Current estimates indicate that nearly a third of the world's population is latently infected with Mycobacterium tuberculosis. Reduced oxygen tension and nitric oxide exposure are two conditions encountered by bacilli in vivo that may promote latency. In vitro exposure to hypoxia or nitric oxide results in bacterial stasis with concomitant induction of a 47-gene regulon controlled by the transcription factor DosR. In this report we demonstrate that both the dosS gene adjacent to dosR and another gene, dosT (Rv2027c), encode sensor kinases, each of which can autophosphorylate at a conserved histidine and then transfer phosphate to an aspartate residue of DosR. Mutant bacteria lacking both sensors are unable to activate expression of DosR-regulated genes. These data indicate that DosR/DosS/DosT comprise a two-component signaling system that is required for the M. tuberculosis genetic response to hypoxia and nitric oxide, two conditions that produce reversible growth arrest in vitro and may contribute to latency in vivo.Tuberculosis (TB) 1 has placed a heavy burden on the global community for centuries, earning such morbid nicknames as The White Plague and The Captain of the Men of Death (1). The causative agent, Mycobacterium tuberculosis (MTB), kills about 2 million people annually making it a leading cause of infectious death worldwide (2, 3). The success of MTB as a pathogen is closely linked with its capacity to persist for years or decades in humans in the absence of any clinical disease symptoms. Current estimates place the number of people latently infected with MTB at nearly 2 billion, or one-third of the Earth's population (3, 4). Eradicating this enormous reservoir of latently infected carriers is complicated by several factors, including the availability, cost, and length of drug therapy required for successful treatment of latent TB.Although TB has been studied for centuries, the triggers that promote and maintain latent infections are still obscure. Two conditions frequently associated with latent TB in vivo are reduced oxygen tension and nitric oxide (NO) exposure (5, 6). Both of these stimuli can induce reversible bacterial stasis in vitro (7,8), and both are encountered by bacilli in vivo (5, 9, 10). Further, although MTB requires oxygen for growth, it can survive without oxygen for surprisingly long periods of time (11,12). Still the evidence linking hypoxia and NO to latent TB in vivo remains circumstantial. Analysis of the MTB response to these factors is needed to define the role they may play in promoting and maintaining TB latency in humans.Previous reports identified a set of 47 MTB genes that are rapidly up-regulated in response to reduced oxygen tension or NO (8, 13). Among the MTB genes induced by hypoxia or exposure to NO is the putative two-component regulatory system dosR-dosS (also called devR-devS, Rv3133c/Rv3132c 2 ) (8, 13). In bacteria, two-component response regulator systems are an important means by which a variety of environmental signals are transduced into a phenotypic respo...

Highlights d Voltage shifting and disulfide locking capture a resting-state structure of Na V Ab d Three gating charges translocate intracellularly through transmembrane electric field d Voltage sensor couples to pore opening by an elbow connecting S4 to the S4-S5 linker d Resting-state structure supports a sliding helix mechanism of gating

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The major facilitator superfamily is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations including both the outward and inward facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.