title: ChIP-Seq and RNA-Seq reveal an AmrZ-mediated mechanism for cyclic di-GMP synthesis and biofilm development by Pseudomonas aeruginosa.
abstract: The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.
authors.affiliation: Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, United States of America; Department of Infection and Immunity and Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Jones
authors.affiliation: Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Newsom
authors.affiliation: Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Kelly
authors.affiliation: Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Irie
authors.affiliation: Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Jennings
authors.affiliation: Department of Microbiology, Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Binjie
authors.initials: B
authors.lastname: Xu
authors.affiliation: Department of Infection and Immunity and Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Dominique H
authors.initials: DH
authors.lastname: Limoli
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Joe J
authors.initials: JJ
authors.lastname: Harrison
authors.affiliation: Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Parsek
authors.affiliation: Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: White
authors.affiliation: Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, United States of America; Department of Microbiology, Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wozniak
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development*; D047369:Chromatin Immunoprecipitation; D002853:Chromatography, Liquid; D006152:Cyclic GMP / Q000096:biosynthesis*; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D013058:Mass Spectrometry; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D011550:Pseudomonas aeruginosa / Q000472:pathogenicity* / Q000502:physiology*; D012329:RNA, Bacterial; D014157:Transcription Factors / Q000378:metabolism; D014774:Virulence / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D014157:Transcription Factors; D006152:Cyclic GMP