Citrate fermentation by Escherichia coli requires the function of the citrate/succinate antiporter CitT (citT gene) and of citrate lyase (citCDEFXG genes). Earlier experiments suggested that the two-component system CitA/CitB, consisting of the membranebound sensor kinase CitA and the response regulator CitB, stimulates the expression of the genes in the presence of citrate, similarly to CitA/CitB of Klebsiella pneumoniae. In this study, the expression of a chromosomal citC-lacZ gene fusion was shown to depend on CitA/CitB and citrate. CitA/CitB is related to the DcuS/DcuR two-component system which induces the expression of genes for fumarate respiration in response to C 4 -dicarboxylates and citrate. Unlike DcuS, CitA required none of the cognate transporters (CitT, DcuB, or DcuC) for function, and the deletion of the corresponding genes showed no effect on the expression of citC-lacZ. The citAB operon is preceded by a DcuR binding site. Phosphorylated DcuR bound specifically to the promoter region, and the deletion of dcuS or dcuR reduced the expression of citC. The data indicate the presence of a regulatory cascade consisting of DcuS/DcuR modulating citAB expression (and CitA/CitB levels) and CitA/CitB controlling the expression of the citC-DEFXGT gene cluster in response to citrate. In vivo fluorescence resonance energy transfer (FRET) and the bacterial two-hybrid system (BACTH) showed interaction between the DcuS and CitA proteins. However, BACTH and expression studies demonstrated the lack of interaction and cross-regulation between CitA and DcuR or DcuS and CitB. Therefore, there is only linear phosphoryl transfer (DcuS¡DcuR and CitA¡CitB) without cross-regulation between DcuS/DcuR and CitA/CitB. E scherichia coli can grow on a wide variety of substrates under aerobic or anaerobic conditions. Citrate fermentation by E. coli requires the presence of an oxidizable cosubstrate, like glucose or glycerol, which is used as an electron donor (28). Citrate is taken up by the citrate/succinate antiporter CitT (39) and cleaved to acetate and oxaloacetate (OAA) by citrate lyase (CL). Holocitrate lyase and the citrate transporter CitT are encoded by the citCDEFXGT gene cluster. Oxaloacetate then is reduced to malate by malate dehydrogenase (Mdh), and malate subsequently is converted to fumarate by fumarase (FumB). Fumarate finally is reduced to succinate by fumarate reductase (FrdABCD). The twocomponent system CitA/CitB of E. coli is supposed to regulate the expression of the genes for citrate fermentation in response to external citrate under anaerobic conditions (20, 52), similarly to the citrate-responsive two-component system CitA/CitB of Klebsiella pneumoniae (6). CitA/CitB represents a typical extracytoplasmic-sensing two-component system consisting of a membrane-bound sensory histidine kinase, CitA, and the cognate response regulator CitB (30, 50). The perception of the stimulus leads to the autophosphorylation of a conserved histidine residue (His 347 ) in the kinase domain of the sensor CitA. The phosphoryl...

