Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins

Type IV pili (T4P) are surface structures that undergo extension/retraction oscillations to generate cell motility. In Myxococcus xanthus, T4P are unipolarly localized and undergo pole-to-pole oscillations synchronously with cellular reversals. We investigated the mechanisms underlying these oscillations. We show that several T4P proteins localize symmetrically in clusters at both cell poles between reversals, and these clusters remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction, respectively, localize to opposite poles with PilB predominantly at the piliated and PilT predominantly at the non-piliated pole, and these proteins oscillate between the poles during reversals. Therefore, T4P pole-to-pole oscillations involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. Fluorescence recovery after photobleaching experiments showed rapid turnover of YFP–PilT in the polar clusters between reversals. Moreover, PilT displays bursts of accumulation at the piliated pole between reversals. These observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. This is the first demonstration that the function of a molecular machine depends on disassembly and reassembly of its individual parts.


Theoretical model for dynamic PilT localization
As shown in the main text, diffusion from pole to pole is expected to take ~7s for a PilT monomer and ~12s for a PilT hexamer. On the other hand, as can be seen from Fig. 7 it takes 50±10s until the intensity of the bleached pole reaches the new plateau value. This suggests that the recovery is not limited by diffusion of PilT in the cytoplasm. Based on this assumption we have developed a theoretical model for the dynamics of PilT in the FRAP experiments. In our model, PilT can bind to either the leading or the lagging pole.
Then, the time-dependence of the concentration of bound protein at the lagging pole (c l ), the concentration of bound protein at the leading pole (c r ) and the concentration of protein in the cytoplasm (c f ) obey the following rate equations: . _ _ ) ( The steady state value of f c is given by i.e. the total amount of bound protein (bound either at the leading or lagging pole) is constant in time as was observed experimentally (Fig. 7BC).
In a typical FRAP experiment one of the poles, say the lagging pole, is bleached giving rise to a concentration Here, It is important to note that the dynamics by which the PilT concentrations at both the bleached and the non-bleached pole reach their new steady state values is characterized by the same rate constant α .
The constant β can be read off from the experimental new steady state fluorescent protein concentrations at the two poles Similarly, α can be determined from the experimental data by plotting the timedependent approach to the new steady state values We used the mathematical model to determine the values of α and β from the FRAP data. α is determined from Eqs. (12) and (13) and β can be determined from Eqs. (10) and (11). By analyzing FRAP data as those shown in Fig. 7 for 10 cells, we find that The mean waiting time for binding of PilT is given by where Eq. (6) has been used. For the ratio between on and off rates one has where Eqs.
(2) and (9) have been used. The ratio between the concentrations of free and bound PilT is found to be c b /(c b +c f )=0.7±0.2 (where the polar regions occupy 20% of the cellular volume). This is in good agreement with the immuno-fluorescence data which yields c b /(c b +c f )=0.58±0.08. With these immuno-fluorescence data the mean waiting time for binding of PilT becomes of the order of 80s. Dissociation of PilT from the poles is even slower than association. Since the concentration of PilT is larger at the poles than in the cytoplasm one has r -<r + and l -<l + and therefore and a degradation product of PilQ (light grey arrow), respectively (Nudleman et al., 2006). Migration of molecular size markers is indicated on the left.  (C) YFP-PilM complements the motility defect in a ∆pilM mutant. Cells were incubated at 32˚ for 24h on 0.5% agar supplemented with 0.5% CTT, and visualized with a Leica MZ8 stereomicroscope. Scale bar: 5 mm. We analyzed the strains for T4P-dependent motility on 0.5% agar, which favors T4P-dependent motility. The wild type DK1622 formed colonies with large rafts of cells at the edge typical of T4P-dependent motility whereas SA3002 (∆pilM) did not form rafts at the edge. However, SA3046 (∆pilM/pilM-yfp) displayed a motility phenotype similar to that of the wild type.

