Functional Determinants of a Small Protein Controlling a Broadly Conserved Bacterial Sensor Kinase

The PhoQ/PhoP two-component system plays a vital role in the regulation of Mg2 + homeostasis, resistance to acid and hyperosmotic stress, cationic antimicrobial peptides, and virulence in Escherichia coli, Salmonella, and related bacteria. Previous studies have shown that MgrB, a 47-amino-acid membrane protein that is part of the PhoQ/PhoP regulon, inhibits the histidine kinase PhoQ. MgrB is part of a negative-feedback loop modulating this two-component system that prevents hyperactivation of PhoQ and may also provide an entry point for additional input signals for the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we analyzed the effects of point mutations, C-terminal truncations, and transmembrane (TM) region swaps on MgrB activity. In contrast to two other known membrane protein regulators of histidine kinases in E. coli, we found that the MgrB TM region is necessary for PhoQ inhibition. Our results indicate that the TM region mediates interactions with PhoQ and that W20 is a key residue for PhoQ/MgrB complex formation. Additionally, mutations of the MgrB cytosolic region suggest that the two N-terminal lysines play an important role in regulating PhoQ activity. Alanine-scanning mutagenesis of the periplasmic region of MgrB further indicated that, with the exception of a few highly conserved residues, most residues are not essential for MgrB's function as a PhoQ inhibitor. Our results indicate that the regulatory function of the small protein MgrB depends on distinct contributions from multiple residues spread across the protein. Interestingly, the TM region also appears to interact with other noncognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new smallprotein modulators of histidine kinases. IMPORTANCE One of the primary means by which bacteria adapt to their environment is through pairs of proteins consisting of a sensor and a response regulator. A small membrane protein, MgrB, impedes the activity of sensor protein PhoQ, thereby affecting the expression of PhoQ regulated virulence genes in pathogenic bacteria. However, it is unknown how such a small protein modulates the activity of PhoQ. Here, we studied the functional determinants of MgrB and identified specific amino acids critical for the protein's inhibitory function. Notably, we find that the membrane-spanning region is important for MgrB interaction with PhoQ. Additionally, this region appears to physically interact with other sensors, a property that may be important for evolving small protein regulators of sensor kinases.