Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity.

The bacterium Myxococcus xanthus uses a G protein cycle to dynamically regulate the leading/lagging pole polarity axis. The G protein MglA is regulated by its GTPase-activating protein (GAP) MglB, thus resembling Ras family proteins. Here, we show structurally and biochemically that MglA undergoes a dramatic, GDP-GTP-dependent conformational change involving a screw-type forward movement of the central ß2-strand, never observed in any other G protein. This movement and complex formation with MglB repositions the conserved residues Arg53 and Gln82 into the active site. Residues required for catalysis are thus not provided by the GAP MglB, but by MglA itself. MglB is a Roadblock/LC7 protein and functions as a dimer to stimulate GTP hydrolysis in a 2:1 complex with MglA. In vivo analyses demonstrate that hydrolysis mutants abrogate Myxococcus' ability to regulate its polarity axis changing the reversal behaviour from stochastic to oscillatory and that both MglA GTPase activity and MglB GAP catalysis are essential for maintaining a proper polarity axis.

Regulation of dynamic polarity switching in bacteria by a Ras-like G-protein and its cognate GAP.

The rod-shaped cells of the bacterium Myxococcus xanthus move uni-directionally and occasionally undergo reversals during which the leading/lagging polarity axis is inverted. Cellular reversals depend on pole-to-pole relocation of motility proteins that localize to the cell poles between reversals. We show that MglA is a Ras-like G-protein and acts as a nucleotide-dependent molecular switch to regulate motility and that MglB represents a novel GTPase-activating protein (GAP) family and is the cognate GAP of MglA. Between reversals, MglA/GTP is restricted to the leading and MglB to the lagging pole defining the leading/lagging polarity axis. For reversals, the Frz chemosensory system induces the relocation of MglA/GTP to the lagging pole causing an inversion of the leading/lagging polarity axis. MglA/GTP stimulates motility by establishing correct polarity of motility proteins between reversals and reversals by inducing their pole-to-pole relocation. Thus, the function of Ras-like G-proteins and their GAPs in regulating cell polarity is found not only in eukaryotes, but also conserved in bacteria.

Reversing cells and oscillating motility proteins.

Cells of the bacterium Myxococcus xanthus organize into two types of patterns depending on their nutritional status, i.e. in the presence of nutrients cells form spreading colonies and in the absence of nutrients cells form fruiting bodies. Formation of both patterns depends on directed cell movements, which, in turn, depend on regulation of motility. M. xanthus cells harbor two motility machines, type IV pili and the A-engine, which act synergistically to generate motive force in the same direction. Periodically, the individual cells reverse their direction of movement. During a reversal the two motility machines switch polarity to generate force in the opposite direction. Recent evidence shows that at the molecular level, reversals involve pole-to-pole oscillations of motility proteins. Between reversals, these proteins localize to the cell poles to stimulate motility and in parallel with a reversal they relocalize between the poles. For two proteins, FrzS and RomR, which are part of the type IV pili and A-engine, respectively, it was directly demonstrated that they oscillate independently of each other but in synchrony, thus, providing evidence that the two motility machines switch polarity independently but synchronously. Protein oscillations are regulated and synchronized by the Frz chemosensory signal transduction system. The correct polarity of the motility systems is likely established by the MglA protein, which is a member of the Ras/Rac/Rho superfamily of small GTPases. In this scenario, MglA establishes the correct polarity of the two motility machines and the Frz-induced synchronized polarity switching maintains the correct polarity of the two motility machines.

PilB and PilT are ATPases acting antagonistically in type IV pilus function in Myxococcus xanthus.

Type IV pili (T4P) are dynamic surface structures that undergo cycles of extension and retraction. T4P dynamics center on the PilB and PilT proteins, which are members of the secretion ATPase superfamily of proteins. Here, we show that PilB and PilT of the T4P system in Myxococcus xanthus have ATPase activity in vitro. Using a structure-guided approach, we systematically mutagenized PilB and PilT to resolve whether both ATP binding and hydrolysis are important for PilB and PilT function in vivo. PilB as well as PilT ATPase activity was abolished in vitro by replacement of conserved residues in the Walker A and Walker B boxes that are involved in ATP binding and hydrolysis, respectively. PilB proteins containing mutant Walker A or Walker B boxes were nonfunctional in vivo and unable to support T4P extension. PilT proteins containing mutant Walker A or Walker B boxes were also nonfunctional in vivo and unable to support T4P retraction. These data provide genetic evidence that both ATP binding and hydrolysis by PilB are essential for T4P extension and that both ATP binding and hydrolysis by PilT are essential for T4P retraction. Thus, PilB and PilT are ATPases that act at distinct steps in the T4P extension/retraction cycle in vivo.

Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus.

Myxococcus xanthus cells harbor two motility machineries, type IV pili (Tfp) and the A-engine. During reversals, the two machineries switch polarity synchronously. We present a mechanism that synchronizes this polarity switching. We identify the required for motility response regulator (RomR) as essential for A-motility. RomR localizes in a bipolar, asymmetric pattern with a large cluster at the lagging cell pole. The large RomR cluster relocates to the new lagging pole in parallel with cell reversals. Dynamic RomR localization is essential for cell reversals, suggesting that RomR relocalization induces the polarity switching of the A-engine. The analysis of RomR mutants shows that the output domain targets RomR to the poles and the receiver domain is essential for dynamic localization. The small GTPase MglA establishes correct RomR polarity, and the Frz two-component system regulates dynamic RomR localization. FrzS localizes with Tfp at the leading pole and relocates in an Frz-dependent manner to the opposite pole during reversals; FrzS and RomR localize and oscillate independently. The Frz system synchronizes these oscillations and thus the synchronous polarity switching of the motility machineries.