title: Transcriptional control by two interacting regulatory proteins: identification of the PtxS binding site at PtxR.
abstract: The PtxS and PtxR regulators control the expression of the glucose dehydrogenase genes from the Pgad promoter in Pseudomonas aeruginosa. These regulators bind to their cognate operators, that are separated by ∼50 nt, within the promoter region and interact with each other creating a DNA-loop that prevents RNA polymerase promoter access. Binding of the 2-ketogluconate effector to PtxS caused PtxS/PtxR complex dissociation and led to the dissolution of the repression loop facilitating the entry of the RNA polymerase and enabling the transcription of the gad gene. We have identified a hydrophobic surface patch on the PtxR putative surface that was hypothesized to correspond to the binding site for PtxS. Two surface-exposed residues in this patch, V173 and W269, were replaced by alanine. Isothermal titration calorimetry assays showed that PtxS does not interact with the mutant variants of PtxR. Electrophoretic mobility shift assay and DNAase I footprinting assays proved that both regulators bind to their target operators and that failure to interact with each other prevented the formation of the DNA-loop. In vitro transcription showed that PtxS per se is sufficient to inhibit transcription from the Pgad promoter, but that affinity of PtxS for its effector is modulated by PtxR.
authors.affiliation: Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Calle Profesor Albareda 1, E-18008 Granada, Spain.
authors.forename: Abdelali
authors.initials: A
authors.lastname: Daddaoua
authors.forename: Tino
authors.initials: T
authors.lastname: Krell
authors.forename: Juan-Luis
authors.initials: JL
authors.lastname: Ramos
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C113064:PtxS protein, Pseudomonas aeruginosa; D014157:Transcription Factors; C073470:LysR protein, Bacteria; D004247:DNA
references.citation: J Mol Recognit. 2012 Nov;25(11):604-15
references.pmid: 23108620
references.citation: Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284-8
references.pmid: 166383
references.citation: Science. 1995 Jun 30;268(5219):1899-902
references.pmid: 7604262
references.citation: Curr Opin Struct Biol. 2004 Feb;14(1):28-35
references.pmid: 15102446
references.citation: Mol Gen Genet. 1998 May;258(3):250-9
references.pmid: 9645431
references.citation: J Bacteriol. 1998 Dec;180(24):6784-8
references.pmid: 9852033
references.citation: Chem Rev. 1997 Aug 5;97(5):1233-1250
references.pmid: 11851449
references.citation: Structure. 1996 Dec 15;4(12):1395-400
references.pmid: 8994966
references.citation: J Bacteriol. 2010 Sep;192(17):4357-66
references.pmid: 20581202
references.citation: Microb Biotechnol. 2008 Mar;1(2):126-36
references.pmid: 21261830
references.citation: Can J Microbiol. 2001 Sep;47(9):820-8
references.pmid: 11683464
references.citation: J Bacteriol. 2003 Aug;185(16):4755-63
references.pmid: 12896994
references.citation: Curr Protoc Immunol. 2004 Feb;Chapter 8:Unit 8.9
references.pmid: 18432935
references.citation: J Bacteriol. 2012 Jan;194(2):406-12
references.pmid: 22081386
references.citation: Annu Rev Phytopathol. 2001;39:259-84
references.pmid: 11701866
references.citation: PLoS One. 2012;7(7):e39390
references.pmid: 22844393
references.citation: J Bacteriol. 2007 Jul;189(14):5142-52
references.pmid: 17483213
references.citation: Cell Mol Life Sci. 2007 Jan;64(1):3-16
references.pmid: 17103112
references.citation: Microbiol Rev. 1992 Dec;56(4):509-28
references.pmid: 1480106
references.citation: Microbiol Rev. 1994 Jun;58(2):268-90
references.pmid: 8078436
references.citation: Proc Natl Acad Sci U S A. 2010 May 18;107(20):9376-81
references.pmid: 20439713
references.citation: Nucleic Acids Res. 2009 Aug;37(14):4545-58
references.pmid: 19474343
references.citation: J Biol Chem. 2010 Jul 9;285(28):21372-81
references.pmid: 20435893
references.citation: Annu Rev Microbiol. 1997;51:593-628
references.pmid: 9343361
references.citation: Gene. 1989 Apr 15;77(1):155-62
references.pmid: 2663653