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

Abstract

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.

Figure 1

MglA is a Ras-like G-protein. (A) Purification of MglB, MglA⁺, MglA^G21V, and MglA^T26/27N of T. thermophilus. Total purified protein separated by SDS–PAGE (5 μg protein loaded per lane). Migration of molecular size markers is indicated on the left. MglB and MglA have calculated molecular masses of 17 and 22 kDa, respectively. (B) MglA binds with high affinity to mant-labelled G-nucleotides. A total of 0.1 μM mGDP, mGTP, mGppNHp, mADP and mATP were titrated with nucleotide-free MglA and binding affinities determined by measuring the relative fluorescence intensities. The K_d's for binding to mGDP, mGTP, and mGppNHp of MglA⁺, MglA^G21V, and MglA^T26/27N are indicated below. (C) MglA has slow intrinsic GTPase activity. Graph depicts the release of ³²P_i using the charcoal assay with [γ-³²P]GTP and 4 μM of MglA⁺, MglA^G21V, and MglA^T26/27N over time (min).

Figure 2

Correlation between MglA localization and cellular behaviour. (A–E) Cells were transferred from exponentially growing cultures to a thin agar pad on a microscope slide, and imaged by time-lapse fluorescence microscopy at 30 s intervals. Red and blue arrows indicate opposite directions of movement. Time in minutes after initiation of the recordings is shown to the left. (A′–D′) The position of the maximum fluorescence signal in the corresponding cell as percentage of cell length is plotted as a function of time. (A″–B″) Quantitative analyses of the fluorescence signals in the corresponding cell over time. Relative integrated fluorescence intensities (arbitrary units) of the polar clusters and the cytoplasmic signal were plotted as function of time. For the colour code see A′″. (A′″) Schematic indicating the three regions for which fluorescence signals were quantified. (A) YFP–MglA⁺ localizes in a cluster at the leading pole between reversals. Fluorescence images are shown of a representative non-reversing cell. Scale bar: 10 μm. (B) YFP–MglA⁺ initiates relocation between the poles before a reversal. Images are shown of a cell that reversed once. Scale bar: 5 μm. (C) YFP–MglA^G21V continuously oscillates between cell poles. Images are shown of a cell that reversed three times. (D) YFP–MglA^G21V relocates between cell poles. Fluorescence images are shown of a stalled cell. White arrows indicate the YFP–MglA^G21V cluster in the upper, stalled cell. Scale bar: 5 μm. (E) YFP–MglA^T26/27N is diffusely localized. Scale bar: 5 μm.

Figure 3

Dynamic localization of MglA is regulated by the Frz system. (A–C) Cells were treated as in Figure 2. Red and blue arrows indicate opposite directions of movement. Time in minutes after initiation of the recordings is shown to the left. (A′–C′) The position of the maximum fluorescence signal in the corresponding cell as percentage of cell length is plotted as a function of time. (A) The Frz system is required for dynamic YFP–MglA⁺ localization. Images are shown of a representative non-reversing cell. Scale bar: 10 μm. (B) The Frz system is sufficient for dynamic YFP–MglA⁺ localization. Scale bar: 4 μm. (C) YFP–MglA^G21V bypasses a frz^lof mutation with respect to reversals. Images are shown of a cell that reversed three times. Scale bar: 5 μm.

Figure 4

MglB is localized to the lagging pole and dynamic during reversals. (A, B, D) Cells were treated as in Figure 2. Red and blue arrows indicate opposite directions of movement. Time in minutes after initiation of the recordings is shown to the left. (A′, B′, D′) The position of the maximum fluorescence signal in the corresponding cell as percentage of cell length is plotted as a function of time. (A″, B″, D″) Quantitative analyses of the fluorescence signals in the corresponding cell over time. Relative integrated fluorescence intensities (arbitrary units) of the polar clusters and the cytoplasmic signal were plotted as function of time. For the colour code see Figure 2A′″. (A) MglB–YFP localizes in a cluster at the lagging pole between reversals. Images are shown of a representative non-reversing cell. Scale bar: 5 μm. (B) MglB–YFP relocates between the poles during a reversal. Images are shown of a cell that reversed once. Scale bar: 5 μm. (C) In the absence of MglA, MglB–YFP localizes symmetrically at both poles. Images are shown of two cells that did not move. The percentage of cells with unipolar or bipolar symmetric localization of MglB–YFP are shown. Numbers in brackets indicate the same percentages in stalled mglA⁺ cells. Scale bar: 5 μm. (D) MglB regulates YFP–MglA⁺ localization. Images are shown of a cell that reversed once. Scale bar: 5 μm.

