Regulation of Aerobic Succinate Transporter dctA of E. coli by cAMP-CRP, DcuS-DcuR, and EIIAGlc: Succinate as a Carbon Substrate and Signaling Molecule.

Introduction: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions.

Asuc_0142 of 130Z is the l-aspartate/C4-dicarboxylate exchanger DcuA.

Anaerobic bacteria often use antiporters DcuB (malate/succinate antiport) or DcuA (l-aspartate/succinate antiport) for the excretion of succinate during fumarate respiration. The rumen bacterium is able to produce large amounts of succinate by fumarate respiration, using the DcuB-type transporter DcuE for l-malate/succinate antiport. Asuc_0142 was annotated as a second DcuB-type transporter.

A signaling complex of adenylate cyclase CyaC of Sinorhizobium meliloti with cAMP and the transcriptional regulators Clr and CycR.

Background: Adenylate cyclases (ACs) generate the second messenger cyclic AMP (cAMP), which is found in all domains of life and is involved in the regulation of various cell physiological and metabolic processes. In the plant symbiotic bacterium Sinorhizobium meliloti, synthesis of cAMP by the membrane-bound AC CyaC responds to the redox state of the respiratory chain and the respiratory quinones. However, nothing is known about the signaling cascade that is initiated by cAMP produced by CyaC.

Fumarate, a central electron acceptor for Enterobacteriaceae beyond fumarate respiration and energy conservation.

C4-dicarboxylates (C4-DCs) such as fumarate, l-malate and l-aspartate are key substrates for Enterobacteria such as Escherichia coli or Salmonella typhimurium during anaerobic growth. In general, C4-DCs are oxidants during biosynthesis, e.g.

C4-dicarboxylate metabolons: interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes.

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA.

Tight Complex Formation of the Fumarate Sensing DcuS-DcuR Two-Component System at the Membrane and Target Promoter Search by Free DcuR Diffusion.

Signaling of two-component systems by phosphoryl transfer requires interaction of the sensor kinase with the response regulator. Interaction of the C4-dicarboxylate-responsive and membrane-integral sensor kinase DcuS with the response regulator DcuR was studied. , the cytoplasmic part of DcuS (PAS-Kin) was employed.

C-Dicarboxylates as Growth Substrates and Signaling Molecules for Commensal and Pathogenic Enteric Bacteria in Mammalian Intestine.

The C-dicarboxylates (C4-DC) l-aspartate and l-malate have been identified as playing an important role in the colonization of mammalian intestine by enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, and succinate as a signaling molecule for host-enteric bacterium interaction. Thus, endogenous and exogenous fumarate respiration and related functions are required for efficient initial growth of the bacteria. l-Aspartate represents a major substrate for fumarate respiration in the intestine and a high-quality substrate for nitrogen assimilation.

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

The sensor kinase DcuS of perceives extracellular fumarate by a periplasmic PAS 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.

Conversion of the Sensor Kinase DcuS to the Fumarate Sensitive State by Interaction of the Bifunctional Transporter DctA at the TM2/PAS-Linker Region.

The membrane-bound C-dicarboxylate (C4DC) sensor kinase DcuS of typically forms a protein complex with the C4DC transporter DctA. The DctA × DcuS complex is able to respond to C4DCs, whereas DcuS without DctA is in the permanent ON state. In DctA, the C-terminal helix 8b (H8b) serves as the site for interaction with DcuS.

C4-dicarboxylates and l-aspartate utilization by Escherichia coli K-12 in the mouse intestine: l-aspartate as a major substrate for fumarate respiration and as a nitrogen source.

C4-dicarboxylates, such as fumarate, l-malate and l-aspartate represent substrates for anaerobic growth of Escherichia coli by fumarate respiration. Here, we determined whether C4-dicarboxylate metabolism, as well as fumarate respiration, contribute to colonization of the mammalian intestinal tract. Metabolite profiling revealed that the murine small intestine contained high and low levels of l-aspartate and l-malate respectively, whereas fumarate was nearly absent.

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 C-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.

Sensing of O and nitrate by bacteria: alternative strategies for transcriptional regulation of nitrate respiration by O and nitrate.

