AmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes

Abstract

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the ␤-sheet for DNA binding, while the ␣-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a ␤-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ ␤-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection.The Pseudomonas aeruginosa transcriptional regulator AmrZ is a proposed member of the ribbon-helix-helix (RHH) family of DNA-binding proteins, sharing structural similarity with the Arc and Mnt repressors of Salmonella enterica serovar Typhimurium bacteriophage P22. This family is grouped by structural similarity and includes several transcriptional regulators found in prokaryotes, archaea and their viruses, and other bacteriophages (2,7,12,20,24,26,34,46). Based on amino acid identity as well as secondary-structure prediction models, AmrZ likely possesses a ribbon-helix-helix motif (i.e., one ␤-strand and two ␣-helices) (Fig. 1B) responsible for DNA-binding activity in this family of proteins (44). RHH proteins function through the oligomerization of the ␣-helices, which allows the two ␤-strands to form an antiparallel ␤-sheet that recognizes and binds in the major groove of the operator site (31, 38). Arc exists as a dimer in solution, while Mnt utilizes an extra carboxy-terminal ␣-helical domain to maintain a tetramer configuration (43). When binding DNA, these oligomers are maintained, and the inhibition of oligomerization negatively impacts DNA-binding activity (44). To facilitate higher-order oligomers at the RHH binding site, operator sites often contain sequences in either a direct repeat or palindromic orientation (34,46). Because there are specific contacts between residues of the DNA-binding ␤-sheet and bases in the operator site, mutations of critical base...

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

“…PCR amplification of the desired psl promoter regions or annealing of complementary oligonucleotides was used to produce DNA fragments. DNA binding was performed as previously published (33). Unless otherwise noted, 5 ng of protein was combined with 150 fmol of DNA fragment in electrophoretic mobility shift assay (EMSA) binding buffer (20 mM Tris-HCl [pH 8.0], 200 mM NaCl, 20 mM MgCl 2 , 20% glycerol) and 750 ng poly(dI-dC) (Sigma) and allowed to bind for 10 min at room temperature.…”

“…Wild-type and R22A-binding-deficient AmrZ proteins were purified as described previously (33). PCR amplification of the desired psl promoter regions or annealing of complementary oligonucleotides was used to produce DNA fragments.…”

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

“…The ribbon-helix-helix domain is conserved in other Arc superfamily proteins, such as Arc, CopG, and MetJ (33)(34)(35)(36). AmrZ tetramerizes in solution, and the AmrZ construct comprising residues 1 to 66 (AmrZ1-66) binds to DNA as a dimer of dimers, suggesting that AmrZ interacts with its DNA targets as oligomers, likely tetramers (32,33,37).…”

“…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). The ribbon-helix-helix domain is conserved in other Arc superfamily proteins, such as Arc, CopG, and MetJ (33)(34)(35)(36).…”

“…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). The ribbon-helix-helix domain is conserved in other Arc superfamily proteins, such as Arc, CopG, and MetJ (33)(34)(35)(36). AmrZ tetramerizes in solution, and the AmrZ construct comprising residues 1 to 66 (AmrZ1-66) binds to DNA as a dimer of dimers, suggesting that AmrZ interacts with its DNA targets as oligomers, likely tetramers (32,33,37).…”