Histidine-containing phosphotransfer protein-B (HptB) regulates swarming motility through partner-switching system in Pseudomonas aeruginosa PAO1 strain

Abstract

The histidine-containing phosphotransfer protein-B (HptB; PA3345) is an intermediate protein involved in transferring a phosphoryl group from multiple sensor kinases to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. The objective of this study was to elucidate the biological significance of the HptB-PA3346 interaction and the regulatory mechanisms thereafter. The transcription profiling analysis of an hptB knock-out mutant showed that the expression of a number of motility-related genes was altered consistent with the non-swarming phenotype observed for the mutant. Domain analysis indicated that the PA3346 C-terminal region (PA3346C) exhibits ∼30% identity with the anti-σ factor SpoIIAB of Bacillus subtilis. The presence of Ser/Thr protein kinase activity targeting an anti-σ antagonist, PA3347, at Ser-56 was confirmed in PA3346C using an in vitro phosphorelay assay. Furthermore, PA3346C and the anti-σ(28) factor FlgM were found to interact with PA3347 individually both in vivo and in vitro. FlgM displaced PA3346C in binding of PA3347 and was then competitively displaced by σ(28) from the PA3347-FlgM complex, forming a phosphorylation-dependent partner-switching system. The significance of PA3347 phosphorylation in linking the partner-switching system and swarming motility was established by analyzing the swarming phenotype of the PA3347 knock-out mutant and its complement strains.

FIGURE 1.

Amino acid sequence alignment of PA3346C with other Ser/Thr protein kinases. PA3346C (from Leu-408 to Ala-571) shows a 65% similarity and 26% identity to RsbW of B. subtilis, a 50% similarity and 28% identity to BtrW of B. bronchiseptica, and a 48% similarity and 32% identity to SpoIIAB of B. subtilis. Motifs labeled N, G1, and G2, which are commonly found in histidine kinases/ATPases, are indicated above the sequences. Amino acid residues with strong similarity are only considered in consensus sequences and are shaded in gray. The identical residues are shaded in black.

FIGURE 2.

PA3346C possesses divalent cation-dependent Ser/Thr protein kinase activity. A, His₆-PA3346C was incubated with [γ-³²P]ATP in the presence of either GST-PA3347 or GST-PA3347-S56A. B, His₆-PA3346C and GST-PA3347 were incubated with [γ-³²P]ATP in the presence of different divalent cations. The proteins were resolved using SDS-PAGE and subjected to autoradiography. ddH₂O, double distilled H₂O.

FIGURE 3.

Interaction of PA3347 with PA3346C and FlgM determined by GST pulldown assay. A, PA3346C and PA3347 interact in vitro. The interaction of His₆-PA3346C with GST-PA3347 either in the presence or absence of ADP was determined by Western blot analysis. A monoclonal anti-His antibody was used to detect His₆-PA3346C in the eluted fraction of the GST pulldown assay. W, PBS wash fraction; E, fraction eluted with 20 mm glutathione. B, PA3347 and FlgM interact in vitro. The interaction between PA3347 and FlgM was monitored using affinity chromatography. Glutathione-Sepharose beads were incubated with affinity-purified GST-PA3347 and GST-PA3347-S56A with His₆-FlgM in a GST pulldown assay. GST was incubated with His₆-FlgM as a negative control. Lane 1, load amount of His₆-FlgM; lane 2, markers; lane 3, wash fraction from the negative control (GST and FlgM proteins); lane 4, eluted fraction from the negative control (GST and FlgM); lane 5, wash fraction from GST-PA3347 and His₆-FlgM; lane 6, eluted fraction from GST-PA3347 and His₆-FlgM; lane 7, wash fraction from GST-PA3347-S56A and His₆-FlgM; lane 8, eluted fraction from GST-PA3347-S56A and His₆-FlgM. Proteins were visualized on the gel using Coomassie Blue staining.

FIGURE 4.

Interaction of PA3347 with FlgM and PA3346C analyzed by bimolecular fluorescence complementation assay. Bright field (left) and fluorescence (right) images of isopropyl β-d-thiogalactopyranoside- and arabinose-induced E. coli BL21(DE3) cells harboring the indicated plasmids taken at 600× magnification are shown. Cells co-expressing the leucine zipper peptide (positive control) displayed bright fluorescence emission, whereas no fluorescence was detected in the negative control cells. Fluorescence was detected in E. coli pNBIFC3347 + pCBIFC3351, E. coli pNBIFC3347M + pCBIFC3351, and E. coli pNBIFC3347 + pHLBIFC46, indicating a positive interaction in the following pairs: PA3347 and FlgM, PA3347-S56A and FlgM, and PA3347 and PA3346C. Scale bar, 15 μm.

