Oligomeric sensor kinase DcuS in the membrane of Escherichia coli and in proteoliposomes: chemical cross-linking and FRET spectroscopy

Abstract

DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C(4)-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by fluorescence resonance energy transfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {f(D) = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer.

FIG. 1.

Influence of oxidizing conditions on the oligomerization of purified His₆-DcuS. Detergent-solubilized and purified variants of His₆-DcuS (1 μg) were subjected to SDS-PAGE in the presence (+) or absence (−) of DTT. DcuS was detected by Western blotting with antiserum against the periplasmic domain of DcuS. The anti-DcuS-positive bands of 61 and 120 kDa correspond to monomeric and dimeric DcuS. Lanes: 1 and 2, DcuS_WT; 3 and 4, DcuS_Cys−; 5 and 6, DcuS_C471S; and 7 and 8, DcuS_C199S. For calibration, PageRuler Plus prestained protein ladder (Fermentas) was used.

FIG. 2.

Determination of the oligomeric state of DcuS by chemical cross-linking. Detergent-solubilized His₆-DcuS, His₆-DcuS reconstituted in liposomes, or preparations of His₆-DcuS embedded in bacterial membranes of the bacteria was cross-linked with disuccinimidyl suberate (DSS). Samples were subjected to SDS-PAGE in the presence of DTT, blotted to nitrocellulose membranes (Protran), and immunostained with anti-DcuS. The anti-DcuS-positive bands of 61, 120 and 240 kDa correspond to monomeric, dimeric, and tetrameric DcuS, respectively. Lanes: 1 and 2, solubilized DcuS_WT (3 μg); 3 and 4, solubilized DcuS_Cys− (3 μg); 5 and 6, reconstituted DcuS_Cys− (3 μg); 7 and 8, bacterial membranes (20 μg) containing DcuS_Cys−; and 9, solubilized DcuS_Cys− (7 μg). For calibration, PageRuler Plus prestained protein ladder (Fermentas) was used.

FIG. 3.

Effect of DcuS_Cys− concentration on dimer and tetramer formation in proteoliposomes (A) and in bacterial membranes of living cells (B). (A) Purified recombinant DcuS_Cys− (8 μg) was reconstituted in liposomes in the following protein/phospholipid ratios: 1:10 (lanes 1 and 2), 1:20 (lanes 3 and 4), 1:50 (lanes 5 and 6), and 1:100 (lanes 7 and 8). Samples were subjected to SDS-PAGE in the presence of DTT before and after cross-linking with disuccinimidyl suberate (DSS), blotted to nitrocellulose membranes (Protran), and immunostained with anti-DcuS. For calibration, PageRuler Plus prestained protein ladder (Fermentas) was used. (B) E. coli JM109(pMW967) (expressing DcuS_Cys−) was grown aerobically in LB broth in the presence of 0 μM (lane 2), 10 μM (lane 3), 50 μM (lane 4), 90 μM (lane 5), 133 μM (lanes 6 and 8), or 333 μM (lane 7) arabinose and cross-linked with DSS. Sixty micrograms (lanes 4 to 8), 180 μg (lane 3), or 230 μg (lane 2) of cell lysates were subjected to SDS-PAGE. DcuS was detected by Western blotting with antiserum against the periplasmic domain of DcuS. Lane 1 contains 1 μg of purified DcuS_Cys−. The anti-DcuS-positive bands of 61, 120, and 240 kDa correspond to monomeric, dimeric, and tetrameric DcuS, respectively.

FIG. 4.

In vitro FRET measurements: mixture series of Alexa-labeled DcuS subunits (A, B) and FRET of reconstituted DcuS (C). (A, B) Detergent-solubilized His₆-DcuS was labeled with either Alexa 488 (donor) or Alexa 594 (acceptor) and mixed at different ratios with known donor fractions [f_D(known)] of 0 to 1. The final protein concentration was 0.5 μM in DcuS-labeling buffer. The recorded spectra, excited at 480 nm and 580 nm, were analyzed (see the supplemental material) to determine the donor fraction [f_D(analysis)] and the transfer efficiency [E(analysis)] of each sample. The values are plotted against the known donor fractions. (C) FRET of reconstituted Alexa-labeled His₆-DcuS in liposomes. Detergent-solubilized His₆-DcuS was labeled with either Alexa 488 (donor) or Alexa 594 (acceptor) and mixed at different ratios. Subsequently, the mixture of Alexa-labeled His₆-DcuS was reconstituted into liposomes. The recorded spectra, excited at 480 nm and 580 nm, were analyzed. Results of FRET of labeled His₆-DcuS in liposomes with (red triangles) or without (blue triangles) Na₂-fumarate (20 mM) are shown. Spectra were measured in Tris buffer (50 mM, pH 7.7) without fumarate. Results are from five independent test series each (in total, n = 25 data points with or without fumarate).

FIG. 5.

Validation of FRET in living cells: mixture series of CFP-expressing cells and YFP-expressing cells in the presence of a fixed amount of E. coli cells. (A) In the presence of a constant amount of non-FP-expressing E. coli cells (volume of E. coli [V_Ecoli], 0.5 ml), CFP-expressing cells and YFP-expressing cells were mixed at different ratios to a total volume (V_CFP + V_YFP + V_Ecoli) of 1 ml. Emission spectra were measured in PBS buffer, pH 7.5, with excitation at 433 nm. Measured spectra (red) were analyzed by multiparameter fitting with equation 1 in the supplemental material (black dashed line). The spectra with increasing fluorescence correspond to mixtures with increasing contents of CFP-expressing cells. (B) The fitting results for parameter e (contribution of CFP) and parameter f (contribution of YFP) are plotted versus the mixing ratio [V_CFP/(V_CFP + V_YFP)].

FIG. 6.

FRET of CFP- and YFP-labeled DcuS in vivo. (A) Coexpression of DcuS-CFP and DcuS-YFP in E. coli cells was induced with arabinose (133 μM) for 1.5 h to 6 h in the absence (blue) or presence (red) of the effector fumarate (fum; 20 mM). Results are from 7 independent test series (in total, n = 52 data points). All samples were measured in PBS buffer, pH 7.5. (B) DcuS-CFP and Tar^1-331-YFP (Tar*-YFP) (17) were coexpressed in E. coli cells for 3 to 6 h. Results are from 6 independent test series (in total, n = 39 data points). The emission spectra of all samples were recorded by excitation at 433 nm and 488 nm, respectively, and subsequently analyzed with equations 1 to 7 in the supplemental material. The average of the DcuS/Tar transfer efficiencies was marked as a background for false-positive results due to CFP/YFP interaction. (C) FRET efficiencies (mean ± standard deviation of the mean) of the FRET pairs CFP and YFP directly coupled in the fusion protein encoded by pMW766, the separate CFP and YFP proteins encoded by pMW762 and pMW765, DcuS-CFP and Tar^1-331-YFP (Tar*-YFP) (17) encoded by pMW408 and pDK108, and DcuS-CFP and DcuS-YFP encoded by pMW408 and pMW407. The proteins were expressed or coexpressed in E. coli JM109. The average of the DcuS/Tar and CFP/YFP transfer efficiencies was marked as a background (dashed horizontal line) for false-positive results due to CFP/YFP interaction.