Large Mg(2+)-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae

Mycobacteria adapt to a decrease in oxygen tension by entry into a non-replicative persistent phase. It was shown earlier that the two-component system, DevR-DevS, was induced in Mycobacterium tuberculosis and Mycobacterium bovis BCG cultures during hypoxia, suggesting that it may play a regulatory role in their adaptation to oxygen limitation. The presence of a homologous genetic system in Mycobacterium smegmatis was predicted by scanning its unfinished genome sequence with devR and devS genes of M. tuberculosis. Rv3134c, which is cotranscribed with devR-devS in M. tuberculosis, was also present in M. smegmatis at a similar location upstream from devR. The expression of all three genes was induced at the RNA and protein levels in M. smegmatis cultures grown under microaerobic and anaerobic conditions. The M. smegmatis genome also contained the hspX gene, encoding chaperone alpha-crystallin, Acr, that was induced during hypoxia. The similarity in sequences and hypoxia-responsive behaviour of devR-devS, Rv3134c and hspX genes in M. smegmatis and M. tuberculosis suggests that the molecular mechanisms involved in the dormancy response are likely conserved in these two species. M. smegmatis could therefore serve as a useful model for the delineation of the hypoxia response in general and DevR-DevS regulated pathways in particular.     

The Residue Threonine 82 of DevR (DosR) Is Essential for DevR Activation and Function in Mycobacterium tuberculosis Despite Its Atypical Location

The DevR (DosR) response regulator initiates the bacterial adaptive response to a variety of signals, including hypoxia in in vitro models of dormancy. Its receiver domain works as a phosphorylation-mediated switch to activate the DNA binding property of its output domain. Receiver domains are characterized by the presence of several highly conserved residues, and these sequence features correlate with structure and hence function. In response regulators, interaction of phosphorylated aspartic acid at the active site with the conserved threonine is believed to be crucial for phosphorylation-mediated conformational change. DevR contains all the conserved residues, but the structure of its receiver domain in the unphosphorylated protein is strikingly different, and key threonine (T82), tyrosine (Y101), and lysine (K104) residues are placed uncharacteristically far from the D54 phosphorylation site. In view of the atypical location of T82 in DevR, the present study aimed to examine the importance of this residue in the activation mechanism. Mycobacterium tuberculosis expressing a DevR T82A mutant protein is defective in autoregulation and supports hypoxic induction of the DevR regulon only very weakly. These defects are ascribed to slow and partial phosphorylation and the failure of T82A mutant protein to bind cooperatively with DNA. Our results indicate that the T82 residue is crucial in implementing conformational changes in DevR that are essential for cooperative binding and for subsequent gene activation. We propose that the function of the T82 residue in the activation mechanism of DevR is conserved in spite of the unusual architecture of its receiver domain.     

Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc

The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis. Hypoxia is a potent stimulator of VEGF expression, and hypoxia-inducible factor-1 (HIF-1) is considered to be critical for this induction. However, we have previously demonstrated that induction of VEGF by hypoxia was preserved when HIF-1alpha was silenced. We sought to better define the molecular basis of this HIF-1-independent regulation. In colon cancer cells, hypoxia stimulated multiple K-ras effector pathways including phosphatidylinositol 3-kinase. VEGF promoter deletion studies identified a novel promoter region between -418 and -223 bp that was responsive to hypoxia in a PI3K/Rho/ROCK-dependent manner. Electrophoretic mobility shift assays identified a fragment between -300 and -251 bp that demonstrated a unique shift only in hypoxic conditions. Inhibition of PI3K or ROCK blocked the formation of this complex. A binding site for c-Myc, a target of ROCK, was identified at -271 bp. A role for c-Myc in the hypoxic induction of VEGF was demonstrated by site-directed mutagenesis of the VEGF promoter and silencing of c-Myc by small interfering RNA. Collectively, these findings suggest an alternative mechanism for the hypoxic induction of VEGF in colon cancer that does not depend upon HIF-1alpha but instead requires the activation of PI3K/Rho/ROCK and c-Myc.     

Co-Expression of DevR and DevRN-Aph Proteins Is Associated with Hypoxic Adaptation Defect and Virulence Attenuation of Mycobacterium tuberculosis

Background

The DevR response regulator is implicated in both hypoxic adaptation and virulence of Mycobacterium tuberculosis (M. tb). DevR regulon genes are powerfully induced in vivo implicating them in bacterial adaptation to host control strategies. A better understanding of DevR function will illumine the way for new strategies to control and treat tuberculosis.

Methodology/Principal Findings

Towards this objective, we used a combination of genetic, microbiological, biochemical, cell biological tools and a guinea pig virulence assay to compare the hypoxic adaptation and virulence properties of two novel M. tb strains, namely, a devR disruption mutant, Mut1, that expresses C-terminal truncated N-terminal domain of DevR (DevR_NTD) as a fusion protein with AphI (DevR_N-Kan), and its complemented strain, Comp1, that expresses intact DevR along with DevR_N-Kan. Comp1 bacteria exhibit a defect in DevR-mediated phosphosignalling, hypoxic induction of HspX and also hypoxic survival. In addition, we find that Comp1 is attenuated in virulence in guinea pigs and shows decreased infectivity of THP-1 cells. While Mut1 bacilli are also defective in hypoxic adaptation and early growth in spleen, they exhibit an overall virulence comparable to that of wild-type bacteria.

Conclusions/Significance

The hypoxic defect of Comp1 is associated to a defect in DevR expression level. The demonstrated repression of DevR function by DevR_N-Kan suggests that such a knockdown approach could be useful for evaluating the activity of DevRS and other two-component signaling pathways. Further investigation is necessary to elucidate the mechanism underlying Comp1 attenuation.     

A two-component regulator of universal stress protein expression and adaptation to oxygen starvation in Mycobacterium smegmatis

We identified a response regulator in Mycobacterium smegmatis which plays an important role in adaptation to oxygen-starved stationary phase. The regulator exhibits strong sequence similarity to DevR/Rv3133c of M. tuberculosis. The structural gene is present on a multigene locus, which also encodes a sensor kinase. A devR mutant of M. smegmatis was adept at surviving growth arrest initiated by either carbon or nitrogen starvation. However, its culturability decreased several orders of magnitude below that of the wild type under oxygen-starved stationary-phase conditions. Two-dimensional gel analysis revealed that a number of oxygen starvation-inducible proteins were not expressed in the devR mutant. Three of these proteins are universal stress proteins, one of which is encoded directly upstream of devR. Another protein closely resembles a proposed nitroreductase, while a fifth protein corresponds to the alpha-crystallin (HspX) orthologue of M. smegmatis. None of the three universal stress proteins or nitroreductase, and a considerably lower amount of HspX was detected in carbon-starved wild-type cultures. A fusion of the hspX promoter to gfp demonstrated that DevR directs gene expression when M. smegmatis enters stationary phase brought about, in particular, by oxygen starvation. To our knowledge, this is the first time a role for a two-component response regulator in the control of universal stress protein expression has been shown. Notably, the devR mutant was 10(4)-fold more sensitive than wild type to heat stress. We conclude that DevR is a stationary-phase regulator required for adaptation to oxygen starvation and resistance to heat stress in M. smegmatis.