The small GTPase MglA together with the TPR domain protein SgmX stimulates type IV pili formation in M. xanthus

Significance Many bacteria move across surfaces using type IV pili (T4P). The piliation pattern varies between species; however, the underlying mechanisms governing these patterns remain largely unknown. Here, we demonstrate that in the rod-shaped Myxococcus xanthus cells, the unipolar formation of T4P at the leading cell pole is the result of stimulation by the small GTPase MglA together with the effector protein SgmX, while MglB, the cognate MglA GTPase activating protein (GAP) that localizes to the lagging cell pole, blocks this stimulation at the lagging pole due to its GAP activity. During reversals, MglA/SgmX and MglB switch polarity, laying the foundation for T4P formation at the new leading cell pole and inhibition of T4P formation at the former leading cell pole.


Supplementary text: Supplementary Materials & Methods
Cell growth and construction of strains. All in-frame deletions and plasmid integrations were verified by PCR. Plasmids were propagated in Escherichia coli Mach1 [∆recA1398 endA1 tonA φ80∆lacZ∆M15 ∆lacX74 hsdR(r K -m K + )]. E. coli cells were grown in LB or on 1.5% agar plates containing LB at 37°C with added antibiotics if appropriate (1). All DNA fragments generated by PCR were verified by sequencing.
Motility assays and determination of reversal frequency. Motility assays were done according to (2). Briefly, in population-based assays, M. xanthus cells from exponentially growing cultures were harvested at 4000 g for 10 min at room temperature (RT) and resuspended in 1% CTT to a calculated density of 7×10 9 cells/ml. 5 µl aliquots of cell suspensions were placed on 0.5% agar plates supplemented with 0.5% CTT for T4Pdependent motility and 1.5% agar plates supplemented with 0.5% CTT for gliding motility (3) and incubated at 32°C. After 24 hrs, colony edges were visualized using a Leica M205FA stereomicroscope and imaged using a Hamamatsu ORCA-flash V2 Digital CMOS camera (Hamamatsu Photonics). For higher magnifications of cells at colony edges on 1.5% agar, cells were visualized using a Leica DM6000B microscope and imaged using a Cascade II 1024 EMCCD camera (Photometrics). To track individual cells moving by T4P-dependent motility, 5 µl of exponentially growing cultures were spotted into a 24-well polystyrene plate (Falcon). After 10 min at RT, cells were covered with 500 µl of 1% methylcellulose in MMC buffer (10 mM MOPS, 4 mM MgSO 4 , 2 mM CaCl 2 , pH 7.6), and incubated at RT for 30 min. Subsequently, cells were visualized for 10 min at 20 s intervals at RT using a Leica DMi8 inverted microscope and imaged using a Leica DFC9000 GT camera. Individual cells were tracked using Metamorph 7.5 (Molecular Devices) and ImageJ 1.52b (4). 20 cells were tracked per strain per biological replicate. For each cell, the distance moved per 20 s interval was determined and the mean speed calculated as µm/min. For reversals, 50 cells were tracked as described and the number of reversals per cell per 10 min determined manually. To calculate mean squared displacement (MSD) of cells, the distance of a cell to its original position at t=0 was determined for every time interval of a time-lapse series, squared and plotted as function of time. Cells were followed for 10 min and recorded every 20 s. For each strain 50 cells were followed.
Transmission electron microscopy. Transmission electron microscopy was used to visualize T4P as described (5). Briefly, 50 µl of exponentially growing M. xanthus cells were placed on Parafilm. A small piece of carbon-coated mica was dipped into the drop for 2 min, allowing cells to adsorb to the surface, excess liquid was soaked off, the film was placed briefly on a drop of distilled water, excess liquid was soaked off again, and the film transferred on a drop of 2% uranyl acetate (wt/vol) for 3-4 seconds and blotted dry. Transmission electron microscopy was performed on a JEOL JEM-1400 electron microscope at calibrated magnifications.
