The polar Ras-like GTPase MglA activates type IV pilus via SgmX to enable twitching motility in Myxococcus xanthus

Significance The type IV pilus (Tfp) is a multipurpose machine found on bacterial surfaces that works by cycles of synthesis/retraction of a pilin fiber. During surface (twitching) motility, the coordinated actions of multiple Tfps at the cell pole promotes single cells and synchronized group movements. Here, directly observing polar Tfp machines in action during motility of Myxococcus xanthus, we identified the mechanism underlying pole-specific Tfps activation. In this process, the Ras-like protein MglA targets a novel essential Tfp-activator, SgmX, to the pole, ensuring both the unipolar activation of Tfps and its switching to the opposite pole when cells reverse their movement. Thus, a dynamic cascade of polar activators regulates multicellular movements, a feature that is likely conserved in other twitching bacteria.

Plasmids pSWU19 carrying P mxan_3192 -pilA and P mxan_1254 -pilA were constructed by Gibson assembly of DNA fragments containing 1000 bp upstream of higly expressed genes mxan_3192 or mxan_1254 (3) and pilA gene. Plasmids were integrated on the chromosome at Mx8 phage attB site by site specific recombination.

(II) Proteins Purification and Pull-Down assay.
Proteins Purification. All proteins were expressed in E. coli BL21(DE3)pLysS strain grown in LB medium. Briefly, cells were grown at 37°C until mid-exponential phase. Protein expression was induced by addition of 1mM IPTG for 4h at 30 °C. Cells were pelleted and stored at -80°C. To purified MglA-His, a pellet of MglA-His expressing cells were resuspended in lysis buffer (BugBuster® Millipore) complemented with Dnase I and a protease inhibitor cocktails (cOmplete Tm Roche). Lysate was clarified by centrifugation and was loaded onto a gravity column prepacked with HisPur Tm Ni-NTA Resin (Thermoscientific) equilibrated with a buffer containing 10 mM Tris (pH7.4), 150 mM NaCl, 10 mM Imidazole, 10 mM MgCl 2 . After 10 min of incubation, the resin was washed with 4-column volume (40 ml) of a buffer containing 10 mM Tris (pH7.4), 150 mM NaCl, 75 mM Imidazole, 10 mM MgCl 2 and MglA-His protein was eluted with a buffer containing 10 mM Tris (pH7.4), 150 mM NaCl, 500 mM Imidazole, 10 mM MgCl 2 . Protein purity was assessed by SDS-polyacrylamide gel and revealed by Coomassie blue staining; protein concentration was measured by Nanodrop. Purified MglA-His protein was directly incubated with 80 µM of GTP or GDP for 25 minutes at 4 °C and processed for pull-down assay experiments.

(IV) Microscopy and Image Analysis.
For standard microscopy, exponentially growing cells grown in CYE media were washed, concentrated in TPM buffer and mounted on microscope slides covered with an 1.5% TPM agarose pad. The cells were imaged on an automated and inverted epifluorescence microscope TE2000-E-PFS (Nikon), with a ×100/1.4 DLL objective and a camera orca flash 4 (Hamamatsu) at room temperature. Mercury fluorescent lamp with Green and Red optical filters was used when necessary. For single cell twitching microscopy, cells grown in CYE media until OD 600nm of 0.3 were directly injected in a preassemble Ibidi sticky-slide VI 0.4 (Ibidi) microfluidic devise sealed with a glass slide, coated with 0.015% carboxymethylcellulose (4). Cells were incubated for 30 min and washed several times with TPM buffer with 1 mM CaCl 2 . The cells were imaged on an automated and inverted epifluorescence microscope TE2000-E-PFS (Nikon), with a ×100/1.4 DLL objective and a camera orca flash 4 (Hamamatsu) at room temperature. Mercury fluorescent lamp with Green and Red optical filters was used when necessary. Images analysis were performed using Fiji plugins MicrobeJ (5) and cell Counter. Pictures and movies were prepared for publication using Fiji (https://fiji.sc/) and Adobe Photoshop.           Used to purifie MglA-His6 protein (7) Kan, Kanamycin bla, β-lactamase Movie S1. Movie_S1.avi Time-lapse series showing cell motility and labelled Tfpa pilin filaments of the strain RM384 (DZ2 att mx8 ::P pilA -pilA D71C ) observed by TIRF microscopy, from which the panels in Fig. 1A were obtained. Fluorescent images were acquired automatically every 2 s for 72 s.  Figure  2f were obtained. Fluorescent images were acquired automatically every 2 s for 2 min. Scale bar: 4 µm.

Movie S6. Movie_S6.avi
Time-lapse series showing cell motility of the strain RM55 (ΔBAR mimA ), from which the panels in SI Appendix Fig. S2C were obtained. Phase-contrast images were acquired automatically every 1 min for 3 hr. Scale bar: 3 µm.

Movie S7. Movie_S7.avi
Time-lapse series showing cell motility of the strain TM500 (ΔBAR), from which the panels in SI Appendix Fig. S2D were obtained. Phase-contrast images were acquired automatically every 1 min for 3 hr. Movie S11. Movie_S11.avi Time-lapse series showing a pole-to-pole dynamics of SgmX-sfGFP in a single reversing cell of the strain RM190 (sgmX-sfGFP), from which the panels in Figure 3C were obtained. Phase-contrast and fluorescent images were acquired automatically every 10 s for 140 s. Scale bar: 2 µm.

Movie S13. Movie_S13.avi
Time-lapse series showing the correlation between the uni or bi-polar localisation of SgmX mimB -mcherry (bottom) and the presence of polar pilin cluster (top) of cells with labelled Tfpa pilin filaments of the strain RM393 (sgmX mimB -mcherry att mx8 ::P pilA -pilA D71C ) observed by TIRF microscopy. Fluorescent images were acquired automatically every 2 s for 2 min. Scale bar: 3 µm.