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.

The cytoplasmic PASC domain of the sensor kinase DcuS of Escherichia coli: role in signal transduction, dimer formation, and DctA interaction.

The cytoplasmic PASC domain of the fumarate responsive sensor kinase DcuS of Escherichia coli links the transmembrane to the kinase domain. PASC is also required for interaction with the transporter DctA serving as a cosensor of DcuS. Earlier studies suggested that PASC functions as a hinge and transmits the signal to the kinase. Reorganizing the PASC dimer interaction and, independently, removal of DctA, converts DcuS to the constitutive ON state (active without fumarate stimulation). ON mutants were categorized with respect to these two biophysical interactions and the functional state of DcuS: type I-ON mutations grossly reorganize the homodimer, and decrease interaction with DctA. Type IIA-ON mutations create the ON state without grossly reorganizing the homodimer, whereas interaction with DctA is decreased. The type IIB-ON mutations were neither in PASC /PASC , nor in DctA/DcuS interaction affected, similar to fumarate activated wild-typic DcuS. OFF mutations never affected dimer stability. The ON mutations provide novel mechanistic insight: PASC dimerization is essential to silence the kinase. Reorganizing the homodimer and its interaction with DctA activate the kinase. The study suggests a novel ON homo-dimer conformation (type IIB) and an OFF conformation for PASC . Type IIB-ON corresponds to the fumarate induced wild-type conformation, representing an interesting target for structural biology.

Regulation of tartrate metabolism by TtdR and relation to the DcuS-DcuR-regulated C4-dicarboxylate metabolism of Escherichia coli.

Escherichia coli catabolizes L-tartrate under anaerobic conditions to oxaloacetate by the use of L-tartrate/succinate antiporter TtdT and L-tartrate dehydratase TtdAB. Subsequently, L-malate is channelled into fumarate respiration and degraded to succinate by the use of fumarase FumB and fumarate reductase FrdABCD. The genes encoding the latter pathway (dcuB, fumB and frdABCD) are transcriptionally activated by the DcuS-DcuR two-component system. Expression of the L-tartrate-specific ttdABT operon encoding TtdAB and TtdT was stimulated by the LysR-type gene regulator TtdR in the presence of L- and meso-tartrate, and repressed by O(2) and nitrate. Anaerobic expression required a functional fnr gene, and nitrate repression depended on NarL and NarP. Expression of ttdR, encoding TtdR, was repressed by O(2), nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR specifically bound to the ttdR-ttdA promoter region. TtdR was also required for full expression of the DcuS-DcuR-dependent dcuB gene in the presence of tartrate. Overall, expression of the ttdABT genes is subject to L-/meso-tartrate-dependent induction, and to aerobic and nitrate repression. The control is exerted directly at ttdA and in addition indirectly by regulating TtdR levels. TtdR recognizes a subgroup (L- and meso-tartrate) of the stimuli perceived by the sensor DcuS, which responds to all C(4)-dicarboxylates; both systems apparently communicate by mutual regulation of the regulatory genes.

Plasticity of the PAS domain and a potential role for signal transduction in the histidine kinase DcuS.

The mechanistic understanding of how membrane-embedded sensor kinases recognize signals and regulate kinase activity is currently limited. Here we report structure-function relationships of the multidomain membrane sensor kinase DcuS using solid-state NMR, structural modeling and mutagenesis. Experimental data of an individual cytoplasmic Per-Arnt-Sim (PAS) domain were compared to structural models generated in silico. These studies, together with previous NMR work on the periplasmic PAS domain, enabled structural investigations of a membrane-embedded 40-kDa construct by solid-state NMR, comprising both PAS segments and the membrane domain. Structural alterations are largely limited to protein regions close to the transmembrane segment. Data from isolated and multidomain constructs favor a disordered N-terminal helix in the cytoplasmic domain. Mutations of residues in this region strongly influence function, suggesting that protein flexibility is related to signal transduction toward the kinase domain and regulation of kinase activity.

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

Escherichia coli contains a versatile respiratory chain which oxidizes ten 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 even two more electron acceptors. The variation is further increased by the presence of isoenzymes for some substrates. Various respiratory pathways can be established by combining the oxidation of different electron donors and acceptors which are linked by respiratory quinones. The enzymes vary largely with respect to architecture, membrane topology, and mode of energy conservation. Most of the energy-conserving dehydrogenases (e.g., FdnGHI, HyaABC, and HybCOAB) and of the terminal reductases (CydAB, NarGHI, and others) form a proton potential (Δp) by a redox loop mechanism. Only two enzymes (NuoA-N and CyoABCD) couple the redox energy to proton translocation by proton pumping. A large number of dehydrogenases (e.g., Ndh, SdhABCD, and GlpD) and of terminal reductases (e.g., FrdABCD and DmsABC) do not conserve the redox energy in a proton potential. For most of the respiratory enzymes, the mechanism of proton potential generation is known from structural and biochemical studies or can be predicted from sequence information. The H+/2e- ratios of proton translocation for most respiratory chains are in the range from 2 to 6 H+/2e-. The energetics of the individual redox reactions and of the respiratory chains is described. In contrast to the knowledge on enzyme function are physiological aspects of respiration such as organization and coordination of the electron transport and the use of alternative respiratory enzymes, not well characterized.