Real-time detection of response regulator phosphorylation dynamics in live bacteria

Abstract

Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct in vivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.

Signal-independent activation reveals two-component regulatory networks

“…Second, intermediary activation of the RelRS and CiaRH pathways suggests buffering mechanisms for TCSs regulating multiple independent loci and integrated cellular response. However, detailed analysis is required to decipher phosphorylation dynamics in each phosphatase-deficient HK + and correlate in vivo RR phosphorylation with regulatory network activation, considering variable factors like the source of RR phosphorylation (kinase activity of the HK + variant, cross-talk by other HK, small metabolites) and specific spontaneous dephosphorylation rates 27,66,67 .…”

Interplay between Two-Component Regulatory Systems Is Involved in Control of Cupriavidus metallidurans Metal Resistance Genes

“…Signaling by two-component regulatory systems accelerates with HK expression but decelerates with RR expression ( 25 ). Expression of czcS and of czcS 2 is upregulated when metal availability changes but not that of czcR (Table S1).…”

“…Two-component regulatory systems are usually composed of a membrane-bound sensor(y) histidine kinase (HK) and cytoplasmic DNA-binding response regulator (RR), which is phosphorylated upon a signal being sensed by the HK ( 24 ). The RR dimerizes within seconds after the HK has received the stimulus and activates transcription ( 25 ). In C. metallidurans , the dominant czc resistance determinant ( Fig.…”

Intermolecular Energy Migration via HomoFRET Captures the Modulation in the Material Property of Phase-Separated Biomolecular Condensates

“…On the contrary, tagging the proteins of interest with fluorescent proteins is an extensively used methodology for studies investigating the expression, localization, functioning, and protein-protein interactions within cells. Along these lines, homoFRET, a special case of FRET, utilizes a single fluorophore with a small Stokes' shift and a significant overlap between its fluorescence excitation and emission spectrum and has been previously captured in the form of fluorescence depolarization or loss in anisotropy as a function of local fluorophore densities [26][27][28][29][30] . Thus, homoFRET can be employed to investigate the proximal and distal molecular packaging within mesoscopic supramolecular assemblies such as phase-separated biomolecular condensates.…”