T4P shear-off assay. T4P were sheared off M. xanthus cells as described (6) with minor modifications. Briefly, cells were grown on 1% CTT/1.5% agar plates at 32°C for 3 days, gently harvested and resuspended in resuspension buffer (100 mM Tris-HCl pH 7.6, 100 mM NaCl). Cells were harvested for 2 min at 13,000 g at RT, resuspended in 200 µl SDS lysis buffer (10% (v/v) glycerol, 60 mM Tris-HCl pH 6.8, 5 mM EDTA, 2% (w/v) SDS, 100 mM DTT, 0.005% bromphenol blue) and immediately denatured at 95°C for 10 min. These samples represent whole cell lysate fractions. To shear-off T4P, cells were vortexed for 10 min and then harvested by centrifugation for 20 min at 13,000 g at 4°C. The supernatant was removed and kept on ice; cells were resuspended in resuspension buffer, vortexed for 5 min and harvested as described. The supernatant removed and combined with the previous supernatant. The combined supernatant was centrifuged twice for 10 min at 13,000 g at 4°C. Subsequently, T4P in the final supernatant were precipitated ON at 4°C by addition of PEG6000 and MgCl 2 to final concentrations of 2% (w/v) and 0.1 M, respectively. Whole cell fraction and sheared fraction were analyzed by immunoblot using α-PilA antibodies (7) and α-PilC antibodies (8) as a loading control.
Pull-down experiments. In experiments involving MglA-His 6 , the protein (final concentration: 2.1 µM) was preloaded with GTP or GDP (final concentration: 104 µM) for 30 min at RT in buffer 1 (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 5 mM MgCl 2 ). To test for direct interactions between MglA-His 6 and SgmX-Strep, the two proteins were incubated in buffer 1 for 120 min at 4°C in a total volume of 500 µl (final concentrations: 2.0 µM and 1.8 µM, respectively) in the presence of the relevant nucleotide (final concentration: 100 µM). Then 100 µl of a Ni 2+ -NTA-agarose resin previously equilibrated in buffer 1 supplemented with the relevant nucleotide were added for 120 min. The resin was washed four times with 1 ml buffer 1 containing the relevant nucleotide at 100 µM.
Proteins were eluted in 100 µl buffer 1 supplemented with 400 mM imidazole. Fractions were analyzed by SDS-PAGE as described (1).
Protein purification. All proteins were expressed in E. coli BL21 (DE3) (B F -ompT gal dcm lon hsdS B (r B -m B -) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB + ] K-12 (λ S )) at 18°C or 30°C. To purify His 6 -tagged proteins, Ni-NTA affinity purification was used. Briefly, cells were washed in wash buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM imidazole, 5% glycerol, 5mM MgCl 2 ) and resuspended in lysis buffer A (50 ml of wash buffer A supplemented with EDTA-free protease inhibitors (Complete Protease Inhibitor Cocktail Tablet EDTA-free (Roche)). Cells were lysed by French press and cell debris removed by centrifugation (48,000 g, 4°C, 30 min). The cleared cell lysate was premixed for 1 hr with 1ml Ni 2+ -NTA-agarose preloaded with NiSO 4 as described by the manufacturer and pre-equilibrated in wash buffer A and sequentially loaded on a gravity column. The column was washed with 50 column volumes of column wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM imidazole, 5mM MgCl 2 ). Proteins were eluted with elution buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 500 mM imidazole) using a linear imidazole gradient from 50-500 mM. Fractions containing purified His 6 -tagged proteins were combined and loaded onto a HiLoad 16/600 Superdex 200 pg (GE Healthcare) gel filtration column that was equilibrated with buffer 1 supplemented with 5% glycerol. Fractions containing His 6 -tagged proteins were pooled, frozen in liquid nitrogen and stored at -80°C. To purify SgmX-Strep, biotin affinity purification was used. Briefly, cells were washed in wash buffer B (100 mM Tris pH 7.5, 150 mM NaCl, 5mM MgCl 2 ) and resuspended in lysis buffer B (30 ml of wash buffer D supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail Tablet (Roche))). Cells were lysed was pre-mixed for 1h with 1ml Strep-Tactin®XT Superflow® (IBA-lifesciences), preequilibrated with wash buffer D and sequentially loaded on a gravity column. The column was washed with 50 column volumes of wash buffer D. Protein was eluted with elution buffer D (150 mM Tris pH 7.5, 150 mM NaCl, 2.5 mM Desthiobiotin). Elution fractions containing SgmX-Strep were loaded onto a HiLoad 16/600 Superdex 200 pg (GE Healthcare) gel filtration column that was equilibrated with buffer D supplemented with 5% flycerol. Fractions with SgmX-Strep were pooled, frozen in liquid nitrogen and stored at -80°C.