Bacteria use membrane-integral sensor histidine kinases (HK) to perceive stimuli and transduce signals from the environment to the cytosol. Information on how the signal is transmitted across the membrane by HKs is still scarce. Combining both liquid-and solid-state NMR, we demonstrate that structural rearrangements in the extracytoplasmic, citrate-sensing Per-Arnt-Sim (PAS) domain of HK CitA are identical for the isolated domain in solution and in a longer construct containing the membrane-embedded HK and lacking only the kinase core. We show that upon citrate binding, the PAS domain contracts, resulting in a shortening of the C-terminal β-strand. We demonstrate that this contraction of the PAS domain, which is well characterized for the isolated domain, is the signal transmitted to the transmembrane (TM) helices in a CitA construct in liposomes. Putting the extracytoplasmic PAS domain into context of the membrane-embedded CitA construct slows down citrate-binding kinetics by at least a factor of 60, confirming that TM helix motions are linked to the citrate-binding event. Our results are confirmation of a hallmark of the HK signal transduction mechanism with atomic resolution on a full-length construct lacking only the kinase core domain.transmembrane receptors | solid-state NMR spectroscopy | transmembrane signaling | integrated structural biology | histidine sensor kinase H istidine kinases (HKs) are key players in prokaryotic signaling and the primary means of processing extracellular stimuli (1-6). Due to their modular organization, HKs can relay a multitude of different input stimuli, including sensing of nutrients, light, physicochemical parameters (e.g., metal ions, ions gradient), temperature, oxygen, and molecules reporting cell density, to accomplish diverse responses. Over the last decade, various structures have been solved for the cytosolic kinase core and a number of signaling domains and more recently even for the complete cytoplasmic region (7-13), but the transmembrane (TM) signaling mechanism itself remains challenging to decipher. Structural information on individual stimulus-receiving domains therefore provides an important tool to identify TM signal transduction schemes.Because extracytoplasmic receiver domains are generally directly connected to TM helices, structural rearrangements within these motifs can be correlated with the TM-signaling mechanism. Different receiver domains have been characterized with and without their ligands and reveal structural differences between the two states that impose restraints on possible motions of the TM helices. Based on such structures, a symmetry-to-asymmetry switch has been postulated for KinB (14), TorT/TorS (15), and LuxPQ (16), implying a possible tilt of TM helices (17). For chemotaxis sensor Tar and for HK NarX a piston movement of the second TM helix has been postulated as a consequence of receiver domain motions (9,(18)(19)(20).Among receiver domains, PAS domains stand out by being able to process diverse input signals, which is reflected by...

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Glucose uptake by the heterofermentative lactic acid bacterium Oenococcus oeni B1 was studied at the physiological and gene expression levels. Glucose-or fructose-grown bacteria catalyzed uptake of [ 14 C]glucose over a pH range from pH 4 to 9, with maxima at pHs 5.5 and 7. Uptake occurred in two-step kinetics in a highand low-affinity reaction. The high-affinity uptake followed Michaelis-Menten kinetics and required energization. It accumulated the radioactivity of glucose by a factor of 55 within the bacteria. A large portion (about 80%) of the uptake of glucose was inhibited by protonophores and ionophores. Uptake of the glucose at neutral pH was not sensitive to degradation of the proton potential, ⌬p. Expression of the genes OEOE_0819 and OEOE_1574 (here referred to as 0819 and 1574), coding for secondary transporters, was induced by glucose as identified by quantitative real-time (RT)-PCR. The genes 1574 and 0819 were able to complement growth of a Bacillus subtilis hexose transport-deficient mutant on glucose but not on fructose. The genes 1574 and 0819 therefore encode secondary transporters for glucose, and the transports are presumably ⌬p dependent. O. oeni codes, in addition, for a phosphotransferase transport system (PTS) (gene OEOE_0464 [0464] for the permease) with similarity to the fructose-and mannose-specific PTS of lactic acid bacteria. Quantitative RT-PCR showed induction of the gene 0464 by glucose and by fructose. The data suggest that the PTS is responsible for ⌬p-independent hexose transport at neutral pH and for the residual ⌬p-independent transport of hexoses at acidic pH.

The thermophilic Geobacillus thermodenitrificans and Geobacillus kaustophilus are able to use citrate or C 4 -dicarboxylates like fumarate or succinate as the substrates for growth. The genomes of the sequenced Geobacillus strains (nine strains) each encoded a two-component system of the CitA family. The sensor kinase of G. thermodenitrificans (termed CitA Gt ) was able to replace CitA of Escherichia coli (CitA Ec ) in a heterologous complementation assay restoring expression of the CitA Ec -dependent citC-lacZ reporter gene and anaerobic growth on citrate. Complementation was specific for citrate. The sensor kinase of G. kaustophilus (termed DcuS Gk ) was able to replace DcuS Ec of E. coli. It responded in the heterologous expression system to C 4 -dicarboxylates and to citrate, suggesting that DcuS Gk is, like DcuS Ec , a C 4 -dicarboxylate sensor with a side-activity for citrate. DcuS Gk , unlike the homologous DctS from Bacillus subtilis, required no binding protein for function in the complementation assay. Thus, the thermophilic G. thermodenitrificans and G. kaustophilus contain citrate and C 4 -dicarboxylate sensor kinases of the CitA and DcuS type, respectively, and retain function and substrate specificity under mesophilic growth conditions in E. coli.