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 Mg 2+ homeostasis, resistance to acid stress, cationic antimicrobial peptides, and virulence in E. 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. Thus, MgrB is part of a negative feedback loop modulating this two-component system, disruption of which is strongly associated with acquired colistin resistance in clinical isolates of Klebsiella pneumoniae. MgrB may also be important for preventing hyperactivation of PhoQ and possibly act as an input for alternate signals in the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we have analyzed the effects of point mutations, C-terminal truncations and domain swaps on MgrB activity. In contrast with two other small membrane protein regulators of histidine kinases in E. coli, we find that the MgrB transmembrane (TM) domain is necessary for PhoQ inhibition. Bacterial two-hybrid assays suggest the TM domain mediates interactions with itself as well as with PhoQ. Additionally, alanine scanning mutagenesis of the periplasmic domain of MgrB 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 is derived via distinct contributions from multiple residues spread across domains. Interestingly, the TM domain 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.

A global data-driven census of Salmonella small proteins and their potential functions in bacterial virulence

“…MgrB also affected the expression of numerous genes that are currently not considered members of the PhoPQ regulon. This suggests additional, PhoPQ-independent roles for MgrB, supported by recent findings of E. coli MgrB interacting with further histidine kinases such as PhoR (Yadavalli et al, 2018). PhoB, the response regulator of the PhoBR TCS, was also detected among the upregulated proteins in our ΔmgrB mutant.…”

Employing scan-SCAM to identify residues that compose transmembrane helix 1 (TM1) of EnvZ

“…We are currently undertaking a rigorous analysis of EnvZ-MzrA and MzrA-MzrA interactions in vivo and building high-resolution models for in silico screening in order to identify potential modulators of PRC signal output. It has recently come to our attention that an analogous approach is currently being undertaken with respect to the membrane-embedded PhoQ/MgrB complex of E. coli lending credibility to this long-term strategy [44]. [15,16], the effect of pH on the cytoplasmic domain [17] and interactions between EnvZ and MzrA in the periplasm [18,19].…”

“…A related structure of the periplasmic domain of Salmonella enteritica serovar Typhimurium PhoQ (PhoQ-PD) possesses a similar hairpin-like β-strand composed of β1 and β2 [43]. Both EnvZ and PhoQ interact with a small biotopic protein, MzrA and MgrB respectively, that consists of a cytoplasmic N-terminus, a single TM helix and a small periplasmic domain suggesting correlations could be drawn between the presence of this hairpin-like β-strand and the existence of membrane-embedded interaction partners [18,19,44] (Figure 7).…”

The inhibitory mechanism of a small protein reveals its role in antimicrobial peptide sensing

“…This disulfide bond would also act as a sensor for the redox state of the bacterial periplasm creating an entry point for redox sensing by the PhoQ/PhoP system [30]. In addition to the two cysteines, eight functionally essential residues have been identified, spreading across the entire molecule [31]. However, the mechanism of MgrB inhibiting PhoQ remained largely unclear.…”