FIGURE 5.

PA3347 interacts with FlgM and PA3346C. A, interaction between PA3347 and FlgM. Lane 1, molecular mass markers; lane 2, P. aeruginosa lysate; lane 3, His₆-FlgM; lane 4, P. aeruginosa lysate incubated with His₆-FlgM; lane 5, E. coli lysate expressing GST-PA3347-S56A; lane 6, His₆-FlgM; lane 7, E. coli lysate expressing GST-PA3347-S56A incubated with His₆-FlgM. B, interaction between PA3347 and PA3346C. Lane 1, molecular mass markers; lane 2, P. aeruginosa lysate; lane 3, His₆-PA3346C; lane 4, P. aeruginosa lysate incubated with His₆-PA3346C; lane 5, E. coli lysate expressing GST-PA3347-S56A; lane 6, His₆-PA3346C; lane 7, E. coli lysate expressing GST-PA3347-S56A incubated with His₆-PA3346C. IP, immunoprecipitation; WB, Western blot.

FIGURE 6.

FliA shows antagonist activity toward PA3347 in FlgM-PA3347 complex. The competitive binding between His₆-FliA and His₆-PA3347 for immobilized GST-FlgM was carried out. The pulldown of GST-FlgM (30 μg/ml) was conducted in the presence of His₆-PA3347 (30 μg/ml) and increasing concentrations of His₆-FliA. Lane 1, markers; lanes 2–4, a constant concentration of GST-FlgM bound to His₆-PA3347 was incubated with increasing amounts of His₆-FliA (0, 20, 100 μg/ml, respectively). The proteins were resolved on an SDS-polyacrylamide gel and stained with Coomassie Blue.

FIGURE 7.

Effect of PA3347 phosphorylation on binding to PA3346 and FlgM. A, pulldown of GST-PA3347 (25 μg/ml) was performed in the presence of a constant concentration of His₆-PA3346C (30 μg/ml) and increasing concentrations of His₆-FlgM. Lane 1, markers; lane 2, the fraction eluted from the GST protein-immobilized column that was incubated with His₆-FlgM and His₆-PA3346C (negative control); lanes 3–5, His₆-PA3346C (30 μg/ml) bound to GST-PA3347 was incubated with increasing amounts of His₆-FlgM (0, 15, and 30 μg/ml, respectively) in the presence of 2 mm ATP and 2 mm MgSO₄; lanes 6–8, same as lanes 3–5 except 2 mm ADP replaced ATP. B, pulldown of GST-PA3347 (25 μg/ml) was performed in the presence of His₆-FlgM (30 μg/ml) and increasing concentrations of His₆-PA3346C. Lane 1, molecular size markers; lane 2, eluted fraction from the GST protein-immobilized column that was incubated with His₆-FlgM and His₆-PA3346C (negative control); lanes 3–5, a constant concentration of His₆-FlgM bound to GST-PA3347 was incubated with increasing amounts of His₆-PA3346C (0, 10, and 20 μg/ml, respectively) in the presence of 2 mm ATP and 2 mm MgSO₄; lanes 6–8, same as lanes 3–5 except 2 mm ADP replaced ATP. The proteins were resolved on an SDS-polyacrylamide gel and stained with Coomassie Blue.

FIGURE 8.

Effects of amino acid substitution at Ser-56 of PA3347 on P. aeruginosa swarming. Shown is the swarming motility of isogenic deletion mutants of PA3347 (A), hptB (B), and PA3346 (C) compared with the strains complemented with pMMB47, pMMB47S56A, and pMMB47S56D and the empty vector pMMB66EH in PAO1.

FIGURE 9.

Proposed model for PA3346–3347-FlgM partner-switching mechanism that regulates P. aeruginosa PAO1 swarming. The N-terminal region of PA3346 can dephosphorylate PA3347. PA3347 binds FlgM, an anti-σ factor known to negatively regulate σ²⁸ activity and affect flagellum structural biogenesis class III gene transcription. The C-terminal domain of PA3346, which is also known to act as a serine kinase, can phosphorylate PA3347, an anti-σ antagonist factor. The absence of PA3347 or the phosphorylation state of PA3347 can activate the PA3346 C terminus (shown as the HATP domain of PA3346 in the figure) to act as an anti-σ factor and inactivate the yet to be identified σ factor in a partner-switching mechanism. REC, receiver domain; PP2C, protein phosphatase 2C domain; HATP, histidine kinase/ATPase domain.