Real-time detection of response regulator phosphorylation dynamics in live bacteria.
Proc Natl Acad Sci U S A
; 119(35): e2201204119, 2022 08 30.
Article
in En
| MEDLINE
| ID: mdl-35994658
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Bacteria
/
Bacterial Proteins
/
Microbial Viability
/
Histidine Kinase
Type of study:
Diagnostic_studies
Language:
En
Journal:
Proc Natl Acad Sci U S A
Year:
2022
Document type:
Article