Supplementary Experimental Procedures
Construction of plasmids and strains. Plasmids were propagated in E. coli TOP10 (F -, mcrA, ∆(mrr-hsdRMS-mcrBC), ϕ80lacZ∆M15, ∆lacX74, deoR, recA1, araD139, ∆(araleu)7679, galU, galK, rpsL, endA1, nupG) unless otherwise stated. Primers used are listed in Table S1 in Supplemental data. All DNA fragments generated by PCR were verified by sequencing. DK1622 was used as the wild type throughout. All strains constructed were confirmed by PCR. The in-frame deletion of pilM (SA3002) was generated as described (Shi et al., 2008) using the primers oPilM-ABCD (A list of all primers used in this work is included in Table S1) after cloning of the appropriate constructs in pBJ114.
To construct pIB75, which contains PpilA-yfp-pilT, the pilT gene was amplified using the primers opilT-4 and opilT-7 and pSL107 as a template giving rise to the full-length pilT with an additional 10 aa linker at the 5'-end (S. Leonardy, pers. communication). The PCR product was digested with EcoRI and HindIII, and cloned in in pBluescript II SKgenerating pIB71. The yfp gene was amplified by PCR using the primers oYFP-1 and oYFP-2 and pSW105-YFP as a template (V. Jakovljevic, pers. communication). The PCR product was digested with SpeI and EcoRI, and cloned in-frame with pilT in pIB71 generating pIB72. The SpeI-HindIII fragment from pIB72 was then re-cloned into pSW105, which contains the pilA promoter giving rise to pIB73. Finally, the NdeI-HindIII fragment of pIB73 containing PpilA-yfp-pilT was cloned into pSWU30, generating pIB75.
To construct pIB74, which contains PpilA-yfp-pilT E205A , plasmid DNA of pSL4TWalkerB (Jakovljevic et al., 2008) was digested with XbaI and HindIII. The resulting fragment, which contained pilT with the E205A substitution, was cloned into pIB75 instead of the wild-type pilT gene, generating pIB74.
To construct pCS8, which contains a PpilA-yfp-pilM construct, M. xanthus chromosomal DNA was amplified with the primers opilM-3 and opilM-4 giving rise to a pilM gene extending from position +1 to +1188. The PCR product was digested with XbaI and HindIII and cloned into pIB75 instead of pilT gene generating pSC8.
Plasmids containing yfp-pilT and yfp-pilM alleles were integrated by site-specific recombination at the Mx8 attB site on the chromosome. Strains containing plasmids integrated at the attB site were constructed by electroporation of the plasmid into the relevant strain (Kashefi and Hartzell, 1995) followed by selection to the relevant antibiotic. All strains were verified by PCR.
To construct pPilC-CD1, which encodes a His6-tagged truncated PilC protein extending from residue 1-185, a pilC PCR fragment generated with the primers oPilC1 and oPilC5 (covering the N-terminal bases 1 to 556 of pilC) was digested with BamHI and EcoRI and cloned into pET-24b+ vector (Novagen).
To construct pCS3, which encodes His6-tagged full-length PilM, a full-length pilM PCR product was generated using the primers oPilM-1(with an EcoRI site followed by six histidine codons and the first 18 nucleotides of pilM) and oPilM-2 (containing the last 21 nucleotides of pilM and a HindIII site) and cloned into pBluescript II SK-giving rise to pIB35. The EcoRI-HindIII fragment was recloned into the pUHE24-2 expression vector (Lanzer and Bujard, 1988) to give pCS3.
Antibody generation and immunoblots. pPilC-CD1 and pIB49 were propagated in E. coli
Cell fractionation. Biochemical fractionation of cells was done as described (Lobedanz and Søgaard-Andersen, 2003). Briefly, cells were grown in CTT. To separate inner membrane and outer membrane proteins from soluble proteins (Lobedanz and Søgaard-Andersen, 2003), cells were resuspended in 50 mM Tris-HCl pH 7.6 supplemented with Complete Mini Protease Inhibitor Cocktail (Roche) (protease inhibitors) as recommended by the supplier (equivalent to 1x concentration of protease inhibitors) and lysed by sonication. Cell debris was removed by centrifugation. The supernatants were centrifuged at 45,000 g for 1 hr at 4°C. The resulting supernatants are enriched in soluble proteins. Pellets containing the crude envelope fractions were resuspended in 50 mM Tris-HCl pH 7.6, 2% Triton X-100 supplemented with protease inhibitors, and subjected to ultracentrifugation as described. The resulting supernatant is enriched in inner membrane proteins, and the pellet is enriched in outer membrane proteins. All fractions were analyzed by immunoblotting. As controls for proper fractionation, fractions were tested with antibodies against PilB in the cytoplasm (Jakovljevic et al., 2008) and PilQ in the outer membrane (Nudleman et al., 2006).
Transmission electron microscopy (TEM). TEM was used to visualize T4P as described (Jakovljevic et al., 2008). Significance was determined using the t test.