Open AccessAmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes
Author(s) -
Elizabeth A. Waligora,
Deborah M. Ramsey,
Edward E. Pryor,
Haiping Lu,
Thomas Hollis,
Gina Parise Sloan,
Rajendar Deora,
Daniel J. Wozniak
Publication year - 2010
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.00711-10
Subject(s) - biology , virulence , repressor , mutant , gene , transcriptional regulation , dna , mutagenesis , pseudomonas aeruginosa , genetics , microbiology and biotechnology , gene expression , bacteria
AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-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 acutePseudomonas 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 β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-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 withamrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Directin vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenicamrZ mutant. Finally, the reduced infection phenotype of theamrZ -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 thein 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.