Figure 5

MglA is a Ras-like G-protein and MglB a MglAGAP. (A) MglB binds to MglA⁺ in its active GTP-bound form (MglA⁺/mGppNHp) and to MglA⁺/GDP in the presence of AlF_x. The K_d for binding of MglB to 1 μM MglA⁺ containing mGDP, mGppNHp, or mGDP/AIF_x is indicated below the graph and was determined by measuring the relative polarization during titration of MglB. (B) MglB stabilizes the transition state of GTP-hydrolysis mimicked by AlF_x. Shown are elution profiles from analytic gel filtration. Upper panel, elution profile of MglB and MglA⁺ bound to GDP and GppNHp, respectively. Lower panel, elution profile after mixing MglB with MglA⁺ bound to GDP, GppNHp, or GDP/AlF_x. On the right side, the corresponding SDS–PAGE are shown of aliquots of the peak maxima (indicated with 1 and 2). (C) MglB stimulates the intrinsic GTPase activity of MglA⁺ and MglA mutants have lost MglB-stimulated GTP hydrolysis. Graph depicts the release of ³²P_i using the charcoal assay in the presence of 4 μM of MglA⁺, MglA^G21V, and MglA^T26/27N bound to 60 nM [γ-³²P]GTP with and without the addition of 0.05 or 4 μM MglB as indicated.

Figure 6

MglA activity establishes correct polarity and regulates dynamic localization of motility proteins. (A–B, D–E, H–I) Cells were treated as in Figure 2. The position of the maximum fluorescence signal of the cells shown in Supplementary Figure S4A and B (A–B), Supplementary Figure S5A and B (D–E), and Supplementary Figure S6A and B (H–I) as percentage of cell length is plotted as a function of time. (A′–B′, D′–E′, H′–I′) Quantitative analyses of the fluorescence signals in the same cells over time. Relative integrated fluorescence intensities (arbitrary units) of the polar clusters and the cytoplasmic signal were plotted as function of time. For the colour code see Figure 2A′″. Red and blue arrows indicate opposite directions of movement. (A) RomR–GFP localization is bipolar, asymmetric between reversals and dynamic during reversals in MglA⁺ cells. (B) MglA^G21V changes RomR–GFP polarity. (C) MglA^T26/27N is unable to establish correct RomR–GFP polarity. Cells of SA3337 were grown on 1.5% agar plates supplemented with 1% CTT, scraped off the agar stained with Cy3 to visualize T4P and inspected by fluorescence microscopy (Cy3, white arrow) and RomR–GFP (GFP, white arrow). Lower panel is the overlay of the fluorescence images. Scale bar: 5 μm. (D) AglZ–YFP localizes in a cluster at the leading pole and is dynamic during reversals. (E) MglA^G21V regulates dynamic AglZ–YFP localization. (F) MglA^T26/27N does not interfere with correct AglZ–YFP polarity. The percentage of cells with unipolar or diffuse localization of AglZ–YFP are shown. Numbers in brackets indicate the same percentages in stalled mglA⁺ cells. Scale bar: 5 μm. (G) Opposite polarity of RomR–mDsRed and AglZ–YFP is absent in mglA^T26/27N mutant. Cells were treated as in (A–B, D–E, H–I). Shown are phase-contrast and fluorescence images as well as the overlays of the fluorescence and phase-contrast images. Scale bar: 4 μm. (H) YFP–PilT localizes in a large cluster at the lagging pole and localization is dynamic during reversals. (I) MglA^G21V regulates dynamic YFP–PilT localization. (J) MglA^T26/27N is unable to establish correct YFP–PilT polarity. The percentage of cells with unipolar, bipolar, asymmetric or bipolar, symmetric localization of YFP–PilT are shown. Numbers in brackets indicate the same percentages in stalled mglA⁺ cells. Scale bar: 4 μm.

Figure 7

Model of temporal and spatial regulation of MglA activity. (A) Temporal regulation of the nucleotide-bound state of MglA. In moving cells, MglA/GTP is present in a low concentration and interacts with effector(s) to stimulate motility. Before a reversal, Frz activity—directly or indirectly—stimulates MglA/GTP accumulation. At the increased concentration, MglA/GTP interacts with effector(s) that stimulate reversals. These effector(s) likely include proteins involved in relocation of MglA and motility proteins. (B) MglA/GTP and MglB set up the leading/lagging polarity axis. In moving cells (upper panel), this axis is stably maintained with the two proteins at opposite poles. At the lagging pole, MglB likely excludes MglA by converting MglA/GTP to MglA/GDP (arrow). In response to Frz activity (second panel), MglA/GTP accumulation is further stimulated at the leading pole followed by release and relocation to the lagging pole (third panel). Here, MglA/GTP interacts shortly with the MglAGAP MglB resulting in a reduction in the MglA/GTP concentration and MglA/GTP binding at the pole (fourth panel). Simultaneously, MglB is excluded from this pole and relocates to the opposite pole (fifth panel). Dashed arrows indicate direction of cell movement.