The polarity of myxobacterial gliding is regulated by direct interactions between the gliding motors and the Ras homolog MglA

Gliding motility in Myxococcus xanthus is powered by flagella stator homologs that move in helical trajectories using proton motive force. The Frz chemosensory pathway regulates the cell polarity axis through MglA, a Ras family GTPase; however, little is known about how MglA establishes the polarity of gliding, because the gliding motors move simultaneously in opposite directions. Here we examined the localization and dynamics of MglA and gliding motors in high spatial and time resolution. We determined that MglA localizes not only at the cell poles, but also along the cell bodies, forming a decreasing concentration gradient toward the lagging cell pole. MglA directly interacts with the motor protein AglR, and the spatial distribution of AglR reversals is positively correlated with the MglA gradient. Thus, the motors moving toward lagging cell poles are less likely to reverse, generating stronger forward propulsion. MglB, the GTPase-activating protein of MglA, regulates motor reversal by maintaining the MglA gradient. Our results suggest a mechanism whereby bacteria use Ras family proteins to modulate cellular polarity.Generating and maintaining polarity is fundamental to the proper functioning of cells. Eukaryotic cells generate polarity for migration and the accurate positioning of macromolecules and organelles (1, 2). For bacteria, polarity is important for motility, division, signal transduction, and pathogenesis (3, 4). The Gram-negative soil bacterium Myxococcus xanthus is a model organism for use in the study of cell polarity for its directed surface motilities.M. xanthus cells move on solid surfaces using two distinct mechanisms. The first mechanism, social motility (S-motility), is powered by the extension and retraction of type IV pili from the leading cell poles (5, 6). In contrast, the second mechanism, gliding motility (adventurous or A-motility), uses proton motive force to power the movement of motor complexes containing flagella stator homologs (7–11). Gliding M. xanthus cells on 1.5% agar plates typically reverse their polarity approximately every 8–12 min (12). The Frz chemosensory pathway regulates the reversal frequency and thus the direction of cell movements of both motility systems (12–16). MglA, a Ras family GTPase, has been identified as the central regulator of cell polarity and the principal responder to Frz pathway signaling (13–15). It has been reported that MglA is connected to the Frz pathway by the response regulator RomR (17–19). Importantly, MglA switches between an active GTP-bound form and an inactive GDP-bound form, which is regulated by MglB, the cognate GTPase-activating protein (GAP) of MglA, providing another layer of regulation (13, 14).The importance of cell polarity in S-motility is obvious, because the S-motility motors localize to cell poles in an MglA-dependent manner (5, 20). In contrast, cell polarity for gliding motility is enigmatic, because the gliding motor complexes, as represented by the MotA homolog AglR and motor-associated proteins, such as AgmU (GltD), localize in blurry patches that move simultaneously in opposite directions along a helical track (7, 8, 10, 11).The gliding complexes consist of the motor proteins AglR, AglQ, and AglS, along with numerous motor-associated proteins that localize in the cytoplasm, inner membrane, and periplasm (21). Genomic analysis has shown that the M. xanthus motor complexes, unlike the MotAB complexes of enteric bacteria, lack peptidoglycan-binding domains and thus are free to move within the membrane (7). Consistent with this idea, the motor protein AglR and the motor-associated protein AgmU (GltD) have been observed to decorate a helical macrostructure that rotates as cells move forward (7, 8). In addition, tracking the movements of single AglR molecules using single-particle photoactivatable localization microscopy (sptPALM) (22) revealed that the gliding motors containing AglR molecules move in helical trajectories. A subpopulation of motors slow down and accumulate into evenly distributed “traffic jam” clusters at the ventral sides of cells, where they contact surfaces. The traffic jam clusters appear to be stationary in relation to the substratum when cells move forward (7). These clusters were originally called “focal adhesion sites” because of some similarities with eukaryotic motility complexes (9, 23).Based on the results of our high-resolution experiments, we proposed a revised model of bacterial gliding (the helical rotor model) that envisions the distance between two adjacent traffic jam sites as corresponding to the period of the helical track (11). According to this model, motors at these sites push against the gliding surface, deform the cell membrane, and exert force against the surface slime (Fig. 1A) (7). This model explains evenly distributed traffic jam sites in gliding cells, without invoking that force is transmitted to the surface by breaching the peptidoglycan barrier (21, 23); however, how the bidirectional motion of gliding motors generates unidirectional cell movements remains unknown.Open in a separate windowFig. 1.Single molecules of AglR-pamCherry reverse their moving directions along cell bodies. (A) The helical rotor model predicts that the gliding motors move simultaneously toward opposite directions along helical tracks. (B) Kymograph of AglR-pamCherry fluorescence in a moving cell. AglR molecules move simultaneously toward the leading and lagging cell poles. Yellow arrows point to the reversal events of AglR. A significant population of AglR molecules also appear stationary (blue dots), which we attribute to the molecules that slow down at traffic jam sites owing to the resistance of the underlying gliding surfaces. The yellow lines mark the positions of the cell poles in the kymograph. An example of the cells is shown in Movie S2.In the present study, we found that MglA directly interacts with AglR. Using a combination of sptPALM and conventional fluorescence microscopy, we showed that MglA localizes not only at the cell poles, but also along the cell bodies, forming a gradient toward the lagging cell poles. We investigated the role of MglA in regulating gliding motility by tracking the movements of motors in various genetic backgrounds. We found that the probability of AglR reversal is positively correlated with the local MglA gradient. Our observations suggest that the MglA gradient dictates the polarity of gliding by triggering the reversal of gliding motors asymmetrically.