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, 190 (7), 2411-21

PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function in Myxococcus Xanthus

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PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function in Myxococcus Xanthus

Vladimir Jakovljevic et al. J Bacteriol.

Abstract

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.

Figures

FIG. 1.
FIG. 1.
Domain structure of secretion ATPases. (A) Scheme of domain structure of PulE, PilB, and PilT proteins. The conserved N-terminal region in PulE and PilB proteins, the N-terminal domain conserved in all secretion ATPases, and the C-terminal domain conserved in all secretion ATPases are indicated. Vertical gray bars in the C-terminal domain indicate the four conserved sequence motifs in secretion ATPases: Walker A box, Asp box, Walker B box, and His box. (B) Alignment of PilB and PilT of M. xanthus with secretion ATPases. PilB (PilBMyxa) and PilT (PilTMyxa) of M. xanthus were aligned with PilB of P. aeruginosa (PilBPsae), PilF (a PilB ortholog) of N. gonorrhoeae (PilFNego), EpsE of V. cholerae (EpsEVich), VirB11 of B. suis (VirB11Brsu), PilT of A. aeolicus (PilTAqae), and PilT of P. aeruginosa (PilTPsae). The conserved Walker A, Asp, Walker B, and His boxes are indicated. The conserved lysine in the Walker A box and the conserved glutamate in the Walker B box that were replaced in this study are indicated with asterisks. White-on-black residues are 100% conserved, white-on-gray residues are 80% conserved, and black-on-gray residues are 60% conserved. Note that the N-terminal extensions of PilB, EpsE, VirB11, and HP0525 are not included.
FIG. 2.
FIG. 2.
Subcellular localization of PilB and PilT in M. xanthus. (A) PilB is a cytoplasmic protein. Cells of the wild-type DK1622GFP were separated into fractions enriched for periplasmic, cytoplasmic, and membrane proteins. Following separation by SDS-PAGE, the fractions were subjected to immunoblot analysis with anti-PilB antibodies. As controls, total cell lysates of DK1622GFP and ΔpilB cells (DK10416) were included. Protein from 5 × 106 cells was loaded per lane. (B) PilT is a cytoplasmic protein. The experiment was done as described for panel A except that anti-PilT antibodies were used and the control included ΔpilT cells (DK10409).
FIG. 3.
FIG. 3.
PilB and PilT of M. xanthus are ATPases. (A) Purification of wild-type and mutant PilB proteins. E. coli JM109/pMS421 containing pSL5 was grown in the absence of IPTG (uninduced) and in the presence of 0.1 mM IPTG (induced). Soluble His6-PilB was purified by Ni2+ affinity chromatography as described in Materials and Methods. His6-PilB(K327A) and His6-PilB(E391A) proteins were purified using a similar procedure. After SDS-PAGE, proteins were stained with Coomassie brilliant blue. Molecular size markers, with sizes in kDa, are included on the left. (B) PilT purification. E. coli JM109/pMS421 containing pSL4 was grown in the absence of IPTG (uninduced) and in the presence of 0.1 mM IPTG (induced). His6-PilT was isolated from inclusion bodies and purified by Ni2+ affinity chromatography as described in Materials and Methods. His6-PilT(K137A) and His6-PilT(E205A) proteins were purified using a similar procedure. After SDS-PAGE, proteins were stained with Coomassie brilliant blue. Molecular size markers, with sizes in kDa, are included on the left. (C) Time dependence of the ATPase activities of purified His6-PilB, His6-PilB(K327A), and His6-PilB(E391A) proteins. His6-PilB proteins were incubated as described in Materials and Methods, and the A360 was followed over time and converted into Pi released. Diamonds, wild type His6-PilB; squares, His6-PilB(K327A); triangles, His6-PilB(E391A); crosses, incubation buffer. (D) ATPase activities of His6-PilT, His6-PilT(K137A), and His6-PilT(E205A) proteins, showing an autoradiogram of labeled adenosine phosphates after incubation of [α-32P]ATP with the indicated His6-PilT proteins or apyrase followed by TLC. The positions of [α-32P]ATP and [α-32P]ADP are indicated.
FIG. 4.
FIG. 4.
T4P-dependent motility of M. xanthus strains containing mutant pilB and pilT alleles. (A) T4P-dependent motility of the indicated strains on 0.5% agar plates. Cells were incubated for 24 h on 0.5% agar supplemented with 0.5% CTT. Strain names and relevant genotypes are indicated above each panel. Bar, 1 mm. (B) Accumulation of PilB and PilT wild-type and mutant proteins. Cells from exponentially growing cultures of the indicated strains were harvested, and total protein was separated by SDS-PAGE and analyzed by immunoblotting using anti-PilB (left panel) or anti-PilT (right panel) antibodies. Protein from 5 × 107 cells was loaded per lane.
FIG. 5.
FIG. 5.
Electron micrographs of M. xanthus strains. Cells from exponentially growing cultures of the indicated strains were directly transferred to a grid, stained with 2% (wt/vol) uranyl acetate, and visualized using transmission electron microscopy. Bar, 0.1 μm.

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