The PrrBA two-component system in Rhodobacter sphaeroides 2.4.1, which is composed of the PrrB histidine kinase and the PrrA response regulator, controls the expression of all of the photosynthesis genes, either directly or indirectly, in response to changes in oxygen tension. In vivo under aerobic conditions it is the cbb(3) cytochrome c oxidase which generates an inhibitory signal preventing the accumulation of activated PrrA. Using purified cbb(3) cytochrome c oxidase, PrrB, and PrrA, we demonstrate in vitro that the cbb(3) oxidase inhibits PrrB activity by apparently increasing the intrinsic PrrB phosphatase activity, which dephosphorylates phosphorylated PrrA without alteration of the PrrB kinase activity. The transmembrane domain of PrrB is required for the enhancement of PrrB phosphatase activity by the cbb(3) oxidase. Full-length PrrB has a significantly greater ability to phosphorylate PrrA than does truncated PrrB lacking the transmembrane domain. This is at least in part due to the lower autophosphorylation rate of the truncated PrrB relative to the full-length PrrB. This finding provides evidence that the sensing domain (transmembrane domain) of PrrB plays an important role not only in optimally sensing the state of the cbb(3) oxidase but also in maintaining the correct conformation of PrrB, providing optimal autokinase activity.

The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O 2 . The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O 2 to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O 2 and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.Mycobacterium smegmatis is a nonpathogenic and fast-growing mycobacterium whose adaptive response to a gradual decrease in oxygen tension and exposure to NO is similar to that of Mycobacterium tuberculosis (7). This adaptive capability of mycobacteria has been suggested to allow them to persist in a latent state in the immune-competent host, especially in the case of M. tuberculosis (14,24,35,52). The hypoxic conditions within granulomas, although this is controversial (1), and the NO synthesized by activated macrophages have been proposed to serve as possible signals for the transition of mycobacteria to the nonreplicating, latent state (35, 50, 52).The DevSR (DosSR) two-component system plays a crucial role in the adaptation of mycobacteria to hypoxic and NO conditions. Approximately 48 genes of M. tuberculosis were reported to be induced under hypoxic conditions, as well as on exposure to NO. The upregulation of these genes is mediated by the DevSR system (35, 38, 39, 52). The DevSR two-component system consists of the DevS histidine kinase (HK) and its cognate response regulator (39, 40). In addition to DevS, the DosT HK was found to cross talk with DevR and to be functional in M. tuberculosis (39,40). DevS and DosT show high sequence similarity to each other over the length of their primary structures. The N-terminal sensory domains of DevS and DosT contain two putative GAF domains. The first GAF domain (GAF-A) serves as a heme-binding domain, while the function of the second one (GAF-B) remains to be revealed (18,22,43,46). It was recently demonstrated that either the binding of O 2 to the ferrous form of hemes of both DevS and DosT or the oxidation of Fe 2ϩ within the heme to...

The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.Oxygen sensing is important for facultative anaerobes to adapt to changes in metabolic necessities during the transition between aerobic and anaerobic conditions. Although Mycobacterium tuberculosis (MTB) is an obligate aerobe, a gradual depletion of O 2 from its culture is known to lead to a drastic change in gene expression (8,21,24,28,34,37,39). Approximately 48 genes of M. tuberculosis were reported to be induced under early hypoxic conditions, which is mediated by the DosSR (DevSR) two-component system (16,24,34). The induction of the DosR regulon is important for survival of M. tuberculosis under hypoxic conditions and for it to enter the nonreplicating dormant state (2, 19). The DosSR two-component system consists of the DosS histidine kinase (HK) and its cognate DosR response regulator (RR) (24,26,29). The DosT HK, which shares high sequence similarity to DosS over the length of their primary structures, was also found to cross talk with DosR (26,30). The N-terminal domains of DosS and DosT contain two tandem GAF domains (GAF-A and GAF-B from their N termini), and the three-dimensional structure of the GAF-A and GAF-B domains was determined (5, 25). A b-type heme is embedded in the GAF-A domain, composed of one five-stranded antiparallel ␤-sheet and four ␣-helices (5, 14, 25, 32). The heme is positioned nearly perpendicular to the ␤-sheet, and H149 and H147 of the polypeptides serve as the proximal axial ligands for DosS and DosT, respectively (5, 25). The ligand-binding state at the distal axial position of heme and the redox state of the heme iron modulate the autokinase activity of DosS and DosT. The O 2 -bound (oxyferrous) and ferric forms of the HKs are inactive, whereas the unliganded ferrous (deoxyferrous) form as well as NO-and CO-bound forms are active (17, 36). The heme iron of Dos...