Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria

Wang，Pengchao1,2; Zhang，Guangming1; Xu，Zeling1; Chen，Zhe1; Liu，Xiaohong3; Wang，Chenyin1; Zheng，Chaogu1; Wang，Jiangyun3; Zhang，Hongmin2; Yan，Aixin1

2022-08-01

10.1016/j.jbc.2022.102258

JOURNAL OF BIOLOGICAL CHEMISTRY   Impact Factor & Quartile

0021-9258

1083-351X

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 FRET between an unnatural amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine, 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 these regulatory systems 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. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis 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.

bacteria-C.elegans interface fluorescent unnatural amino acid FRET genetic codon expansion promoter transcription factor transcription regulation two-component system

SCI ; EI

English

NI Journal Papers

Corresponding

Office of Research Infrastructure Programs, National Institutes of Health[P40 OD010440Z];

Biochemistry & Molecular Biology

Biochemistry & Molecular Biology

WOS:000848198300008

ELSEVIER

20223312577954

Amino acids ; Bacteriophages ; Escherichia coli ; Fluorophores ; Nucleic acids ; Signal transduction ; Transcription

Biological Materials and Tissue Engineering:461.2 ; Biology:461.9 ; Light/Optics:741.1 ; Biochemistry:801.2 ; Physical Chemistry:801.4 ; Organic Compounds:804.1

BIOLOGY & BIOCHEMISTRY

2-s2.0-85136322318

Scopus

1.School of Biological Sciences,The University of Hong Kong,Hong Kong,Hong Kong
2.Department of Biology,School of Life Sciences,Southern University of Science and Technology,Shenzhen,China
3.Institute of Biophysics,Chinese Academy of Sciences,Beijing,China

Wang，Pengchao,Zhang，Guangming,Xu，Zeling,et al. Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria[J]. JOURNAL OF BIOLOGICAL CHEMISTRY,2022,298(8).

Wang，Pengchao.,Zhang，Guangming.,Xu，Zeling.,Chen，Zhe.,Liu，Xiaohong.,...&Yan，Aixin.(2022).Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria.JOURNAL OF BIOLOGICAL CHEMISTRY,298(8).

Wang，Pengchao,et al."Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria".JOURNAL OF BIOLOGICAL CHEMISTRY 298.8(2022).