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

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 have analyzed the effects of point mutations, C-terminal truncations and transmembrane region swaps on MgrB activity. In contrast with two other known membrane protein regulators of histidine kinases in E. coli, we find 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 indicates 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 non-cognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new small protein modulators of histidine kinases.

Adaptation and compensation in a bacterial gene regulatory network evolving under antibiotic selection

“…The fifth isolate (TMPR5) harbored a single nucleotide deletion in the coding region of mgrB that resulted in a frame-shift and altered sequence of the C-terminal domain of the protein ( Figure 1A , Table 1 ). The C-terminus of MgrB is necessary for its inhibitory activity against PhoQ and mutations in this region alter the ability of MgrB to bind to PhoQ ( Yadavalli et al, 2020 ). Sanger sequencing of the mgrB gene and its promoter from isolates TMPR6–10 confirmed the presence of similar mutations in these bacteria as well ( Figure 1A ), suggesting that lower MgrB expression or activity may be advantageous for E. coli in the presence of trimethoprim.…”

Adaptation and Compensation in a Bacterial Gene Regulatory Network Evolving Under Antibiotic Selection

“…The fifth isolate (TMPR5) harboured a single nucleotide deletion in the coding region of mgrB that results in stop-codon readthrough and altered sequence of the C-terminal domain of the protein (Figure 1A, S5, Table 1). The C-terminus of MgrB is necessary for its inhibitory activity against PhoQ [34]. We therefore argued that lower MgrB expression or activity may be advantageous for E. coli in the presence of trimethoprim.…”

Diversity in Sensing and Signaling of Bacterial Sensor Histidine Kinases

“…Whereas the periplasmic region of SafA is sufficient for PhoQ activation, UgtL requires both its periplasmic and transmembrane regions to activate PhoQ [65]. In contrast, PhoQ activity is repressed by another small membrane protein, MgrB [67][68][69][70]. Similar to UgtL, the periplasmic, TM, and cytoplasmic domains are all involved in the interaction between PhoQ and MgrB (more details are provided in Section 3).…”

“…Inhibition of PhoQ requires the TM domain of MgrB. Trp20 within the TM domain is the key residue for PhoQ/MgrB complex formation [69]. However, in the case of MgrB, the short cytoplasmic N-terminal and periplasmic regions are also involved in the repression of PhoQ activity.…”