Journal of Biological Chemistry
Volume 289, Issue 40, 3 October 2014, Pages 27899-27910
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Microbiology
A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis*

https://doi.org/10.1074/jbc.M114.596221Get rights and content
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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.

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*

This work was supported by Deutsche Forschungsgemeinschaft Grant GE2164/3-1 and the Fonds der Chemischen Industrie (to the S. G. laboratory) and Deutsche Forschungsgemeinschaft Grant Exc114-2 (to the K. J. laboratory).

3

T. Mascher and C. Fang, personal communication.

4

S. Dintner, T. Mascher, and S. Gebhard, unpublished observations.

2

The abbreviations used are:

    TCS

    two-component system

    ABC

    ATP-binding cassette

    HK

    histidine kinase

    UPP

    undecaprenyl-pyrophosphate

    MIC

    minimal inhibitory concentration

    DDM

    n-dodecyl β-d-maltoside

    SPR

    surface plasmon resonance

    NHS

    N-hydroxysuccinimide

    BACTH

    bacterial two-hybrid

    β-ME

    β-mercaptoethanol

    Ni2+-NTA

    Ni2+-nitrilotriacetic acid

    TM

    transmembrane.