Employing genetically encoded fluorescent amino acid to probe the transcriptional regulation and signal transduction in bacteria

Abstract

The genetic code expansion technique has been widely used in biological sciences studies. In my Ph. D. thesis, I aimed to use genetically encoded unnatural amino acids as probes to study the transcriptional regulation and signal transduction in bacteria. 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, I 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. I 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, I 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. Sensing and responding to environmental stresses efficiently and precisely are important for bacterial adaptation and survival. CusSR is a prototype two-component system (TCS) that senses the heavy metals Cu(I) and Ag(I) and responds by activating the Cu(I)/Ag(I) efflux pump CusCFBA. A complete signal transduction pathway within the membrane sensor kinase CusS has not yet been revealed. In the second part of my thesis, I characterized the structural dynamics of CusS using the FRET. To achieve this, I designed and constructed seven constructs expressing CusS variants with fluorescent UAA p-cyanophenylalanine (pCNF) introduced in the positions that near the Förster radius (~17 Å) with an endogenous tryptophan (Trp) residue, i.e., F43, F175, S195. The conformational changes can be examined by pCNF-Trp located across the transmembrane (TM) helices and cytosolic signal transduction domain. The FRET efficiency is increased upon supplementation with the Ag in the CusS variants containing the FRET pairs of W148-pCNF43, W148-pCNF175, W33-pCNF195, W18-pCNF195, W18-pCNF200, and W18-pCNF207, whereas that of W33-pCNF175 is reduced upon supplementation with Ag, indicating the conformational changes in the transmembrane domain upon signal sensing. These studies demonstrated new techniques to study signal transduction in vivo and advanced our understanding of bacterial stress adaptation.