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DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA

(RECEIVED March 22, 2007; FINAL REVISION May 7, 2007; ACCEPTED May 9, 2007)

Exposure of Mycobacterium tuberculosis to hypoxia is known to alter the expression of many genes, including ones thought to be involved in latency, via the transcription factor DevR (also called DosR). Two sensory kinases, DosT and DevS (also called DosS), control the activity of DevR. We show that, like DevS, DosT contains a heme cofactor within an N-terminal GAF domain. For full-length DosT and DevS, we determined the ligand-binding parameters and the rates of ATP reaction with the liganded and unliganded states. In both proteins, the heme state was coupled to the kinase such that the unliganded, CO-bound, and NO-bound forms were active, but the O₂-bound form was inactive. Oxygen-bound DosT was unusually inert to oxidation to the ferric state (half life in air >60 h). Though the kinase activity of DosT was unaffected by NO, this ligand bound 5000 times more avidly than O₂ to DosT (K_d [NO] 5 nM versus K_d [O₂] = 26 µM). These results demonstrate direct and specific O₂ sensing by proteins in M. tuberculosis and identify for the first time a signal ligand for a sensory kinase from this organism. They also explain why exposure of M. tuberculosis to NO donors under aerobic conditions can give results identical to hypoxia, i.e., NO saturates DosT, preventing O₂ binding and yielding an active kinase.

Keywords: FixL; GAF domain; heme-based sensor; histidine-protein kinase; host-microbe interactions; oxygen sensor; sensor kinase; response regulator; signal transduction

Reprint requests to: Marie-Alda Gilles-Gonzalez, Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038, USA; e-mail: marie-alda.gilles-gonzalez{at}utsouthwestern.edu; fax: (214) 648-8856.

Abbreviations: DosT, M. tuberculosis sensor kinase encoded by the Rv2027c gene; DevR, M. tuberculosis response regulator encoded by Rv3133c and also called DosR; DevS, M. tuberculosis sensor kinase encoded by Rv3132c and also called DosS; BjFixL, Bradyrhizobium japonicum FixL; RmFixL, Sinorhizobium meliloti FixL; GAF, regulatory domain originally named for its association with cGMP-regulated cyclic nucleotide phosphodiesterases, adenylate cyclases, and the bacterial transcriptional regulator FhlA; PAS, signal-detection domain originally named for its association with the Per, ARNT, and Sim proteins; deoxy, Fe^II form; oxy, Fe^IIO₂ form; carbonmonoxy, Fe^IICO form; nitrosyl, Fe^IINO form.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072897707.

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