The dynamics of regulation of the PhoQ sensor kinase by small proteins in Escherichia coli

Enterobacteria rely primarily on protein two-component systems (TCS) to sense host conditions and activate their virulence system at the correct host location. In E. coli, the PhoQ/PhoP TCS senses host-associated signals and is crucial for virulence in pathogenic E. coli. Two membrane-located small proteins regulate the sensor kinase PhoQ: MgrB inhibits PhoQ while SafA activates PhoQ. MgrB expression is regulated by the PhoQ/PhoP TCS, and SafA expression is regulated by the EvgS/EvgA TCS. The regulation of PhoQ by MgrB and SafA is presumably coordinated to allow optimal signal transduction. However, the coordination and dynamics of regulation by these two small proteins have not been studied systematically. Additionally, it is unclear how the regulation of PhoQ by MgrB and SafA plays a role under physiological conditions where both small proteins are expressed. Here, we use a combination of transcription assays, Förster resonance energy transfer (FRET) and functional assays to address these open questions. We analysed the expression dynamics of MgrB and SafA and quantified their native levels. The obtained results show that SafA expression is upregulated very rapidly after activation of EvgS and upregulation of PhoP-regulated genes follows after a short time period. safA and mgrB are transcribed at similar amount, but MgrB protein levels are much higher. To understand how PhoQ can integrate the opposing regulation of MgrB and SafA we identified possible competition of the small proteins. Our results indicate that SafA and MgrB influence each other in binding to PhoQ depending on the relative expression levels and the more abundant protein dominates PhoQ regulation. To define additional factors that underlie this competition apart from expression levels we determined the binding affinity of MgrB and SafA to PhoQ and identified additional modulators of this interaction. We identified divalent cations (Mg2+ and Ca2+) as a modulator of PhoQ-MgrB interaction and showed that they increase binding which has functional relevance. Furthermore, we laid the experimental foundation to study how our findings relate to the survival of pathogenic E. coli in macrophages, an environment where presumably both MgrB and SafA are expressed. We identified an enteropathogenic E. coli strain that is suitable for macrophage infection and established the infection protocol. In summary, our data suggests that regulation of PhoQ by MgrB and SafA is dependent on the amounts of the small proteins which change dynamically upon encounter of activating stimuli, but is also directly modulated by external factors.