Many bacteria are able to use O and nitrate as alternative electron acceptors for respiration. Strategies for regulation in response to O and nitrate can vary considerably. In the paradigmatic system of E.

L-Aspartate as a high-quality nitrogen source in Escherichia coli: Regulation of L-aspartase by the nitrogen regulatory system and interaction of L-aspartase with GlnB.

Escherichia coli uses the C4-dicarboxylate transporter DcuA for L-aspartate/fumarate antiport, which results in the exploitation of L-aspartate for fumarate respiration under anaerobic conditions and for nitrogen assimilation under aerobic and anaerobic conditions. L-Aspartate represents a high-quality nitrogen source for assimilation. Nitrogen assimilation from L-aspartate required DcuA, and aspartase AspA to release ammonia.

Fumarate dependent protein composition under aerobic and anaerobic growth conditions in Escherichia coli.

In the absence of sugars, C4-dicarboxylates (C4DC) like fumarate represent important substrates for growth of Escherichia coli. Aerobically, C4DCs are oxidized to CO whereas anaerobically, C4DCs are used for fumarate respiration. In order to determine the impact of fumarate under aerobic and anaerobic conditions, proteomes of E.

Control of the bifunctional O -sensor kinase NreB of Staphylococcus carnosus by the nitrate sensor NreA: Switching from kinase to phosphatase state.

The NreB-NreC two-component system of Staphylococcus carnosus for O sensing cooperates with the accessory nitrate sensor NreA in the NreA-NreB-NreC system for coordinated sensing and regulation of nitrate respiration by O and nitrate. ApoNreA (NreA in the absence of nitrate) interacts with NreB and inhibits NreB autophosphorylation (and activation). NreB contains the phosphatase motif DxxxQ.

CyaC, a redox-regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme-B membrane anchor domain.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases.

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 C -dicarboxylate transporter for the main anaerobic C -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 C -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.

Cellular Concentrations of the Transporters DctA and DcuB and the Sensor DcuS of Escherichia coli and the Contributions of Free and Complexed DcuS to Transcriptional Regulation by DcuR.

In , the catabolism of C-dicarboxylates is regulated by the DcuS-DcuR two-component system. The functional state of the sensor kinase DcuS is controlled by C-dicarboxylates (like fumarate) and complexation with the C-dicarboxylate transporters DctA and DcuB, respectively. Free DcuS (DcuS) is known to be constantly active even in the absence of fumarate, whereas the DcuB-DcuS and DctA-DcuS complexes require fumarate for activation.

The E. coli dicarboxylic acid transporters DauA act as a signal transducer by interacting with the DctA uptake system.

The Slc26A/SulP family of ions transporter is ubiquitous and widpsread in all kingdon of life. In E. coli, we have demonstrated that the Slc26 protein DauA is a C-dicarboxilic acids (C-diC) transporter active at acidic pH.

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.

C4-Dicarboxylate Utilization in Aerobic and Anaerobic Growth.

C4-dicarboxylates and the C4-dicarboxylic amino acid l-aspartate support aerobic and anaerobic growth of Escherichia coli and related bacteria. In aerobic growth, succinate, fumarate, D- and L-malate, L-aspartate, and L-tartrate are metabolized by the citric acid cycle and associated reactions. Because of the interruption of the citric acid cycle under anaerobic conditions, anaerobic metabolism of C4-dicarboxylates depends on fumarate reduction to succinate (fumarate respiration).

Conversion of the sensor kinase DcuS of Escherichia coli of the DcuB/DcuS sensor complex to the C -dicarboxylate responsive form by the transporter DcuB.

The sensor kinase DcuS of Escherichia coli co-operates under aerobic conditions with the C -dicarboxylate transporter DctA to form the DctA/DcuS sensor complex. Under anaerobic conditions C -dicarboxylate transport in fumarate respiration is catalyzed by C -dicarboxylate/fumarate antiporter DcuB. (i) DcuB interacted with DcuS as demonstrated by a bacterial two-hybrid system (BACTH) and by co-chromatography of the solubilized membrane-proteins (mHPINE assay).

Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles.

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.

Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: A long-range piston-type displacement of transmembrane helix 2.

The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane-water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21-41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states.