title: Pseudomonas aeruginosa AmrZ Binds to Four Sites in the algD Promoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation.
abstract: During late stages of cystic fibrosis pulmonary infections, Pseudomonas aeruginosa often overproduces the exopolysaccharide alginate, protecting the bacterial community from host immunity and antimicrobials. The transcription of the alginate biosynthesis operon is under tight control by a number of factors, including AmrZ, the focus of this study. Interestingly, multiple transcription factors interact with the far-upstream region of this promoter (PalgD), in which one AmrZ binding site has been identified previously. The mechanisms of AmrZ binding and subsequent activation remain unclear and require more-detailed investigation. In this study, in-depth examinations elucidated four AmrZ binding sites, and their disruption eliminated AmrZ binding and promoter activation. Furthermore, our in vitro fluorescence resonance energy transfer experiments suggest that AmrZ holds together multiple binding sites in PalgD and thereafter induces the formation of higher-order DNA-AmrZ complexes. To determine the importance of interactions between those AmrZ oligomers in the cell, a DNA phasing experiment was performed. PalgD transcription was significantly impaired when the relative phase between AmrZ binding sites was reversed (5 bp), while a full-DNA-turn insertion (10 bp) restored promoter activity. Taken together, the investigations presented here provide a deeper mechanistic understanding of AmrZ-mediated binding to PalgD IMPORTANCE: Overproduction of the exopolysaccharide alginate provides protection to Pseudomonas aeruginosa against antimicrobial treatments and is associated with chronic P. aeruginosa infections in the lungs of cystic fibrosis patients. In this study, we combined a variety of microbiological, genetic, biochemical, and biophysical approaches to investigate the activation of the alginate biosynthesis operon promoter by a key transcription factor named AmrZ. This study has provided important new information on the mechanism of activation of this extremely complex promoter.
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, Ohio, USA Center for Microbial Interface Biology, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Binjie
authors.initials: B
authors.lastname: Xu
authors.affiliation: Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Soukup
authors.affiliation: Department of Microbial Infection and Immunity, The Ohio State University, Columbus, Ohio, USA Center for Microbial Interface Biology, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Jones
authors.affiliation: Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Wexner Medical Center and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Fishel
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, Ohio, USA Department of Microbial Infection and Immunity, The Ohio State University, Columbus, Ohio, USA Center for Microbial Interface Biology, The Ohio State University, Columbus, Ohio, USA Daniel.Wozniak@osumc.edu.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wozniak
mesh_terms: D000464:Alginates; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006603:Hexuronic Acids; D020723:Glucuronic Acid