MotI (DgrA) acts as a molecular clutch on the flagellar stator protein MotA in Bacillus subtilis

Abstract

Stator elements consisting of MotAMotB complexes are anchored to the cell wall, extend through the cell membrane, and interact with FliG in the cytoplasmic C ring rotor of the flagellum. The cytoplasmic loop of MotA undergoes proton-driven conformational changes that drive flagellar rotation. Functional regulators inhibit motility by either disengaging or jamming the stator-rotor interaction. Here we show that the YcgR homolog MotI (formerly DgrA) of inhibits motility like a molecular clutch that disengages MotA. MotI-inhibited flagella rotated freely by Brownian motion, and suppressor mutations in MotA that were immune to MotI inhibition were located two residues downstream of the critical force generation site. The 3D structure of MotI bound to c-di-GMP was solved, and MotI-fluorescent fusions localized as transient MotA-dependent puncta at the membrane when induced at subinhibitory levels. Finally, subinhibitory levels of MotI expression resulted in incomplete inhibition and proportional decreases in swimming speed. We propose a model in which flagellar stators are disengaged and sequestered from the flagellar rotor when bound by MotI.

Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary among Bacillus subtilis Subpopulations

“…Instead, the recent development of cell-type-specific fluorescent reporters allows investigators to examine features of B. subtilis subpopulations and their relationship to c-di-GMP effector proteins, DGCs, and PDEs. (51)(52)(53).…”

“…In many instances, however, c-di-GMP regulates phenotypic outputs at the posttranslational level (78)(79)(80). This is exemplified in B. subtilis motile cells, where the binding of MotI to c-di-GMP directs MotI to directly disengage MotA from the rotor of the flagellar apparatus (51). It is possible that c-di-GMP posttranslationally regulates YdaK as well, and this interaction activates production of the unknown exopolysaccharide (EPS).…”

“…S1 in the supplemental material) (49,50). MotI is a PilZ domain-containing protein that is thought to inhibit flagellar motility by acting as a molecular clutch on the flagellar stator element MotA, to disengage and sequester it from the flagellar rotor FliG (51). Genetic evidence also supports a direct relationship between c-di-GMP and the regulation of motility through MotI and PdeH (49,50).…”

Structural conservation and diversity of PilZ-related domains

“…In the past 12 years, more than 30 structures of various PilZ domains have been solved and deposited in the Protein DataBank (PDB), see Table S1. These include high-resolution crystal structures of the stand-alone PilZ domains, as well as PilZ domains from the bacterial cellulose synthase (BcsA and AcsAB), alginate production protein Alg44, flagellar brake proteins YcgR, VCA0042, and MotI, and the transcriptional regulator MrkH (22, 25-27, 34, 35, 38, 39, 41-43). As noted in the respective reports, most of these domains are very similar in sequence and structure.…”

“…These proteins are generally similar to the previously described (16) alphaproteobacterial proteins with tandemly duplicated PilZ domains, such as B. diazoefficiens Bll4394 and Blr5568, but show various structural changes in their N-terminal PilZ-like domains. Based on the similar lengths and sequence similarity to the experimentally characterized flagellar brake proteins YcgR and MotI (22, 39, 41, 49), these proteins are often referred to as YcgR or YcgR-like. However, sequence similarity between these proteins is mostly limited to their common C-terminal PilZ domains.…”