title: A large family of anti-activators accompanying XylS/AraC family regulatory proteins.
abstract: AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.
authors.affiliation: Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA.
authors.forename: Araceli E
authors.identifier: 0000-0003-3988-0853
authors.initials: AE
authors.lastname: Santiago
authors.affiliation: Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Yan
authors.affiliation: Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA.
authors.forename: Minh
authors.initials: M
authors.lastname: Tran
authors.affiliation: Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA, USA.
authors.forename: Nathan
authors.initials: N
authors.lastname: Wright
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, School of Medicine, Charlottesville, VA, USA.
authors.forename: Deborah H
authors.initials: DH
authors.lastname: Luzader
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, School of Medicine, Charlottesville, VA, USA.
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Kendall
authors.affiliation: Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA.
authors.forename: Fernando
authors.initials: F
authors.lastname: Ruiz-Perez
authors.affiliation: Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA.|Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, School of Medicine, Charlottesville, VA, USA.
authors.forename: James P
authors.initials: JP
authors.lastname: Nataro
mesh_terms: D051777:AraC Transcription Factor / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005797:Genes, araC / Q000235:genetics* / Q000502:physiology; D006090:Gram-Negative Bacteria / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014774:Virulence / Q000235:genetics
chemical_list: D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015534:Trans-Activators; D014157:Transcription Factors