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Resources » Paper

Wang P et al. (2022) J Biol Chem "Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria."

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  • Comments on Wang P et al. (2022) J Biol Chem "Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria." (0)

  • Overview

    PMID:
    35839853
    DOI:
    10.1016/j.jbc.2022.102258
    Status:
    Publication type:
    Journal_article
    WormBase ID:
    WBPaper00064476

    Wang P, Zhang G, Xu Z, Chen Z, Liu X, Wang C, Zheng C, Wang J, Zhang H, & Yan A (2022). Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria. J Biol Chem, 102258. doi:10.1016/j.jbc.2022.102258

    Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF-promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Forster resonance energy transfer (FRET) between an unnatural amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (CouA), which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all regulatory systems tested based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Lastly, we show that the FRET system is applicable for single-cell analysis and for monitoring TF activities in E. coli colonizing a C. elegans host. In conclusion, we established a tractable and sensitive TF-promoter binding assay which not only complements currently available approaches for DNA-protein interactions, but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

    Authors: Wang P, Zhang G, Xu Z, Chen Z, Liu X, Wang C, Zheng C, Wang J, Zhang H, Yan A


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