Properties of transmembrane helix TM1 of the DcuS sensor kinase of Escherichia coli, the stator for TM2 piston signaling.

The sensor kinase DcuS of Escherichia coli perceives extracellular fumarate by a periplasmic PASP sensor domain. Transmembrane (TM) helix TM2, present as TM2-TM2' homo-dimer, transmits fumarate activation in a piston-slide across the membrane. The second TM helix of DcuS, TM1, is known to lack piston movement. Structural and functional properties of TM1 were analyzed. Oxidative Cys-crosslinking (CL) revealed homo-dimerization of TM1 over the complete membrane, but only the central part showed α-helical +3/+4 spacing of the CL maxima. The GALLEX bacterial two-hybrid system indicates TM1/TM1' interaction, and the presence of a TM1-TM1' homo-dimer is suggested. The peripheral TM1 regions presented CL in a spacing atypical for α-helical arrangement. On the periplasmic side the deviation extended over 11 AA residues (V32-S42) between the α-helical part of TM1 and the onset of PASP. In the V32-S42 region, CL efficiency decreased in the presence of fumarate. Therefore, TM1 exists as a homo-dimer with α-helical arrangement in the central membrane region, and non-α-helical arrangement in the connector to PASP. The fumarate induced structural response in the V32-S42 region is suggested to represent a structural adaptation to the shift of TM2 in the TM1-TM1'/TM2-TM2' four-helical bundle.

Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase.

Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains an LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type TM signaling by the TM2 homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation.

DcuA of aerobically grown Escherichia coli serves as a nitrogen shuttle (L-aspartate/fumarate) for nitrogen uptake.

DcuA of Escherichia coli is known as an alternative C4 -dicarboxylate transporter for the main anaerobic C4 -dicarboxylate transporter DcuB. Since dcuA is expressed constitutively under aerobic and anaerobic conditions, DcuA was suggested to serve aerobically as a backup for the aerobic (DctA) transporter, or for the anabolic uptake of C4 -dicarboxylates. In this work, it is shown that DcuA is required for aerobic growth with L-aspartate as a nitrogen source, whereas for growth with L-aspartate as a carbon source, DctA was needed. Strains with DcuA catalyzed L-aspartate and C4 -dicarboxylate uptake (like DctA), or an L-aspartate/C4 -dicarboxylate antiport (unlike DctA). DcuA preferred L-aspartate to succinate in transport (KM = 43 and 844 µM, respectively), whereas DctA has higher affinity for C4 -dicarboxylates like succinate compared to L-aspartate. When L-aspartate was supplied as the sole nitrogen source together with glycerol as the carbon source, L-aspartate was taken up by the bacteria and fumarate (or L-malate) was excreted in equimolar amounts. Both reactions depended on DcuA. L-Aspartate was taken up in amounts required for nitrogen metabolism but not for carbon metabolism. Therefore, DcuA catalyzes an L-aspartate/C4 -dicarboxylate antiport serving as a nitrogen shuttle for nitrogen supply without net carbon supply.

Bone cement allocation analysis in artificial cancellous bone structures.

