AmrZ Modulates Pseudomonas aeruginosa Biofilm Architecture by Directly Repressing Transcription of thepslOperon

Abstract

e Pseudomonas aeruginosa strains recovered from chronic pulmonary infections in cystic fibrosis patients are frequently mucoid. Such strains express elevated levels of alginate but reduced levels of the aggregative polysaccharide Psl; however, the mechanistic basis for this regulation is not completely understood. Elevated pslA expression was observed in an amrZ null mutant and in strains expressing a DNA-binding-deficient AmrZ. AmrZ is a transcription factor that positively regulates twitching motility and alginate synthesis, two phenotypes involved in P. aeruginosa biofilm development. AmrZ bound directly to the pslA promoter in vitro, and molecular analyses indicate that AmrZ represses psl expression by binding to a site overlapping the promoter. Altered expression of amrZ in nonmucoid strains impacted biofilm structure and architecture, as structured microcolonies were observed with low AmrZ production and flat biofilms with amrZ overexpression. These biofilm phenotypes correlated with Psl levels, since we observed elevated Psl production in amrZ mutants and lower Psl production in amrZ-overexpressing strains. These observations support the hypothesis that AmrZ is a multifunctional regulator mediating transition of P. aeruginosa biofilm infections from colonizing to chronic biofilms through repression of the psl operon while activating the algD operon.

Which Bacterial Biofilm Exopolysaccharide Is Preferred, Psl or Alginate?

“…It is likely that these three proteins do interact with PalgD, either individually or in some combination, to induce looping of the promoter region under certain physiological conditions that have yet to be determined. The current work by Jones et al (1) shows that AmrZ is able to act as an activator on algD as it represses pslA. These data indicate that either an AlgUdependent gene somehow modifies AmrZ or increases the amount of AmrZ such that it becomes an activator of PalgD.…”

“…One model that could be proposed based upon the current literature is that Pel is induced initially by the Las quorum-sensing system, followed by Psl, as the organisms encounter starvation or other stresses. The data from Jones et al (1) indicate that AmrZ would facilitate the transition from Psl to alginate production by repressing Psl and activating alginate, potentially when membrane-perturbing (AlgU-inducing) conditions ensue. There are likely still more regulatory mechanisms yet to be discovered.…”

“…n this month's issue of the Journal of Bacteriology, Jones et al (1) examine the relationship between the Pseudomonas aeruginosa chronically produced exopolysaccharide, alginate, and a polysaccharide that is produced during the initial stages of biofilm formation, Psl. Psl, composed of mannose, rhamnose, and glucose, was identified as one of the first exopolysaccharides in nonmucoid biofilm matrices (2).…”

Pseudomonas aeruginosa AmrZ Binds to Four Sites in the algD Promoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation

“…All bacterial strains, plasmids, and DNA oligonucleotides are listed in Table S1 in the supplemental material. Strains of Escherichia coli and Pseudomonas aeruginosa were incubated as described previously (30). All E. coli strains were cultured in LB (1 liter LB contains 10 g tryptone, 5 g yeast extract, and 5 g sodium chloride) or on LA (LB with 1.5% [wt/vol] agar).…”

“…Well-studied targets include those involved in exopolysaccharide production (alginate, Psl, and Pel), flagella, twitching motility, and the metabolism of bis-(3=,5=)-cyclic diguanylate (4,13,(27)(28)(29)(30)(31). AmrZ is composed of a flexible N terminus (residues 1 to 11), a DNA binding ribbonhelix-helix domain (residues 12 to 66), and a C-terminal tetramerization domain (CTD) (residues 67 to 108) (32,33).…”