The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM

Abstract

Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery ofMyxococcus xanthus PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 ofPseudomonas aeruginosaformed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex.

Keywords: ATPase; Myxococcus xanthus; cell motility; membrane reconstitution; membrane transport; secretion; type IV pili.

FIGURE 1.

Expression, purification, and reconstitution of PilC. A, Coomassie-stained SDS-PAGE of membranes isolated from E. coli Rosetta 2 (DE3) strains expressing no PilC (control), His₆-YbeL-TEV-StrepII-PilC (β:PilC), and His₆-YbeL-TEV-StrepII-PilC-expressing membranes treated with TEV protease (StrepII-PilC). Representative figure is of at least 10 biological replicates. B, Coomassie-stained SDS-PAGE of purified His₆-YbeL-TEV-StrepII-PilC (β:PilC) and StrepII-PilC. Representative figure is of at least 10 biological replicates. C, Blue-Native PAGE analysis of purified His₆-YbeL-TEV-StrepII-PilC (β:PilC) and StrepII-PilC. Representative figure is of three biological replicates. Non-native (A and B) and native (C) molecular mass markers are indicated. D, reconstitution of purified StrepII-PilC was analyzed by sucrose density gradient centrifugation. Solubilized and reconstituted PilCs were loaded onto a sucrose step gradient. Samples from each layer were analyzed for the amount of phospholipid-derived phosphate (upper graph) and the presence of StrepII-PilC by immunoblot analyses using α-PilC antibody (lower lanes). The indicated error bars represent the average of two biological replicates containing two technical replicates. E, orientation of PilC in proteoliposomes was analyzed by incubation of PilC-containing proteoliposomes with (+) or without (−) 1 mg/ml proteinase K in the presence (+) and absence (−) of 0.5% n-decyl-β-d-maltopyranoside (DM), followed by Western blotting using an α-PilC antibody, directed against the 185 N-terminal cytoplasmic amino acids. Representative figure is of two biological replicates with two technical replicates.

FIGURE 2.

PilB:Hcp1 fusion forms hexamers and binds MANT-ATP in a cooperative manner. A, Coomassie-stained SDS-PAGE of cell lysates of E. coli BL21 Star expressing PilB:Hcp1 with (induced) and without (uninduced) induction with IPTG. PilB:Hcp1 was further purified from induced cells using nickel-affinity chromatography and size exclusion chromatography (purified). Representative figure is of at least 10 biological replicates. B, size exclusion chromatography of purified His₁₀-PilB:Hcp1. Elution volumes of molecular mass markers and the void volume are indicated. Representative figure is of at least 10 biological replicates. C, relative increase in fluorescence of increasing MANT-ATP concentrations upon addition of 10 μm PilB:Hcp1 (closed dots) or 10 μm PilB (open dots). Data were fitted to the Hill equation y = (axⁿ/(bⁿ + xⁿ)) resulting in PilB:Hcp1 (continuous curve) and PilB (dashed curve) in best fits with Hill coefficients (n) of 2.8 and 0.9, respectively. Representative figure of two biological replicates with three technical replicates. Standard deviation was calculated from the technical replicates. D, His₆-PilB (left panel) or His₁₀-PilB:Hcp1 (right panel) was incubated with either liposomes (−) or proteoliposomes containing PilC (+). After 20 min, samples were subjected to ultracentrifugation, and the pellet (P) and supernatant (S) fractions were analyzed by Western blotting using the antibodies indicated. Representative figure is of at least two biological replicates with two technical replicates. E, ATP hydrolysis activity of PilB:Hcp1 (200 ng/ml, circles) and PilB (150 ng/ml, triangles) was determined in the presence of increasing concentrations of detergent-solubilized StrepII-PilC (open circles and triangles) or equivalent concentrations of the detergent-containing buffer without StrepII-PilC (closed circles and triangles). F, similar as in E, but now PilB:Hcp1 (200 ng/ml, circles) and PilB (150 ng/ml, triangles) were incubated with increasing concentrations of StrepII-PilC reconstituted into liposomes (open circles and triangles) and empty liposomes (closed circles and triangles) as a control. E and F, representative figures of two biological replicates with three technical replicates. Standard deviation was calculated from three technical replicates.

FIGURE 3.

PilM-N(1–16) binds to PilB but not to PilC. A, Coomassie-stained SDS-PAGE of cell lysates of E. coli BL21 Star expressing PilM-N(1–16) with (induced) and without (uninduced) induction with IPTG. PilM-N(1–16) was further purified from induced cells using nickel-affinity chromatography and size exclusion chromatography (purified). Representative figure is of five biological replicates. B, size exclusion chromatography of purified PilM-N(1–16). Elution volumes of molecular mass markers and the void volume are indicated. Representative figure is of five biological replicates. C, fluorescence of 1 μm TNP-ATP was determined as a function of increasing concentrations of PilM-N(1–16). The wavelength of the maximum emission (closed dots) and the relative fluorescence increase at 545 nm (open dots) are depicted. The data for the relative fluorescence increase was fitted to the Hill equation y = (axⁿ/(bⁿ + xⁿ)) resulting in a Hill coefficient (n) of 1.06 (dashed line). Representative figure is of two biological replicates with three technical replicates. Standard deviation was calculated from the technical replicates. D, relative fluorescence emission at 445 nm of 1 μm TNP-ATP and 1.2 μm PilM-N(1–16) was determined in the presence of increasing concentrations of ATP. Standard deviation was calculated from three independent experiments. The curve was fitted to a sigmoidal dose-response curve F = 1/(1 + 10̂(log(IC₅₀)-[ATP])). Representative figure is of two biological replicates with three technical replicates. Standard deviation was calculated from the technical replicates. E, PilM-N(1–16) was incubated with either liposomes (−) or proteoliposomes containing PilC (+). After 20 min, samples were subjected to ultracentrifugation, and the pellet (P) and supernatant (S) fractions were analyzed by Western blotting using the antibodies indicated. Representative figure is of two biological replicates with three technical replicates. F, tagless PilM-N(1–16) was either expressed alone (left panels) or together with His₁₀-tagged PilB:Hcp1 (right panels). Cell lysates were loaded onto nickel-immobilized metal affinity chromatography tips (load, L), washed (wash, W) and eluted with 500 mm imidazole (elution, E). Fractions were analyzed by immunoblotting using antibodies indicated. Representative figure is of two biological replicates with three technical replicates.

FIGURE 4.

Interaction between PilB, PilM-N(1–16), and PilC. PilM-N(1–16) was incubated with PilB (left panel) or PilB:Hcp1 (right panel) with either liposomes (−) or proteoliposomes containing PilC (+). After 20 min, the sample was subjected to ultracentrifugation, and pellet (P) and supernatant (S) were analyzed by Western blotting using the antibodies indicated. Representative figure is of two biological replicates with three technical replicates.