BACKGROUND: One of the most serious adverse events potentially occurring during vertebroplasty is cement leakage. Associated risks for the patient could be reduced if cement filling is preoperatively planned. This requires a better understanding of cement flow behaviour. Therefore, the aim of the present study was to investigate bone cement distribution in artificial inhomogeneous cancellous bone structures during a simulated stepwise injection procedure. METHODS: Four differently coloured 1-mL cement portions were injected stepwise into six open-porous aluminum foam models with simulated leakage paths. Each model was subsequently cross-sectioned and high-resolution pictures were taken, followed by anatomical site allocation based on the assumption about a posterior insertion of the cannula. A radial grid consisting of 36 equidistant beams (0°-350°) was applied to evaluate the cement flow along each beam by measuring the radial length of each cement portion (total length) and of all four portions together (distance to border). Independently from the injection measurements, the viscosity of 20 cement portions was measured at time points corresponding to the start of the first and the end of the last injection. RESULTS: Despite some diffuse colour transitions at the borderlines, no interfusion between the differently coloured cement portions was observed. The two highest values for total length of each of the first three injected cement portions and for distance to border were indicated in directions anterior bilateral to the cannula along the 120°, 240° and 250° beams and posterolateral along the 60° beam. The two highest total lengths for the fourth cement portion were registered in the direction of the cannula along the 170° and 180° beams. Standard deviations of total length for each of the last three injected portions and for distance to border were with two highest values in directions anterior bilateral to the cannula along the 120°, 150°, 240° and 250° beams and opposite to the direction of the cannula along the 10° beam. The two highest values for the first cement portion were registered posterior bilateral to the cannula along the 70° and 350° beams. The values for averaged standard deviations of the total length of the fourth cement portion and the distance to border were significantly higher in comparison to the first cement portion (p ≤ 0.020). Dynamic viscosity at the start of the first injection was 343 ± 108 Pa∙s and increased to 659 ± 208 Pa∙s at the end of the fourth injection. CONCLUSION: The simulated leakage path seemed to be the most important adverse injection factor influencing the uniformity of cement distribution. Another adverse factor causing dispersion of this distribution was represented by the simulated bone marrow. However, the rather uniform distribution of the totally injected cement amount, considered as one unit, could be ascribed to the medium viscosity of the used cement. Finally, with its short waiting time of 45 s, the stepwise injection procedure was shown to be ineffective in preventing cement leakage.

Bone cement flow analysis by stepwise injection through medical cannulas.

Cement leakage is a serious adverse event potentially occurring during vertebroplasty. Pre-operative in-silico planning of the cement filling process can help reducing complication rates related to leakage. This requires a better understanding of the cement flow along the whole injection path. Therefore, the aim of the present study was to analyze bone cement flow behavior by stepwise injections through medical cannulas. Sixteen cannulas were assigned to four groups for stepwise injection of differently colored cement portions of 1ml volume. Each group differed in the amount of injected cement portions with a range of 1-4ml. After cement curing longitudinal cross-sections of the cannulas were performed and high-resolution pictures taken. Based on these pictures, quadratic polynomial interpolation was applied to the marked intersections between the last two injected cement portions to calculate the leading coefficients. Leading coefficients in the groups with three cement portions (0.287 ± 0.078), four portions (0.243 ± 0.041) and two portions (0.232 ± 0.050) were comparable and significantly higher than the group with one cement portion (0.0032 ± 0.0004), p ≤ 0.016. Based on these findings, cement flow through medical cannulas can be considered as predictable and can therefore be excluded as a source of risk for possible cement leakage complications during vertebroplasty procedures.

Polar localization of a tripartite complex of the two-component system DcuS/DcuR and the transporter DctA in Escherichia coli depends on the sensor kinase DcuS.

The C4-dicarboxylate responsive sensor kinase DcuS of the DcuS/DcuR two-component system of E. coli is membrane-bound and reveals a polar localization. DcuS uses the C4-dicarboxylate transporter DctA as a co-regulator forming DctA/DcuS sensor units. Here it is shown by fluorescence microscopy with fusion proteins that DcuS has a dynamic and preferential polar localization, even at very low expression levels. Single assemblies of DcuS had high mobility in fast time lapse acquisitions, and fast recovery in FRAP experiments, excluding polar accumulation due to aggregation. DctA and DcuR fused to derivatives of the YFP protein are dispersed in the membrane or in the cytosol, respectively, when expressed without DcuS, but co-localize with DcuS when co-expressed at appropriate levels. Thus, DcuS is required for location of DctA and DcuR at the poles and formation of tripartite DctA/DcuS/DcuR sensor/regulator complexes. Vice versa, DctA, DcuR and the alternative succinate transporter DauA were not essential for polar localization of DcuS, suggesting that the polar trapping occurs by DcuS. Cardiolipin, the high curvature at the cell poles, and the cytoskeletal protein MreB were not required for polar localization. In contrast, polar localization of DcuS required the presence of the cytoplasmic PAS(C) and the kinase domains of DcuS.

Differentiation of DctA and DcuS function in the DctA/DcuS sensor complex of Escherichia coli: function of DctA as an activity switch and of DcuS as the C4-dicarboxylate sensor.

