A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis - PubMed (original) (raw)

A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis

Sebastian Dintner et al. J Biol Chem. 2014.

Abstract

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.

Keywords: ABC Transporter; Antimicrobial Peptide (AMP); Bacitracin; Histidine Kinase; Membrane Protein; Protein-Protein Interaction.

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Figures

FIGURE 1.

Working model for the BceRS-BceAB bacitracin resistance system of B. subtilis. Bacitracin is bound directly by the transporter BceAB. BceAB and BceS interact to form a sensory complex in the membrane. ATP hydrolysis by the transporter triggers the activation of BceS, which in turn leads to phosphorylation of BceR. Activation of the target promoter (P_bceA_) by BceR then induces increased production of BceAB to ensure resistance. Interactions between proteins are marked by double-headed arrows; events relating to transcription are labeled with dotted arrows; the potential interaction of BceR with the sensory complex of BceS and BceAB is indicated with a question mark.

FIGURE 2.

Bacterial two-hybrid analysis of the BceRS-BceAB module. Hybrids consisting of B. pertussis CyaA T18 or T25 domains and each individual Bce protein or the complete transporter (BceAB) or TCS (BceRS) were introduced into E. coli BTH101. Cells were spotted onto LB plates containing X-Gal (40 μg/ml), isopropyl 1-thio-β-

-galactopyranoside (0.5 m

), and antibiotics for selection. Pictures were taken after 48 h of incubation at 30 °C. The blue colonies indicating positive results for interaction are depicted as dark gray in the grayscale image.

FIGURE 3.

A, signal transduction activities of affinity-tagged BceAB and BceS. Exponentially growing cells of strains carrying the P_bceA-luxABCDE_ reporter were challenged with 30 μg/ml Zn2+-bacitracin (+) or left untreated (−). Luminescence (relative luminescence units, RLU) was measured at 48 min post-induction. Luminescence was normalized to cell density and is expressed as relative luminescence units/_A_600. The left graph shows complementation of bceS deletion by untagged (left) and His-tagged (right) BceS. The right graph shows complementation of bceAB deletion by untagged (left) and Strep-tagged (right) BceAB. Data are shown as the mean ± S.D. of three to four independently performed experiments. B, affinity purification of BceAB, BceS, and BceR from E. coli C43(DE3) cells. Left panel, BceS with a C-terminal His8 tag was purified with a Ni2+-NTA column. Center panel, BceAB carrying an N-terminal Strep II®-tag on the ATPase domain was purified via a Strep-Tactin® column. Right panel, BceR carrying a C-terminal His10 tag was purified with a Ni2+-NTA column. Proteins were analyzed using SDS-PAGE, and gels were stained with Coomassie Brilliant Blue. Purified proteins are indicated on the right by the last letter of their name. A molecular size marker is indicated on the left in kDa. MF, membrane fraction; S, solubilized fraction; E, elution; CF, cytoplasmic fraction.

FIGURE 4.

Size exclusion analysis of the BceAB complex. A, conserved domain analysis of BceB. TM helices are shown as black rectangles. FtsX domains are indicated by dashed boxes. Predictions were done using the SMART database (41). B and C, size exclusion analysis of BceB (B) and BceAB (C) on a Superdex 200 10/30 column. Left panels show the chromatograms. The vertical dashed line indicates the void volume (_V_0) of the column. The calculated molecular mass corresponding to the major eluted peaks is shown in parentheses, and eluted proteins are given by the last letter of their name. The right panels show the corresponding SDS-PAGE analysis of the fractions indicated by numbers below the chromatograms. A molecular size marker is indicated on the left in kDa; proteins are indicated on the right by last letter of their name. mAU, milli absorbance units.

FIGURE 5.

Binding of bacitracin to BceB. SPR spectroscopy was used to quantify interactions between BceB and Zn2+-bacitracin or nisin. BceB-Strep was captured onto a Strep-Tactin® XT-coated chip before increasing concentrations of Zn2+-bacitracin or nisin (control) were injected. A, single cycle binding kinetic of bacitracin binding to BceB. The gray line shows the recorded sensorgram, and the black line shows the fitted sensorgram. B, steady-state affinity of bacitracin (black) and nisin (gray) to BceB. RU, response units.

FIGURE 6.

_In vitr_o pull-down assay using NHS-activated Sepharose. Each panel shows the fraction containing all proteins eluted from the beads (Pull-down), as well as the purified protein tested for interaction with the beads (Prey). A, NHS-conjugated BceS (NHS-BceS) and the last washing step before elution (Wash) are also shown. The protein conjugated to the beads (Bait) is given below each panel. A and B, NHS-conjugated BceS incubated with purified BceAB (A) or BSA (B). C, NHS-conjugated BSA incubated with purified BceAB. D, NHS-Sepharose blocked with Tris and incubated with purified BceAB. BceAB eluting specifically from NHS-BceS beads is indicated by asterisk. Proteins were analyzed using SDS-PAGE, and gels were stained with Coomassie Brilliant Blue. A molecular size marker is indicated on the left in kDa, and the protein bands are labeled on the right.

FIGURE 7.

In vitro phosphorylation of Bce module proteins. Purified, detergent-solubilized BceS-His8 was mixed in equimolar ratios with BceAB, BceR, or both as indicated on the left. A, phosphorylation was started at t = 0 min by adding [γ-32P]ATP. At the indicated times, reactions were stopped; the samples were subjected to SDS-PAGE, and phosphorylated proteins were detected by phosphorimaging. Representative autoradiographs of three to four independently performed experiments are shown. B and C, band intensities of BceS-32P (B) and BceR-32P (C) were quantified and plotted over time. The protein combinations in each assay are given on the right by the last letter of their names. Data are shown as the mean ± S.D. of three to four independently performed experiments.

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A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis - PubMed (original) (raw)