The AlgT-dependent transcriptional regulator AmrZ (AlgZ) inhibits flagellum biosynthesis in mucoid, nonmotile Pseudomonas aeruginosa cystic fibrosis isolates

Abstract

Pseudomonas aeruginosa is a microorganism associated with the disease cystic fibrosis. While environmental P. aeruginosa strains are generally nonmucoid and motile, isolates recovered from the cystic fibrosis lung frequently display a mucoid, nonmotile phenotype. This phenotypic conversion is mediated by the alternative sigma factor AlgT. Previous work has shown that repression of fleQ by AlgT accounts for the loss of flagellum biosynthesis in these strains. Here, we elucidate the mechanism involved in the AlgT-mediated control of fleQ. Electrophoretic mobility shift assays using purified AlgT and extracts derived from isogenic AlgT(+) and AlgT(-) strains revealed that AlgT inhibits fleQ indirectly. We observed that the AlgT-dependent transcriptional regulator AmrZ interacts directly with the fleQ promoter. To determine whether AmrZ functions as a repressor of fleQ, we mutated amrZ in the mucoid, nonmotile P. aeruginosa strain FRD1. Unlike the parental strain, the amrZ mutant was nonmucoid and motile. Complementation of the mutant with amrZ restored the mucoid, nonmotile phenotype. Thus, our data show that AlgT inhibits flagellum biosynthesis in mucoid, nonmotile P. aeruginosa cystic fibrosis isolates by promoting expression of AmrZ, which subsequently represses fleQ. Since fleQ directly or indirectly controls the expression of almost all flagellar genes, its repression ultimately leads to the loss of flagellum biosynthesis.

FIG. 1.

AlgT represses fleQ indirectly. (A) Autoradiograph of a radiolabeled 250-bp fleQ promoter fragment which was incubated with protein extracts derived from isogenic AlgT⁺ or AlgT⁻ P. aeruginosa or with purified AlgT-His₆ and separated by polyacrylamide gel electrophoresis under nondenaturing conditions. Lane 1, free fleQ promoter DNA; lane 2, AlgT⁺ extract (3 μg); lane 3, AlgT⁻ extract (3 μg); lanes 4 to 9, increasing amounts of AlgT-His₆ (25, 50, 100, 250, 500, and 750 ng, respectively). (B) Binding of extracts from AlgT⁺ to the fleQ promoter. Lane 1, free fleQ promoter DNA; lanes 2 to 9, increasing amounts of AlgT⁺ extract (50, 100, 250, 500, 750, 1,500, 3,000, and 5,000 ng, respectively). (C) Binding of protein in extracts from AlgT⁺ P. aeruginosa to the fleQ promoter is conserved among mucoid, nonmotile CF isolates. Lane 1, free fleQ promoter DNA; lanes 2, 4, and 6, extracts (3 μg) derived from mucoid, nonmotile AlgT⁺ P. aeruginosa CF isolates; lanes 3, 5, and 7, extracts (3 μg) derived from the corresponding isogenic algT mutants.

FIG. 2.

AmrZ binds to the fleQ promoter. (A) Mutation of amrZ in AlgT⁺ P. aeruginosa abolishes fleQ binding activity. Lane 1, free fleQ promoter DNA; lane 2, AlgT⁺ extract (3 μg); lane 3, AlgT⁻ extract (3 μg); lane 4, AlgT⁺ AlgB⁻ extract (3 μg); lane 5, AlgT⁺ AlgR⁻ extract (3 μg); lane 6, AlgT⁺ AmrZ⁻ extract (3 μg). (B) Complementation of the amrZ mutant restores binding to fleQ promoter DNA. Lane 1, free fleQ promoter DNA; lane 2, AlgT⁺ extract (3 μg); lane 3, AlgT⁺ AmrZ⁻ extract (3 μg); lane 4, extract derived from an AlgT⁺ AmrZ⁻ strain complemented with amrZ (3 μg). (C) AmrZ binds to the fleQ promoter. Lane 1, free fleQ promoter DNA; lane 2, AlgT⁺ extract (3 μg); lane 3, AlgT⁺ AmrZ⁻ extract (3 μg); lanes 4 to 9, increasing amounts of recombinant AmrZ (1, 5, 10, 25, 50, and 100 ng, respectively). (D) Mutation of critical residues abolishes the ability of AmrZ to bind to fleQ. Lane 1, free fleQ promoter DNA; lanes 2 to 4, increasing amounts of recombinant AmrZ (20, 40, and 60 ng, respectively); lanes 5 to 7, increasing amounts of recombinant AmrZ K18A (20, 40, and 60 ng, respectively); lanes 8 to 10, increasing amounts of recombinant AmrZ R22A (20, 40, and 60 ng, respectively).

FIG. 3.

AmrZ inhibits flagellum biosynthesis. (A) fleQ::lacZ fusions were integrated into the chromosomes of mucoid, nonmotile P. aeruginosa CF isolate FRD1 (open bar) and its isogenic amrZ mutant (hatched bar) at the neutral attB site. Promoter activity was measured by β-galactosidase assays with ONPG (o-nitrophenyl-β-d-galactopyranoside) used as a substrate and is expressed as the amount of ONPG hydrolyzed per minute as a function of cell density. Shown are the averages of four independent experiments and standard deviations. (B) Whole-cell lysates of the indicated strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined for AmrZ and flagellin expression by Western blotting with AmrZ (top) and flagellin B (bottom) antiserum. Lane 1, AlgT⁺ P. aeruginosa; lane 2, AlgT⁻ P. aeruginosa; lane 3, AlgT⁺ AmrZ⁻ P. aeruginosa; lane 4, AlgT⁺ AmrZ⁻ P. aeruginosa complemented with amrZ. As a loading control, a second gel containing comparable amounts of total protein was simultaneously prepared and processed by Coomassie staining. (C) TEM of nonflagellated AlgT⁺ P. aeruginosa (I) and its isogenic, flagellated amrZ mutant (II). Magnification, ×15,600.

FIG. 4.

Proposed model for AlgT-mediated inverse regulation of flagellum synthesis and alginate production in mucoid, nonmotile P. aeruginosa CF isolates. Under most physiological conditions, the activity of the alternative sigma factor AlgT is inhibited by the anti-sigma factor MucA. Unique environmental conditions in CF airways result in mutations of mucA and, subsequently, deregulation of AlgT. AlgT is now free to up-regulate expression of the ribbon-helix-helix-protein AmrZ, which has dual functions as a repressor of fleQ and an activator of algD.