Identification of the AmrZ regulon from the TM-active “edge” sub-population.

<p>(A.) Venn diagram of the AmrZ regulon under the TM-promoting condition. 112 genes in the red box were included in the AmrZ regulon as they displayed differential expression between both <i>amrZ</i> mutants (Δ<i>amrZ</i> and AmrZV20A) and strains encoding WT AmrZ (PAO1 and the complemented strain Δ<i>amrZ</i>/<i>amrZ</i>). The other three numbers indicate the total number of genes that exhibited distinct expression from PAO1, respectively. (B.) Representative virulence pathways regulated by AmrZ. Red arrows indicate activation, while green bars represent repression.</p

Cellophane-air interface stimulates <i>P</i>. <i>aeruginosa</i> twitching motility.

<p>(A.) Illustration of the cellophane-based TM-promoting condition. (B.) Quantification of <i>pilA</i> transcript levels from indicated growth conditions via qRT-PCR. “Broth” refers to overnight broth culture (stationary phase). Transcript levels of <i>pilA</i> were normalized to those isolated from single colonies on agar surfaces (“agar”) with <i>rpoD</i> as the reference gene. Unpaired two-tailed student t-test was used for statistical analysis of three independent experiments. *: P<0.05; **: P<0.01; ***: P<0.001.</p

TM-proficient and TM-deficient strains retained their TM phenotypes on cellophane.

<p>(A.) TM measurement of four <i>P</i>. <i>aeruginosa</i> strains by subsurface twitching assays. Scale bar: 5 mm. (B.) Phase contrast microscopy of edge sub-populations of the same strains above cellophane sheets. Dashed lines illustrated tendril-like structures. Scale bar: 100 μm.</p

Development of a Novel Method for Analyzing <i>Pseudomonas aeruginosa</i> Twitching Motility and Its Application to Define the AmrZ Regulon

<div><p>Twitching motility is an important migration mechanism for the Gram-negative bacterium <i>Pseudomonas aeruginosa</i>. In the commonly used subsurface twitching assay, the sub-population of <i>P</i>. <i>aeruginosa</i> with active twitching motility is difficult to harvest for high-throughput studies. Here we describe the development of a novel method that allows efficient isolation of bacterial sub-populations conducting highly active twitching motility. The transcription factor AmrZ regulates multiple <i>P</i>. <i>aeruginosa</i> virulence factors including twitching motility, yet the mechanism of this activation remains unclear. We therefore set out to understand this mechanism by defining the AmrZ regulon using DNA microarrays in combination with the newly developed twitching motility method. We discovered 112 genes in the AmrZ regulon and many encode virulence factors. One gene of interest and the subsequent focus was <i>lecB</i>, which encodes a fucose-binding lectin. DNA binding assays revealed that AmrZ activates <i>lecB</i> transcription by directly binding to its promoter. The <i>lecB</i> gene was previously shown to be required for twitching motility in <i>P</i>. <i>aeruginosa</i> strain PAK; however, our <i>lecB</i> deletion had no effect on twitching motility in strain PAO1. Collectively, in this study a novel condition was developed for quantitative studies of twitching motility, under which the AmrZ regulon was defined.</p></div

The <i>lecB</i> gene is required for TM in <i>P</i>. <i>aeruginosa</i> strain PAK, but not in strain PAO1.

<p>(A.) TM of the parental strain PAK and its derivative <i>lecB</i>::Gm^R via subsurface twitching assays [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136426#pone.0136426.ref026" target="_blank">26</a>]. (B.) Subsurface twitching assays of strains in PAO1 background. In the Δ<i>amrZ</i> strain with the p<i>lecB</i> plasmid, 1% arabinose was added as the inducer. Scale bar: 5 mm.</p

Verification of representative AmrZ-dependent genes.

