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

Abstract

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 (DcuS_F) 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 DcuS_F 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 (DcuS_F) and the complexed (DcuB-DcuS) states. Unexpectedly, DcuS_F levels were always low (<2% of total DcuS), ruling out earlier models that show DcuS_F as the major species under noninducing conditions. In the absence of fumarate, when DcuS_F 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 (DcuS_F) 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.

Keywords: C4-dicarboxylate sensing; DctA; DcuB; DcuS; Escherichia coli; SRM; absolute quantification; sensor complex.

FIG 1

Absolute quantification of DctA, DcuB, and DcuS in the membrane fraction of E. coli W3110 wild type. The amounts of DctA, DcuB, and DcuS were determined after growth under control conditions (0 Fum) and after the addition of fumarate (20 Fum or 50 Fum). In all cases, average values and standard deviations from three biological replicates are presented, and the ratios of the protein amounts depending on fumarate addition were calculated.

FIG 2

Direct comparison of changes of DcuB in contents and activity upon fumarate induction. (A) Absolute contents of DcuB in E. coli wild type grown without and with fumarate induction as given in Fig. 1. (B) Uptake of [¹⁴C]fumarate in cells of strain IMW658 of E. coli containing only DcuB (but not DcuA and DcuC) was determined by a filtration assay (37).

FIG 3

Absolute quantification of DctA, DcuB, and DcuS in E. coli IMW503/pMW1194 or E. coli IMW503/pMW2334 grown anaerobically. The amounts were determined under four different conditions by cultivation in the additional presence of 100 μM glucose (100Glc), 0 μM arabinose (0Ara), 1 μM arabinose (1Ara), and 10 μM arabinose (10Ara). Absolute amounts of DctA and DcuS (A and B) or DcuB and DcuS (C and D) were measured in the membrane fraction of E. coli IMW503/pMW1194 or E. coli IMW503/pMW2334, respectively. Average values and standard deviations from five (A and B) or from three or four (C and D) biological replicates are presented.

FIG 4

Correlation of dcuB-lacZ or dcuS expression (black) and control factor (red, diamonds) with total contents of DcuB. The normalized DcuS (MU/DcuS per cell) activity in the presence (○, dashed line) or absence (□, dotted line) of fumarate (10 mM) is plotted against the copy number of DcuB. Strain IMW503/pMW2334, carrying dcuB-phoA under the control of the arabinose-inducible pBAD promoter, was grown with 50 mM glycerol and 20 mM DMSO. Levels of DcuB were varied by addition of glucose (100 μM, ④) or l-arabinose (0 μM, ②; 1 μM, ③; or 10 μM ⑤) during cultivation. ① corresponds to the ΔdcuB mutant strain. The β-galactosidase activity was taken from Fig. 2B of reference but normalized for the cellular copy number of DcuS. The ratio between DcuS activity with and without fumarate or the control factor (red line) is given on the red axis. The expression of dcuS-lacZ of E. coli IMW237 (wild-type dcuB, chromosomal) showed normalized DcuS activities of 2.0 and 28.1 MU/DcuS per cell without and with fumarate (expression activity from reference , normalized for DcuS contents of E. coli W3110 [Fig. 1]).

FIG 5

Expression of dcuB-lacZ by DcuS under standby (left) and induced (right) conditions. (A) Modeled expression profile of dcuB-lacZ. (B) Underlying regulation and prevalence of DcuS and DcuB. The total levels of DcuS and DcuB (presented as number molecules per cell) correspond to the levels in wild-type E. coli measured in the absence or the presence of fumarate (20 mM; circles) in this work. The portions of the free (DcuS_F and DcuB_F) and the complexed (DcuS₂/DcuB₄) forms are given, and the supporting calculation and a detailed description of the scheme are presented in the text. + and ++ indicate weak and strong expression, respectively.

FIG 6

Scheme showing the control of DcuS function by the DcuB-DcuS complex formation (functional switch) and the activity regulation of DcuB-DcuS by C₄-dicarboxylates. Formation of the DcuB-DcuS complex converts DcuS from the permanent active (on) to a state where C₄-dicarboxylates control activity (activity regulation of DcuS function). The activity of DcuS consists in autophosphorylation; the phosphoryl group is then transferred to the response regulator DcuR. DcuS_F (free or noncomplexed DcuS) is present in low concentrations and autophosphorylates in a C₄-dicarboxylate-independent manner. The DcuB (light orange) levels exceed those of DcuS_F (light orange), and DcuB is directly used for DcuB-DcuS (blue) formation, while the other (larger) parts serve as C₄-dicarboxylate transporters (DcuB_T; dark orange). Transcriptional activation of dcuB by DcuS_F and DcuB-DcuS at low or lacking C₄-dicarboxylate concentrations results in standby expression of dcuB, activation by DcuB-DcuS in the presence of C₄-dicarboxylate in the induced state (see Fig. 5).