Asymmetry and Inequity in the Inheritance of a Bacterial Adhesive

Pseudomonas aeruginosa is an opportunistic human pathogen that forms biofilm infections in a wide variety of contexts. Biofilms initiate when bacteria attach to a surface, which triggers changes in gene expression leading to the biofilm phenotype.Wehave previously shown, for the P. aeruginosa lab strain PAO1, that the self-produced polymer Psl is the most dominant adhesive for attachment to the surface but that another self-produced polymer, Pel, controls the geometry of attachment of these rod-shaped bacteria—strains that make Psl but not Pel are permanently attached to the surface but adhere at only one end (tilting up off the surface), whereas wild-type bacteria that make both Psl and Pel are permanently attached and lie down flat with very little or no tilting (Cooley et al 2013 Soft Matter 9 3871–6). Here we show that the change in attachment geometry reflects a change in the distribution of Psl on the bacterial cell surface. Bacteria that make Psl and Pel have Psl evenly coating the surface, whereas bacteria that make only Psl have Psl concentrated at only one end.Weshow that Psl can act as an inheritable, epigenetic factor. Rod-shaped P. aeruginosa grows lengthwise and divides across the middle.Wefind that asymmetry in the distribution of Psl on a parent cell is reflected in asymmetry between siblings in their attachment to the surface. Thus, Pel not only promotes P. aeruginosa lying downWe thank Professor Matthew Parsek (University of Washington, Seattle) for his generous gift of bacterial PAO1 strains.Wealso thank Professor Marvin Whiteley (University of Texas at Austin) forWTandΔpsl polysaccharide preparations. SIM imaging (for figure 1) was performed in the Microscopy Core Facility within the Institute for Cellular and Molecular Biology atUTAustin, with the assistance of Julie Hayes. This work was funded by startup funds fromUTAustin and a gift from ExxonMobil to VDG, and by a grant from the Human Frontiers Science Program (HFSP RGY0081/2012-GORDON).Center for Nonlinear Dynamic

The Transcription Factor AmrZ Utilizes Multiple DNA Binding Modes to Recognize Activator and Repressor Sequences of Pseudomonas aeruginosa Virulence Genes

AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation

The extended N-terminus of AmrZ.

<p>The N-terminus of AmrZ is ordered only when in contact with the DNA (shown as wheat colored tubes). It forms a looped structure which is stabilized by Glu25 from chain B and Tyr11 from chain A. This region positions the side chains of two residues, Ser13 and Arg14 in the major groove of DNA. Ser13 forms hydrogen bonding contacts to the phosphate backbone, while Arg14 is not positioned to contact any nucleotides. This loop also interacts with the DNA binding residue Arg22 via the hydrogen bond between the backbone carbonyl of Ser13 and the εN of the arginine side chain.</p

Transcriptional activation of <i>algD</i> containing <i>amrZ1</i>-repressor binding sequence.

<p>Transcriptional activation of <i>algD</i> containing <i>amrZ1</i>-repressor binding sequence.</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

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 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

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

Crystallographic data collection and refinement statistics.

a<p>Rmerge = (Σ|I−<i>|)/ΣI, where I is the observed intensity and <i> is the average intensity.</i></i></p><i><i>b<p>Rfactor = Σ∥F_o|−|F_c∥/Σ|F_o|. Rfree is calculated with the same equation, but with 5% of reflections not used in the refinement.</p>c<p>Ramachandran statistics are given as the number of amino acids that lie within each region, and the percentage is given in parenthesis.</p><p>Values in parenthesis are for the outermost resolution shell (3.15 Å – 3.1 Å).</p></i></i