<p>Bacteria growth conditions and RNA isolations were the same as for microarray analyses. Gene expression in the Δ<i>amrZ</i> mutant was compared to the parental strain PAO1 by qRT-PCR. The horizontal dashed line represented normalized mRNA levels as seen in the parental strain PAO1. Unpaired two-tailed student t-test was used for statistical analysis of three independent experiments. *: P<0.05; **: P<0.01; ***: P<0.001.</p

Structural overview of AmrZ - <i>amrZ1</i> complex.

<p>(A) The Δ42 AmrZ protein binds to the 18 bp <i>amrZ1</i> binding site as a dimer of dimers. One dimer is composed of chains A and B (green/cyan), while the other dimer is composed of chains C and D (magenta/orange). (B) The superposition of AmrZ dimers show no major structural differences between them (Cα RMSD = 0.381 Å). (C) The dimer - dimer interface is created by a network of hydrogen bonds between the residues in the loop region between α-helix 1 and α-helix 2 of chains A and C. (D) Secondary structure representation of one AmrZ ribbon-helix-helix monomer. Residues forming hydrogen bonds to DNA are indicated by the purple triangles, while residues forming hydrophobic interactions to DNA are indicated by purple squares. Residues forming the dimer interface between each monomer are underlined in red, and the residues which form the dimer-dimer interface are overlined in red. (E) Schematic of both the sequence dependent and sequence independent interactions between AmrZ and <i>amrZ1</i>. Hydrogen bonding interactions to the DNA are illustrated with a short dashed line, while hydrophobic interactions are illustrated with a vertical dashed line. Nucleotides involved in sequence specific interactions are represented in orange. The peptide chain for each residue is labeled in parentheses, and residues that make contacts to more than one nucleotide are notated with an asterisk.</p

AmrZ binding to the <i>algD</i> activator site.

<p>(A) Alignment of the <i>amrZ1</i> and <i>algD</i> DNA sequences that have been derived from previous footprinting experiments <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002648#ppat.1002648-Baynham1" target="_blank">[6]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002648#ppat.1002648-Ramsey2" target="_blank">[9]</a>. The two binding half sites on the <i>amrZ1</i> sequence are boxed, while nucleotides that were mutated in the <i>algD</i> sequence are shown in red. (B) Results from the scanning mutagenesis of the <i>algD</i> site (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002648#ppat-1002648-t003" target="_blank">Table 3</a>). Only mutations of guanine nucleotides at positions 6 (forward strand), and 7 and 8 (reverse strand) resulted in a noticeable decrease in binding affinity of AmrZ compared to the WT <i>algD</i> sequence. Mutations to the right side binding half site resulted in no major decrease in binding affinity of AmrZ. Percent affinity was calculated by dividing the K_d for the WT <i>algD</i> binding site by the K_d for each mutant binding site.</p

AmrZ affinity for <i>amrZ1</i> binding site mutants.

a<p>The two AmrZ binding sites on the wild type <i>amrZ1</i> sequence are represented by the underlined nucleotides. Mutations to the wild type <i>amrZ1</i> binding site are notated by the bolded nucleotides in each mutant sequence.</p>b<p>The K_d was calculated by fitting the hyperbolic equation for a single ligand binding model with saturation (eq 2) to the data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002648#ppat.1002648.s002" target="_blank">Figure S2</a>, which were averaged from four independent experiments.</p>c<p>Fold over (wild type) WT <i>amrZ1</i> is defined by (K_d of sample)/(K_d of wild type) for each sample.</p

AmrZ affinity for <i>algD</i> binding site mutants.

a<p>Mutations to the wild type <i>algD</i> binding site are notated by the bolded nucleotides in each mutant sequence.</p>b<p>The K_d was calculated by fitting the hyperbolic equation for a single ligand binding model with saturation (eq 2) to the data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002648#ppat.1002648.s003" target="_blank">Figure S3</a>, which were averaged from four independent experiments.</p>c<p>Fold over (wild type) WT <i>algD</i> is defined by (K_d of sample)/(K_d of wild type) for each sample.</p