PT  - JOURNAL ARTICLE
AU  - Pengchao Wang
AU  - Guangming Zhang
AU  - Zeling Xu
AU  - Zhe Chen
AU  - Xiaohong Liu
AU  - Chenyin Wang
AU  - Chaogu Zheng
AU  - Jiangyun Wang
AU  - Hongmin Zhang
AU  - Aixin Yan
TI  - FRET monitoring of transcription factor activities in living bacteria
AID  - 10.1101/2022.01.14.476424
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.01.14.476424
4099  - http://biorxiv.org/content/early/2022/01/15/2022.01.14.476424.short
4100  - http://biorxiv.org/content/early/2022/01/15/2022.01.14.476424.full
AB  - Bacteria adapt to the constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor the in situ TF-promoter interactions in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Förster resonance energy transfer (FRET) between a fluorescent unnatural amino acid CouA which is genetically encoded into defined sites in TFs and the live cell fluorescent nucleic acid stain SYTO 9. We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems with specificity and sensitivity. Furthermore, the assay is applicable to identify novel modulators of the regulatory systems of interest and monitor TF activities in bacteria colonized in C. elegans. In conclusion, we established a tractable and sensitive TF-promoter binding assay in living bacteria 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.Competing Interest StatementThe authors have declared no competing interest.