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Abstract

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.