AmrZ beta-sheet residues are essential for DNA binding and transcriptional control of Pseudomonas aeruginosa virulence genes.
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 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 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 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.
Waligora
Elizabeth A
EA
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, North Carolina 27157, USA.
Ramsey
Deborah M
DM
Pryor
Edward E
EE
Lu
Haiping
H
Hollis
Thomas
T
Sloan
Gina P
GP
Deora
Rajendar
R
Wozniak
Daniel J
DJ
D000464:Alginates; D000595:Amino Acid Sequence; D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D014158:Transcription, Genetic; D014774:Virulence
D000464:Alginates; D004269:DNA, Bacterial; D006603:Hexuronic Acids; D020723:Glucuronic Acid