PilZ Domain Protein FlgZ Mediates Cyclic Di-GMP-Dependent Swarming Motility Control in Pseudomonas aeruginosa

Abstract

The second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. In Pseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation of P. aeruginosa's polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility by P. aeruginosa and support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator.

Importance: The regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility in P. aeruginosa remains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility by P. aeruginosa We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities.

FIG 1

FlgZ and Pel polysaccharide contribute to swarming motility repression. (A) Top panel: representative swarm plates of the indicated strains. Bottom panel: Western blot probed with anti-His antibody to detect FlgZ-His expression in the ΔbifA ΔpelA ΔflgZ::flgZ-His strain in which the flgZ gene deletion is complemented by allelic replacement, resulting in expression of a His epitope-tagged FlgZ protein. (B) Representative swarm plates of the indicated strains. (C) Representative swarm plates of the indicated strains. The values below the swarm plates indicate the percentages (means ± standard errors of the means [SEM] of the results determined in three independent experiments performed with six plates each) of plate surface coverage of the mutant strains relative to that of the WT strain (set at 100%). Significance was determined by analysis of variance and Dunnett's posttest comparison for differences relative to the WT. *, P < 0.05 (compared to WT).

FIG 2

Conserved residues in the c-di-GMP binding domain are important for FlgZ stability and function. (A) Multiple-sequence alignment of the predicted c-di-GMP binding region of FlgZ in P. aeruginosa (Pa) with orthologs from P. putida (Pp), P. fluorescens (Pf), S. enterica (Se), E. coli (Ec), and B. subtilis (Bs) along with other PilZ domain-containing proteins from C. crescentus (Cc) and V. cholerae (Vc). The sequence alignment was generated by Clustal Omega (59, 60) using the complete PilZ domain of each protein as predicted by SMART (61, 62). A portion of the alignment is shown here. Clustal Omega determined conservation of residues. ★, a fully conserved residue; :, a residue with strongly similar properties. The boxed conserved residues were targeted for site-directed mutagenesis. Numbers correspond to the amino acid residues in the P. aeruginosa FlgZ full-length protein. (B) Top panel: representative swarm assays of the indicated strains. Bottom panel: protein levels determined using Western blotting and anti-His antibody to detect expression of the wild-type strain and mutant FlgZ-His variants.

FIG 3

Detection of interaction between FlgZ and MotC by bacterial two-hybrid analysis. (A and B) Full-length flgZ (A) and flgZ (R140A) (B), as well as the flagellar motor genes motA, motC, fliG, and fliM, were cloned into vector pKNT25, pKT25, pUT18, or pUT18C and cotransformed into E. coli BTH101 cells. The coexpressed fusion protein combinations for each transformation are indicated on the left. The transformants were 10-fold serial diluted, spotted (2 μl) on LB agar containing Cb, Kan, X-Gal, and IPTG, and then incubated for 40 h at 30°C. Cells cotransformed with empty vectors served as negative controls, and cells cotransformed with leucine zipper vectors provided by the manufacturer (T18-zip and T25-zip) served as positive controls. The degradation of X-Gal (blue) indicates a positive protein-protein interaction. (C) Bacterial two-hybrid interactions were quantified by measuring β-galactosidase activity in transformants grown in LB broth supplemented with Cb and Kan overnight at 30°C. The data represent results of three independent experiments performed with three or four biological replicates each, and values are reported as means ± SEM. Significance was determined by analysis of variance and Dunnett's posttest comparison for differences relative to the negative control (T18 + T25). n.s., not significant; ***, P < 0.001 (the positive control [T18-zip + T25-zip] was not included in the statistical analysis).

FIG 4

Immunoprecipitation analysis to assess FlgZ and MotC interaction. Immunoprecipitations (Co-IP) with a nickel-chelating resin were performed with cell lysates expressing FlgZ-HA and MotA-His or MotC-His. Western blots of precipitate (top panel) and input (bottom panel) were probed with anti-HA. Immunoprecipitations were performed with and without 5 μM c-di-GMP (cdG), as indicated.

FIG 5

Localization of GFP-FlgZ is impacted by c-di-GMP levels and MotCD. (A) Representative images of WT, ΔbifA, and ΔmotCD strains expressing GFP-FlgZ. (B) For each strain, more than 2,000 bacteria from three independent experiments were analyzed, and values are reported as means ± SEM. Significance was determined by analysis of variance and Dunnett's posttest comparison for differences relative to WT. ***, P < 0.001.

FIG 6

MotCD overexpression restores c-di-GMP-inhibited swarming only in the absence of Pel. (A) Representative swarm plates of the indicated strains carrying either an empty vector or a MotCD-His-expressing plasmid (pMotCD-His). Swarm plates contained 0.2% arabinose. (B) Protein levels determined by the use of Western blotting and anti-His antibody to detect expression of pMotCD-His.