Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor.

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 ß-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.

CitA (citrate) and DcuS (C4-dicarboxylate) sensor kinases in thermophilic Geobacillus kaustophilus and Geobacillus thermodenitrificans.

The thermophilic Geobacillus thermodenitrificans and Geobacillus kaustophilus are able to use citrate or C4-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 CitAGt) was able to replace CitA of Escherichia coli (CitAEc) in a heterologous complementation assay restoring expression of the CitAEc-dependent citC-lacZ reporter gene and anaerobic growth on citrate. Complementation was specific for citrate. The sensor kinase of G. kaustophilus (termed DcuSGk) was able to replace DcuSEc of E. coli. It responded in the heterologous expression system to C4-dicarboxylates and to citrate, suggesting that DcuSGk is, like DcuSEc, a C4-dicarboxylate sensor with a side-activity for citrate. DcuSGk, 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 C4-dicarboxylate sensor kinases of the CitA and DcuS type, respectively, and retain function and substrate specificity under mesophilic growth conditions in E. coli.

The sensor kinase DctS forms a tripartite sensor unit with DctB and DctA for sensing C4-dicarboxylates in Bacillus subtilis.

The DctSR two-component system of Bacillus subtilis controls the expression of the aerobic C4-dicarboxylate transporter DctA. Deletion of DctA leads to an increased dctA expression. The inactivation of DctB, an extracellular binding protein, is known to inhibit the expression of dctA. Here, interaction between the sensor kinase DctS and the transporter DctA as well as the binding protein DctB was demonstrated in vivo using streptavidin (Strep) or His protein interaction experiments (mSPINE or mHPINE), and the data suggest that DctA and DctB act as cosensors for DctS. The interaction between DctS and DctB was also confirmed by the bacterial two-hybrid system (BACTH). In contrast, no indication was obtained for a direct interaction between the transporter DctA and the binding protein DctB. Activity levels of uptake of [(14)C]succinate by bacteria that expressed DctA from a plasmid were similar in the absence and the presence of DctB, demonstrating that the binding protein DctB is not required for transport. Thus, DctB is involved not in transport but in cosensing with DctS, highlighting DctB as the first example of a TRAP-type binding protein that acts as a cosensor. The simultaneous presence of DctS/DctB and DctS/DctA sensor pairs and the lack of direct interaction between the cosensors DctA and DctB indicate the formation of a tripartite complex via DctS. It is suggested that the DctS/DctA/DctB complex forms the functional unit for C4-dicarboxylate sensing in B. subtilis.

Role of secondary transporters and phosphotransferase systems in glucose transport by Oenococcus oeni.

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 high- and 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, Δp. 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 Δp 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 Δp-independent hexose transport at neutral pH and for the residual Δp-independent transport of hexoses at acidic pH.