The small G-protein MglA connects the motility machinery to the bacterial actin cytoskeleton

Motility of Myxococcus xanthus cells is powered by two distinct engines: S-motility allows grouped cells movement and is driven by type IV pili (T4P) at the leading cell pole that use ATP for their function and pull the cell forward upon their retraction. Single cell movement is called gliding or A-...

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Bibliographic Details
Main Author: Hot, Edina
Contributors: Sogaard-Andersen, Lotte (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:English
Published: Philipps-Universität Marburg 2013
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Summary:Motility of Myxococcus xanthus cells is powered by two distinct engines: S-motility allows grouped cells movement and is driven by type IV pili (T4P) at the leading cell pole that use ATP for their function and pull the cell forward upon their retraction. Single cell movement is called gliding or A-motility and its AglQ/R/S engine is powered by proton-motive force and is incorporated at focal adhesion complexes in the cell. The control of motility and its direction is accomplished by cells rapidly switching their leading into lagging cell pole (cellular reversal), a process regulated by the small Ras-like G-protein MglA and its cognate GTPase activating protein (GAP) MglB. Using fluorescence microscopy it was previously shown that MglA localizes at the leading cell pole and MglB at the lagging cell pole and both proteins dynamically switch polarity during cellular reversal. Further, recent experiments showed that an A-motility protein AglZ, and A-motility engine AglQ/R/S localize at clusters distributed along the cell body that stay fixed relative to the substratum as the cell moves forming focal adhesion complexes (FACs). Based on the in vivo experiments it has been proposed that gliding motility machinery assembles at the leading cell pole and that it is guided by the cytoskeletal element to the lagging cell pole, where it disassembles. In this work we investigated the function of MglA during gliding motility. First, we demonstrate that MglA in its active state forms a focal adhesion cluster, which co-localizes with AglZ and AglQ, thus showing that active MglA is a component of the FACs. We show that MglA is essential for incorporation of AlgQ in the FACs, and that MglA GTPase cycle regulates the number of AglQ clusters. Further, we provide evidence that the GTPase negative MglA variant MglAQ82A leads to regularly reversing cells after movement of only one cell length, and that MglA GTPase cycle regulates the disassembly of the FACs at the lagging cell pole. Fluorescent YFP-MglAQ82A forms a focal adhesion cluster which appears to regularly oscillate between the poles, and causes the cell to move in a pendulum-like manner. Unlike wildtype MglA, MglAQ82A is insensitive to the GAP activity of MglB, and upon reaching the lagging cell pole where MglB localizes, it causes a cellular reversal by starting to oscillate in the opposite direction. The co-localizing YFP-MglAQ82A/AglZ-mCherry and YFP-MglAQ82A/AglQ-mCherry FAC also appear to continuously oscillate between the poles suggesting that the gliding motility machinery coupled to active MglA needs to be disassembled at the lagging cell pole by MglB GAP, and in this way allow uni-directional motility for distances longer than one cell length. Furthermore, in this work we demonstrate that active wt MglA and MglAQ82L variant interact directly with filament forming MreB actin homolog. Additionally, our results show that the formation and localization of FACs depend on intact MreB, thus indicating that MreB acts as a scaffold for the assembly of gliding motility machinery. The addition of antibiotics which inhibit peptidoglycan (PG) synthesis and reduce the dynamics of MreB in other bacteria did not inhibit single cell motility and did not cause mislocalization of MglA and AglQ. This strongly suggests that the major proposed function of MreB as a scaffold for PG elongation machinery is not coupled to its essential role during gliding motility in M. xanthus. Thus, we demonstrate that MreB is required for MglA, AglZ and AglQ localization at FACs during gliding, and this function of MreB is separable from its major proposed function in PG synthesis.
DOI:10.17192/z2014.0066