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. Under laboratory conditions, addition of C4-dicarboxylate at concentrations corresponding to the levels of the C4-dicarboxylates in the small intestine (2.6 mmol kg-1 dry weight) induced the dcuBp-lacZ reporter gene (67% of maximal) in a DcuS-DcuR-dependent manner. In addition to its role as a precursor for fumarate respiration, l-aspartate was able to supply all the nitrogen required for anaerobically growing E. coli. DcuS-DcuR-dependent genes were transcribed in the murine intestine, and mutants with defective anaerobic C4-dicarboxylate metabolism (dcuSR, frdA, dcuB, dcuA and aspA genes) were impaired for colonizing the murine gut. We conclude that l-aspartate plays an important role in providing fumarate for fumarate respiration and supplying nitrogen for E. coli in the mouse intestine.

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 Escherichia coli, the catabolism of C4-dicarboxylates is regulated by the DcuS-DcuR two-component system. The functional state of the sensor kinase DcuS is controlled by C4-dicarboxylates (like fumarate) and complexation with the C4-dicarboxylate transporters DctA and DcuB, respectively. Free DcuS (DcuSF) is known to be constantly active even in the absence of fumarate, whereas the DcuB-DcuS and DctA-DcuS complexes require fumarate for activation. To elucidate the impact of the transporters on the functional state of DcuS and the concentrations of DcuSF and DcuB-DcuS (or DctA-DcuS), the absolute levels of DcuS, DcuB, and DctA were determined in aerobically or anaerobically grown cells by mass spectrometry. DcuS was present at a constant very low level (10 to 20 molecules DcuS/cell), whereas the levels of DcuB and DctA were higher (minimum, 200 molecules/cell) and further increased with fumarate (12.7- and 2.7-fold, respectively). Relating DcuS and DcuB contents with the functional state of DcuS allowed an estimation of the proportions of DcuS in the free (DcuSF) and the complexed (DcuB-DcuS) states. Unexpectedly, DcuSF levels were always low (<2% of total DcuS), ruling out earlier models that show DcuSF as the major species under noninducing conditions. In the absence of fumarate, when DcuSF is responsible for basal dcuB expression, up to 8% of the maximal DcuB levels are formed. These suffice for DcuB-DcuS complex formation and basal transport activity. In the presence of fumarate (>100 µM), the DcuB-DcuS complex drives the majority of dcuB expression and is thus responsible for induction.IMPORTANCE Two-component systems (TCS) are major devices for sensing by bacteria and adaptation to environmental cues. Membrane-bound sensor kinases of TCS often use accessory proteins of unknown function. The DcuS-DcuR TCS responds to C4-dicarboxylates and requires formation of the complex of DcuS with C4-dicarboxylate transporters DctA or DcuB. Free DcuS (DcuSF) is constitutively active in autophosphorylation and was supposed to have a major role under specific conditions. Here, absolute concentrations of DcuS, DcuB, and DctA were determined under activating and nonactivating conditions by mass spectrometry. The relationship of their absolute contents to the functional state of DcuS revealed their contribution to the control of DcuS-DcuR in vivo, which was not accessible by other approaches, leading to a revision of previous models.

Conversion of the sensor kinase DcuS of Escherichia coli of the DcuB/DcuS sensor complex to the C4 -dicarboxylate responsive form by the transporter DcuB.

The sensor kinase DcuS of Escherichia coli co-operates under aerobic conditions with the C4 -dicarboxylate transporter DctA to form the DctA/DcuS sensor complex. Under anaerobic conditions C4 -dicarboxylate transport in fumarate respiration is catalyzed by C4 -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). (ii) In the DcuB/DcuS complex only DcuS served as the sensor since mutations in the substrate site of DcuS changed substrate specificity of sensing, and substrates maleate or 3-nitropropionate induced DcuS response without affecting the fumarate site of DcuB. (iii) The half-maximal concentration for induction of DcuS by fumarate (1 to 2 mM) and the corresponding Km for transport (50 µM) differ by a factor of 20 to 40. Therefore, the fumarate sites are different in transport and sensing. (iv) Increasing levels of DcuB converted DcuS from the permanent ON (DcuB deficient) state to the fumarate responsive form. Overall, the data show that DcuS and DcuB form a DcuB/DcuS complex representing the C4 -dicarboxylate responsive form, and that the sensory site of the complex is located in DcuS whereas DcuB is required for converting DcuS to the sensory competent state.