The C4-dicarboxylate responsiveness of the sensor kinase DcuS is only provided in concert with C4-dicarboxylate transporters DctA or DcuB. The individual roles of DctA and DcuS for the function of the DctA/DcuS sensor complex were analysed. (i) Variant DctA(S380D) in the C4-dicarboxylate site of DctA conferred C4-dicarboxylate sensitivity to DcuS in the DctA/DcuS complex, but was deficient for transport and for growth on C4-dicarboxylates. Consequently transport activity of DctA is not required for its function in the sensor complex. (ii) Effectors like fumarate induced expression of DctA/DcuS-dependent reporter genes (dcuB-lacZ) and served as substrates of DctA, whereas citrate served only as an inducer of dcuB-lacZ without affecting DctA function. (iii) Induction of dcuB-lacZ by fumarate required 33-fold higher concentrations than for transport by DctA (Km = 30 µM), demonstrating the existence of different fumarate sites for both processes. (iv) In titration experiments with increasing dctA expression levels, the effect of DctA on the C4-dicarboxylate sensitivity of DcuS was concentration dependent. The data uniformly show that C4-dicarboxylate sensing by DctA/DcuS resides in DcuS, and that DctA serves as an activity switch. Shifting of DcuS from the constitutive ON to the C4-dicarboxylate responsive state, required presence of DctA but not transport by DctA.

The Aerobic and Anaerobic Respiratory Chain of Escherichia coli and Salmonella enterica: Enzymes and Energetics.

Escherichia coli contains a versatile respiratory chain that oxidizes 10 different electron donor substrates and transfers the electrons to terminal reductases or oxidases for the reduction of six different electron acceptors. Salmonella is able to use two more electron acceptors. The variation is further increased by the presence of isoenzymes for some substrates. A large number of respiratory pathways can be established by combining different electron donors and acceptors. The respiratory dehydrogenases use quinones as the electron acceptors that are oxidized by the terminal reductase and oxidases. The enzymes vary largely with respect to their composition, architecture, membrane topology, and the mode of energy conservation. Most of the energy-conserving dehydrogenases (FdnGHI, HyaABC, HybCOAB, and others) and the terminal reductases (CydAB, NarGHI, and others) form a proton potential (Δp) by a redox-loop mechanism. Two enzymes (NuoA-N and CyoABCD) couple the redox energy to proton translocation by proton pumping. A large number of dehydrogenases and terminal reductases do not conserve the redox energy in a proton potential. For most of the respiratory enzymes, the mechanism of proton potential generation is known or can be predicted. The H+/2e- ratios for most respiratory chains are in the range from 2 to 6 H+/2e-. The energetics of the individual redox reactions and the respiratory chains is described and related to the H+/2e- ratios.

Interaction of the Escherichia coli transporter DctA with the sensor kinase DcuS: presence of functional DctA/DcuS sensor units.

The aerobic Escherichia coli C(4) -dicarboxylate transporter DctA and the anaerobic fumarate/succinate antiporter DcuB function as obligate co-sensors of the fumarate responsive sensor kinase DcuS under aerobic or anaerobic conditions respectively. Overproduction under anaerobic conditions allowed DctA to replace DcuB in co-sensing, indicating their functional equivalence in this capacity. In vivo interaction studies between DctA and DcuS using FRET or a bacterial two-hybrid system (BACTH) demonstrated their interaction. DctA-YFP bound to an affinity column and was able to retain DcuS. DctA shows substantial sequence and secondary structure conservation to Glt(Ph), the Na(+)/glutamate symporter of Pyrococcus horikoshii with known 3D structure. Topology studies of DctA demonstrated the presence of eight transmembrane helices in an arrangement similar to that of Glt(Ph) . DctA contains an additional predicted amphipathic helix 8b on the cytoplasmic side of the membrane that is specific for DctA and not present in Glt(Ph). Mutational analysis demonstrated the importance of helix 8b in co-sensing and interaction with DcuS, and the isolated helix 8b showed strong interaction with DcuS. In DcuS, deletion and mutation of the cytoplasmic PAS(C) domain affected the interaction between DctA and DcuS. It is concluded that DctA forms a functional unit or sensor complex with DcuS through specific interaction sites.