DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis

Oxygen is an especially important signal molecule to living organisms, and heme-based sensor proteins play a central role in sensing this ligand (Gilles-Gonzalez and Gonzalez 2005). Pathogenic and symbiotic bacteria should logically possess O2 sensors to guide their survival, since the availability of O2 often changes dramatically from the primary site of attachment to the location in the host where the bacteria can best persist. For example, in the obligate aerobe Mycobacterium tuberculosis, infection begins with entry into the host via the richly aerated lungs; a macrophage then engulfs this bacterium and confines it into a phagosome, or ultimately a granuloma (Wayne and Sohaskey 2001; Kohler et al. 2002; Schnappinger et al. 2003). While information on the O2 concentration in tuberculous human lesions is limited, there is clear evidence that O2 is depleted from blocked human cavities to a sufficient degree that conditions corresponding to those seen in the Wayne-in vitro model of M. tuberculosis nonreplicative persistence are attained (Haapanen et al. 1959; Wayne and Hayes 1996; Wayne and Sohaskey 2001). The transcription-factor DevR governs nearly all genetic responses of M. tuberculosis to O2 limitation (Dasgupta et al. 2000; Sherman et al. 2001; Saini et al. 2002, 2004a,b; Florczyk et al. 2003; Parish et al. 2003; Park et al. 2003; Timm et al. 2003; Malhotra et al. 2004; Muttucumaru et al. 2004; Roberts et al. 2004; Talaat et al. 2004; Voskuil et al. 2004; Bagchi et al. 2005; Matsoso et al. 2005; Sohaskey 2005; Sharma et al. 2006; Singh et al. 2006; Reed et al. 2007). Given that about two billion people worldwide are infected with M. tuberculosis, it is urgently important to understand processes that are thought to assist its survival in its human host (Dye et al. 1999). Among these are the adaptive responses of this pathogen to hypoxia (Wayne and Hayes 1996; Wayne and Sohaskey 2001). Rhizobia also experience a large drop in the O2 concentration as they move from their initial site of attachment on a root hair to the site of chronic persistence in a newly formed symbiotic root nodule (Fischer 1994; Dixon and Kahn 2004; Brencic and Winans 2005). In these bacteria, in vitro hypoxia initiates gene-expression cascades similar to those induced during symbiosis with leguminous plants (Fischer 1994; Dixon and Kahn 2004; Brencic and Winans 2005). This leads to the expression of nitrogen-fixation genes as well as genes essential for surviving O2 deprivation, such as those encoding alternative terminal oxidases for respiration in low O2 (Fischer 1994; Dixon and Kahn 2004; Brencic and Winans 2005). The biochemical mechanisms of hypoxic induction are well described for Sinorhizobium meliloti and Bradyrhizobium japonicum. In both cases, a classical prokaryotic two-component regulatory system made up of the FixL and FixJ proteins governs a direct response to O2 (David et al. 1988; Sciotti et al. 2003). In FixL, a protein-histidine kinase activity is coupled to a neighboring heme-binding domain, such that O2 switches off the kinase and hypoxia switches it on (Gilles-Gonzalez et al. 1991; Gilles-Gonzalez and Gonzalez 2005). Under hypoxic conditions, ferrous FixL has no O2 bound to its heme and is called deoxy-FixL. Deoxy-FixL catalyzes the transfer of a γ-phosphoryl group from ATP to FixJ, a response-regulating transcription factor that functions as FixL's regulatory partner (Gilles-Gonzalez and Gonzalez 1993; Tuckerman et al. 2002; Dunham et al. 2003). The phosphorylation of FixJ causes it to dimerize and bind to target sites in DNA (Da Re et al. 1999). An intermediate of the phosphoryl-transfer reaction is a FixL that becomes transiently phosphorylated. Binding of O2 to the heme is known to inhibit formation of the phospho-FixL intermediate (Sousa et al. 2005). FixL has been noted to discriminate against unwanted ligands at the “switching” rather than the “binding” step (Gilles-Gonzalez and Gonzalez 2005). This stands in contrast to a sensor such as the mammalian soluble guanylyl cyclase, which specifically binds NO and rejects the false signal O2 by using a heme pocket that simply excludes the latter. Oxygen binds less avidly than NO and CO to FixL, yet O2 specifically regulates FixL. Therefore FixL achieves its rejection of false signals such as NO, not by failing to bind those ligands, but by ignoring any bound ligand that is not a true signal. Several lines of evidence indicate that, like the rhizobial FixJ, the DevR transcription factor of M. tuberculosis is controlled by one or more sensory protein-histidine kinases that directly detect O2. Genetic experiments suggest that DevR is important for survival of the pathogen during hypoxia in vitro (Sherman et al. 2001; Florczyk et al. 2003; Parish et al. 2003; Park et al. 2003; Timm et al. 2003; Malhotra et al. 2004; Roberts et al. 2004; Saini et al. 2004a; Talaat et al. 2004; Voskuil et al. 2004; Sharma et al. 2006; Reed et al. 2007). Two closely related protein-histidine kinases, DosT and DevS (62.5% sequence identity) are thought to activate DevR, and the DevS protein is known to contain heme (Roberts et al. 2004; Saini et al. 2004a,b; Sardiwal et al. 2005; Ioanoviciu et al. 2007). The domain organization of DevS is reminiscent of that in FixL, with one notable difference being that FixL binds its heme within a PAS rather than a GAF domain (Fig. 1) (Gong et al. 1998). Thus far, only versions of DosT and DevS lacking heme, either because of deletion of their N-terminal region or treatment with denaturants, have been shown to phosphorylate themselves or DevR in vitro (Roberts et al. 2004; Saini et al. 2004a,b). Does the DosT protein contain heme, and if so, does the heme-GAF domain in DosT or DevS direct a response to gaseous ligands? In the current work, we isolate full-length soluble DosT and DevS, each with its full complement of heme, and examine switching in these sensors. Figure 1. A heme prosthetic group in full-length DosT and DosT1–208. (A) Absorption spectrum of full-length ferrous DosT in air (red), and under anerobic conditions (blue). This protein was found, by a pyridine hemochromogen assay, to contain a single heme ...