Construction of plasmids: pLC51 (plasmid for generation of in-frame deletion of sgmX): up-(5766 A/5766 B) and downstream fragments (5766 C/5766 D) were amplified from genomic DNA of M. xanthus DK1622. Subsequently, the AB and CD fragments were used as template for overlapping PCR (5766 A/5766 D) to generate the AD fragment. AD fragment was digested with HindIII+EcoRI, cloned in pBJ114 and sequenced. pIB123 (plasmid for generation of in-frame deletion of pilT): up-(oPilT-A/oPilT-B) and downstream fragments (oPilT-C/oPilT-D) were amplified from genomic DNA of M. xanthus DK1622/ Subsequently, the AB and CD fragments were used as template for overlapping PCR (oPilT-A/oPilT-D) to generate the AD fragment. AD fragment was digested with HindIII+EcoRI, cloned in pBJ114 and sequenced. pAP12 (plasmid for expression of P nat pilB-mCherry from the attB site): Pnat pilB (PilB nat fw / PilB rev) fragment was amplified from genomic DNA of M. xanthus DK1622 and digested with NdeI +BamHI. Subsequently, fragment was cloned in pNG062 carrying Cterminal mCherry and sequenced. pAP16 (plasmid for expression of P nat mCherry-pilM from the attB site): Pnat (PilM nat fw / PilM nat rev) and pilM (PilM fw/PilM rev) fragments were amplified from genomic DNA of M. xanthus DK1622 and digested with NdeI+BglII and XbaI+HindIII, respectively. Subsequently, fragments were cloned in pNG063 carrying N-terminal mCherry and sequenced.
pSC73, pSC74 (C-and N-terminal fusions of pilM with T25 fragment of B. pertussis adenylate cyclase, respectively) and pSC61, pSC62 (C-and N-terminal fusions of pilM with T18 fragment of B. pertussis adenylate cyclase, respectively): pilM (opilM-pUT18fw/opilM-pUT18Crv for pSC73/pSC62 and opilM-pUT18fw/opilM-pUT18Crv for pSC74/pSC62) was amplified from genomic DNA of M. xanthus DK1622. Both fragments were digested with BamHI+EcoRI, cloned into pKT25, pKNT25, pUT18 and pUT18C vectors in proper combinations and sequenced.    (E) Immunoblot analysis of PilQ-sfGFP accumulation. Total cell lysates were loaded from the same number of cells per lane. Molecular size markers are indicated on the left. Calculated molecular mass of monomeric PilQ-sfGFP is indicated on the right and a molecular size marker on the left. All samples were analyzed on the same blot but lanes were removed for presentation purposes.

Figure S5. Localization of PilQ-sfGFP by epi-fluorescence microscopy.
Cells were treated, imaged and analyzed as in Fig. 3B. The data for WT are the same as in Fig. 7. N = 150 cells for each strain. Scale bars, 5 µm.

Figure S6. MglA does not detectably interact with PilB,-T or -M in a bacterial two hybrid assay.
Full-length MglA, MglB, PilB, PilT and PilM were fused to variants of the Bordetella pertussis adenylate cyclase and co-expressed as indicated in E. coli BTH101 (17). In constructs labelled 18-P and P-18 the fusions were created in the plasmids pUT18C and pUT18, respectively. In constructs labelled 25-P and P-25 the fusions were created in the plasmids pKT25 and pKNT25, respectively. In the positive control (+, upper right panel), pKT25-zip and pUT18C-zip were co-transformed. In the negative control (-, upper right panel), empty vectors encoding T25 and T18 fragments were cotransformed. In the lower panel, each MglA, MglB, PilB, PilT and PilM construct was coexpressed with pKT25-zip or pUT18C-zip to rule out possible false positive interactions. An interaction is indicated by a blue color and no interaction by a white color.  (18). Functional annotation of flanking genes is included. Together MXAN_5764 and MXAN_5763 are predicted to make up a TamA/TamB system for efficient secretion of autotransporters (18,19). Orientation of arrows indicates direction of transcription. Cross indicates that the corresponding gene is lacking. Two parallel, slanted lines indicate the presence of two unrelated genes in Vulgatibacter incomptus.