The Pseudomonas aeruginosa ribbon-helix-helix DNA-binding protein AlgZ (AmrZ) controls twitching motility and biogenesis of type IV pili

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content, P. aeruginosa utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an algZ deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type P. aeruginosa and P. aeruginosa with the algZ deletion, the amount of PilA on the surface of the cells was drastically reduced in the algZ mutant. Analysis of algZ and algD mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that algZ (PA3385) be designated amrZ (alginate and motility regulator Z).

FIG. 1.

TM analysis of amrZ and algD strains. A thin 1% agar plate containing LA with no sodium chloride and tetrazolium red without (A) or with (B) 2% arabinose supplementation was stab inoculated to the bottom of the plate and incubated for 24 to 48 h at 37°C. The central growth of the colony on top of the agar is seen in all cases, while TM is shown as a lighter circle surrounding the colony. The following strains were analyzed: strain 1, WFPA205 (amrZ deletion); strain 2, PAO1 (wild type, TM⁺); strain 3, WFPA203 (amrZ deletion with an arabinose-inducible amrZ at attB). (C) TM analysis of algD and amrZ strains performed as described above. The following strains were analyzed: strain 1, WFPA1 (algD deletion); strain 2, WFPA205 (amrZ deletion).

FIG. 2.

TEM analysis of P. aeruginosa strains. Bacteria were harvested from plates cultured overnight, negatively stained using 2% uracyl acetate, and visualized using a Philips TEM 400. Strains analyzed were (A) PAO1 (wild type), (B) AWO (pilA deletion), and (C) WFPA205 (amrZ deletion).

FIG. 3.

Analysis of PilA production and localization via Western blotting. Whole-cell extracts (A) with identical samples in a stained-gel control (C) and sheared-surface TFP (B) were visualized via Western blotting with enhanced chemiluminescence (ECL) detection. The primary antipilin antibody was a gift from Randall Irvin and was used at a 1:5,000 dilution. Antiflagellin antibody at 1:20,000 was used as a loading control for the surface samples (D) with detection via ECL reagents. The following strains were analyzed: strain 1, PAO1 (wild type); strain 2, AWO (pilA deletion); strain 3, WFPA205 (amrZ deletion); strain 4, WFPA203 (amrZ deletion with an arabinose-inducible amrZ at attB) without arabinose supplementation; strain 5, WFPA203 with 3% arabinose supplementation.

FIG. 4.

Amino acid residues K18 and R22 are absolutely required for binding of AmrZ to the algD promoter. (A) Amino acid alignment between Arc (residues 1 to 53) and AmrZ (residues 10 to 62). Amino acid residues that correspond to the beta-strand (Arc residues 9 to 13), alpha helix A (Arc residues 16 to 28), or alpha helix B (Arc residues 33 to 47) are indicated in bold. Amino acids conserved between the two proteins are underlined. Numerical references for a portion of the amino acids are listed above their corresponding residues. (B to E) Electrophoretic mobility shift assays with the following amounts of the wild-type (B), K18A (C), V20A (D), or R22A (E) N-terminal domain (NTD) six-His-tagged AmrZ incubated with end-labeled DNA containing the AmrZ-binding site at algD: lane 1, 1.22 pmol; lane 2, 2.44 pmol; lane 3, 3.66 pmol; lane 4, 4.88 pmol; lane 5, 6.10 pmol; lane 6, 12.2 pmol; lane 7, 24.4 pmol; lane 8, 36.6 pmol; lane 9, 48.8 pmol.

FIG. 5.

TM analysis of amrZ alleles with a mutation in the DNA-binding domain. Strains analyzed were PAO1 (wild type), WFPA510 (amrZ deletion complemented with WT amrZ), WFPA511 (amrZ K18A), WFPA512 (amrZ V20A), WFPA513 (amrZ R22A), and WFPA205 (amrZ deletion).

FIG. 6.

TEM analysis of P. aeruginosa strains containing DNA-binding-domain mutations. Bacteria were harvested from plates cultured overnight, negatively stained using 2% uracyl acetate, and visualized using a Philips TEM 400. Strains analyzed were all PAO1 derivatives: (A) WFPA510 (complemented strain, amrZΔ amrZ⁺), (B) WFPA511 (amrZ K18A), (C) WFPA512 (amrZ V20A), and (D) WFPA513 (amrZ R22A).