Quantitative Kinetic Analyses of Shutting Off a Two-Component System

Abstract

Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo, which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro. We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression.IMPORTANCE Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.KEYWORDS PhoBR, autoregulation, computer modeling, histidine kinase, phosphatases, systems biology, transcriptional regulation, two-component regulatory systems B acteria often respond to diverse environmental perturbations through twocomponent systems (TCSs) that involve a conserved phosphotransfer between a histidine kinase (HK) sensor and a cognate response regulator (RR) (1, 2). The HK possesses autokinase, phosphotransferase, and, often, phosphatase activities that regulate the phosphorylation level of its cognate RR. Activation of the pathway occurs through stimulus-mediated control of t...

Counterbalancing Regulation in Response Memory of a Positively Autoregulated Two-Component System

“…1B) to the rate of YFP synthesis, is used to indicate the real-time promoter activities. The promoter activity quickly rose upon Pi starvation and reached a steady state thereafter, recapitulating the kinetics of PhoB phosphorylation (17). PhoBR levels correlate well with the steady-state level as well as with the rising rate of promoter activity ( Fig.…”

“…The PhoB level was halved for every doubling of bacterial growth, and it took longer than 3 h for the PhoB concentration to return to approximately the prestimulus level. The levels of PhoA and the phoA-yfp operon reporter have also been discovered to be growth diluted upon stimulus removal (17). An instantaneous dephosphorylation of PhoBϳP has been observed to shut off the pathway, following which there was no new synthesis either of PhoB or of proteins whose expression is regulated by PhoB.…”

“…7A). Kinetics of PhoB phosphorylation were derived from previous Phos-tag analyses (17,28) and used as system inputs (Fig. 7C).…”

“…Its memory-like learning behavior (4) is not necessarily dependent on autoregulation but is best understood as phenotypic memory that results from transmission of stable proteins through a limited number of bacterial generations for an altered response to the recurrence of stimulus. Such short-term memory could be beneficial, as suggested in other systems (6,9), or only incidental, due to the fact that the time scale of protein dilution is much greater than the time scale of the fast kinetics of shutting off the pathway through PhoB dephosphorylation (17).…”

Quantitative Analysis of Intracellular Response Regulator Phosphatase Activity of Histidine Kinases

“…The importance of HK sensing and transmembrane domains in regulating HK activities raises questions about the signaling state(s) represented by isolated domains. Perhaps due to the absence of the regulatory domains or the natural membrane environment that may be required to maintain the phosphatase signaling state, phosphatase activities measured in vitro using cytoplasmic fragments have been found to be substantially less than those estimated for intact HKs in cellular studies (Gao & Stock, 2017). Furthermore, cellular studies have the advantage of allowing comparison of enzymatic activities in the presence and absence of stimuli if environmental conditions that lead to activation are known, even for systems in which specific ligands or physical parameters have not been identified, or in which sensing is mediated by auxiliary proteins.…”