Inverse levels of alginate and Psl in mucoid and nonmucoid strains. Polysaccharides were extracted from 1.0 ml of a 1.0 OD₆₀₀ culture to normalize for cell number. Results are reported as micrograms of target polysaccharide per milliliter of OD₆₀₀ culture ± standard deviation. Alginate levels were determined via a carbazole assay (see Materials and Methods). Psl concentrations were determined via an ELISA and visualized with an immunoblot. Concentrations are the average of four independent assays performed in triplicate. P. aeruginosa PAO1 and FRD1 ΔalgT strains are nonmucoid; PAO1 mucA22 and FRD1 strains are their respective isogenic mucoid counterpart. Comparisons of polysaccharide production were determined via Student's t test. α-Psl, anti-Psl antibody.

Deletion of algT and amrZ leads to an increase of Psl in P. aeruginosa FRD1. Psl was extracted from 1.0 ml of a 1.0 OD₆₀₀ culture to normalize for cell number. Results are reported as μg of target polysaccharide per ml of OD₆₀₀ culture. All strains are derivatives of the mucoid clinical isolate FRD1. Psl concentrations are the averages of four independent assays performed in triplicate, with the standard deviations represented by error bars. A one-way analysis of variance (ANOVA) with Tukey post hoc test was used to determine statistical significance. All values are compared to the values for FRD1 (∗∗, P < 0.01; ∗∗∗, P < 0.001).

AmrZ correlates with inverse transcript levels of algD and pslA. (A) AmrZ Western blot of whole-cell lysates. Nonspecific cross-reactive band were included as a loading control. (B) Observation of pslA and algD mRNA levels via quantitative reverse transcription real-time PCR. All samples were normalized to the level of expression in the mucoid isolate FRD1. The graph represents mean expression of three independent experiments performed in triplicate. Error bars represent standard deviations. (C) A one-way ANOVA with Tukey post hoc test was used to determine statistical differences in . All comparisons are between the indicated strain and FRD1, and statistical significance of the differences is indicated as follows: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ns, not significant.

AmrZ binds to the psl promoter region. (A) Increasing amounts of purified AmrZ or AmrZ R22A were incubated with fluorescently labeled fragments of DNA overlapping the algD or psl promoter. Lanes: 1, free DNA; 2, 4.0 nM wild-type (WT) AmrZ; 3, 2.8 nM WT AmrZ; 4, 1.1 nM WT AmrZ; 5, 0.6 nM WT AmrZ; 6, 0.3 nM WT AmrZ; 7, 4.0 nM AmrZ R22A; 8, 0.3 nM AmrZ R22A. The position of free DNA is indicated by an arrow, while DNA bound by AmrZ is indicated by a bracket. (B) AmrZ binding fragments containing the psl promoter from 400 bp to 110 bp. Lanes contain either free DNA of the fragment size noted (−) or DNA of the size indicated and 4.0 nM WT AmrZ protein (+). (C) Sequence of the 200-bp region upstream of the pslA coding sequence. The ATG codon of pslA is located at bases 201 to 203. The putative AmrZ binding site is indicated in red (, ). Gray and black bars indicate the sequences of fragment 1 and fragment 2, respectively. These fragments were used in an EMSA to verify AmrZ binding. Irie et al. () identified the transcription start site (+1) at base 53. Overhage et al. () mapped an alternative transcription start site (+1) at base 160. Sequence alignment of the consensus AmrZ binding site (, ) to binding sites of fragment 1 (red sequence) and mutant fragment 2 of the pslA promoter is shown. DNA fragments used in and were produced utilizing 5′-FAM-labeled fragments, while those in utilized unlabeled fragments that were stained with the SYBR green EMSA stain.

Modulation of AmrZ and Psl production impacts biofilm structure. (A) Confocal scanning laser microscopy of 24-h biofilms grown in flow cell chambers. Top down images (top left of image) and orthogonal views (right side and bottom of image) allow visualization of biofilm thickness and structure. Red scale bars represent 20 μm. Green fluorescence is Syto9 stain to indicate biomass, while the red staining represents antibody detection of Psl. (B) Model of the AmrZ-mediated effect of Psl regulation on biofilms. Elevated AlgT leads to high AmrZ, which represses Psl. Biofilms formed by Psl-deficient bacteria are thin, flat, and devoid of tall microcolonies compared to wild-type biofilms.