricarboxylic acid (TCA) program. Alterations in the biomass composition indicated an increase in the cell wall metabolites required for cell-wall growth, as well as heightened accumulation of triacylglycerol in preparation for a low-nutrient, low metabolic activity life style. In contrast, the gene expression program in the deletion mutant of dosR, which encodes the immediate hypoxic response regulator, failed to adapt to low-oxygen stress. Our predictions were compatible with recent experimental observations of M. tuberculosis activity under hypoxic and anaerobic conditions. Importantly, alterations in the flow and accumulation of a particular metabolite were not necessarily directly linked to differential gene expression of the enzymes catalyzing the related metabolic reactions.     

Analysis of expression and regulatory functions of the ribosome-binding protein TypA in Mycobacterium tuberculosis under stress conditions

In many bacterial species, the translational GTPase TypA acts as a global stress- and virulence regulator and also mediates resistance to the antimicrobial peptide BPI. On the chromosome of M. tuberculosis, typA is located next to narGHJI, which plays a role in adaptation of the pathogen to various environmental conditions. Here, we show that Mycobacterium tuberculosis is sensitive to P2, a derivative of BPI. Using a typA mutant of M. tuberculosis, we found this phenotype to be independent of TypA. We further tested typA expression in M. tuberculosis under defined stress conditions, such as oxygen- and nutrient depletion, low pH, heat shock, antibiotic stress and the presence of P2, and found that typA expression remains unaffected by any of these conditions. Analysis of growth and whole-genome expression revealed similar growth kinetics and gene expression profiles of the wild type and the mutant under normal growth conditions as well as under stress conditions. Our results suggest that in contrast to the findings in other bacteria, TypA does not act as a global stress- and virulence regulator in M. tuberculosis.     

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H₂O₂ and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.