Regulierung der Polarität des A-Bewegungssystems in Myxococcus xanthus

The cells of the rod-shaped bacterium Myxococcus xanthus move by gliding, using two different motility systems. The S-motility system depends on retraction of type IV pili (Tfp), which are localized in a unipolar pattern and only present at the leading cell pole. For the A-motility system two models have been suggested. In the first model, motility depends on extrusion of slime form polar nozzle like structures. Slime is hypothesized to occur from the lagging cell pole, i.e. the pole opposite to that containing Tfp. In the second model, force generation involves multiple adhesion complexes, which are defined by the AglZ protein and distributed along the cell body. M. xanthus cells regularly undergo cellular reversals, in which the old lagging pole becomes the new leading cell pole. To resume gliding after a cellular reversal cells have to switch the directionality and, thus, the polarity of both engines. To investigate the molecular mechanism underlying polarity switching of the A-engine, we analyzed the two proteins RomR and MglA in detail.The response regulator RomR is crucial for A-motility and consists of a N-terminal receiver domain of two component systems and a Pro-rich output domain. We found that RomR localizes in an asymmetric bipolar pattern with a large cluster at the lagging cell pole and a small cluster at the leading cell pole. In parallel with a cellular reversal, the large RomR cluster switches to the new lagging cell pole, indicating that RomR stimulates a part of the A-machinery at the lagging pole. Genetic evidence suggests that the dynamic RomR localization depends on phosphorylation of the RomR receiver domain and that it is regulated by the Frz chemosensory system, which controls the reversal frequency. The analysis of mutant RomR proteins in a hypo-reversing frz mutant let us assume that RomR acts downstream of the Frz system to induce cellular reversals in the A-motility system. The MglA protein, which has sequence similarity to small GTPases of the Ras/Rac/Rho superfamily, is essential for A-motility. A mutant MglA protein containing a substitution which supposedly locks MglA in the GDP-bound form (MglAlof) is unable to support A-motility. On the other hand, a mutant MglA protein which contains a substitution that is predicted to lock MglA in the GTP-bound form (MglAgof) restores A-motility and also causes hyper-reversals. Additionally we found that the native YFP-MglA protein localizes to the leading pole and switches between the poles when the cell reverses the direction of movement. However, the YFP-MglAgof protein constantly oscillates between the poles, whereas the YFP-MglAlof protein is homogenously distributed in the cell. These findings indicate that a certain level of MglA in the GTP-bound form is required for the stimulation of A-motility and for the localization of MglA at the leading cell pole. Furthermore, we hypothesize that a high level of MglA in the GTP-bound form leads to the polar release and the transfer of MglA to the lagging pole. Finally, the completed localization of MglA at the lagging cell pole induces a cellular reversal in the A-system and the cell starts moving in the opposite direction. Genetic evidence suggests that the Frz system and MglA act in the same pathway to induce reversals in the A-motility system and that MglA act downstream of the Frz system in this pathway. Additionally, the RomR protein seems to be downstream of the MglA protein concerning the stimulation of reversals. However, our results imply that there is a feedback between RomR and MglA, since the localization of RomR depends on MglA and vice versa. On the basis of our data, we propose the following model: Initially, the Frz system senses a signal. This results in the stimulation of GTP binding to MglA. The stimulated GTP binding to MglA leads to a high level of MglA in the GTP-bound form and thus to the transfer of MglA from the leading pole to the lagging pole. Presumably, there MglA-GTP interacts with the unphosphorylated RomR protein. It remains possible that the unphosphorylated RomR protein act as a GTPase activating protein (GAP) and that MglA-GTP mediates the phosphorylation of RomR. Subsequently, RomR is transferred from the old lagging pole to the new lagging pole, where it stimulates a part of the A-machinery. In parallel the promoted GTP hydrolysis leads again to a low level of MglA in the GTP-bound form and MglA binds to the new leading pole. Finally, MglA localizes at the leading pole, RomR at the lagging pole and the cell moves in the opposite direction. The results of this work suggest that MglA is a prokaryotic small GTPase of the Ras/Rac/Rho superfamily which is required for cell polarity by setting up the correct localization of RomR and probably other A-motility proteins.