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Abstract:
Well-defined front-rear cell polarity is essential for directional cell movement. The rod-shaped Myxococcus xanthus cells move using two motility systems and with defined front-rear polarity. Both systems are polarized, i.e. type IV pili assemble at the leading pole while the Agl/Glt gliding motility complexes assemble at the leading, translocate rearward to propel the cell, and disassemble at the lagging pole. During cellular reversals, which are induced by the Frz chemosensory system, polarity of the motility systems is inverted. The Ras-like GTPase MglA together with MglB, the cognate MglA GTPase activating protein (GAP) and the RomR response regulator constitute a module that determine front-rear polarity. MglA-GTP and MglB localize to and define the leading and lagging pole, respectively. MglA-GTP and MglB depend on polarly localized RomR for correct polar targeting. During the Frz system-induced reversals, MglA, MglB and RomR switch poles. Here, using a comparative genomics approach together with experimental work, we identify RomX and RomY as integral components of the polarity module. RomX localizes asymmetrically to the poles with a large cluster at the lagging pole. In vitro data analyses demonstrated that RomX not only interacts directly with RomR alone and MglA-GTP alone but that RomR, RomX and MglA together form a heteromeric RomR/RomX/MglA-GTP complex. In this complex, RomX is sandwiched between RomR and MglA-GTP. Moreover, our data provide evidence that the RomR/RomX complex has MglA GEF activity. In vivo, polarly localized RomR recruits RomX and, in turn, the RomR/RomX complex recruits MglA-GTP to the leading cell pole. Thus, the RomR/RomX complex has dual functions in establishing front-rear polarity for motility in M. xanthus, it is a GEF that stimulates the accumulation of MglA-GTP, the active form of MglA, and it is a polar recruitment factor that recruits MglA-GTP to the leading cell pole. Both activities contribute to a high local concentration of MglA-GTP at this pole. At the leading pole, the RomR/RomX/MglA-GTP complex stimulates the assembly of Agl/Glt gliding motility complexes and is also incorporated into these complexes. However, in the absence of MglB, RomR/RomX is not essential for assembly of these complexes. Importantly, the Agl/Glt complexes assembled in the absence of RomR/RomX are less stable and transfer less directionally towards the lagging pole. These results suggest that RomR/RomX at the leading and MglB at the lagging cell pole establish leading lagging polarity axis for efficient gliding motility.RomY localizes unipolarly with a cluster at the lagging cell pole. In vivo experiments demonstrated, that RomY regulates reversals and cell polarity similarly to MglB. Moreover, RomY localization depends on MglB suggestíng a functional connection between these two proteins. Protein-protein interaction analyses suggested that RomY directly interacts with MglA and RomX. Moreover, RomX and RomR are not essential for gliding motility in the absence of RomY. Based on these data we suggests that RomY either stimulates MglB GAP activity or MglA GTPase activity.
