title: The flagellar hierarchy of Rhodobacter sphaeroides is controlled by the concerted action of two enhancer-binding proteins.
abstract: The expression of the bacterial flagellar genes follows a hierarchical pattern. In Rhodobacter sphaeroides the flagellar genes encoding the hook and basal body proteins are expressed from sigma54-dependent promoters. This type of promoters is always regulated by transcriptional activators that belong to the family of the enhancer-binding proteins (EBPs). We searched for possible EBPs in the genome of R. sphaeroides and mutagenized two open reading frames (ORFs) (fleQ and fleT), which are in the vicinity of flagellar genes. The resulting mutants were non-motile and could only be complemented by the wild-type copy of the mutagenized gene. Transcriptional fusions showed that all the flagellar sigma54-dependent promoters with exception of fleTp, required both transcriptional activators for their expression. Interestingly, transcription of the fleT operon is only dependent on FleQ, and FleT has a negative effect. Both activators were capable of hydrolysing ATP, and were capable of promoting transcription from the flagellar promoters at some extent. Electrophoretic mobility shift assays suggest that only FleQ interacts with DNA whereas FleT improves binding of FleQ to DNA. A four-tiered flagellar transcriptional hierarchy and a regulatory mechanism based on the intracellular concentration of both activators and differential enhancer affinities are proposed.
authors.affiliation: Departamento de Biología Molecular y Biotecnología. Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, 04510 México D. F., México.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Poggio
authors.forename: Aurora
authors.initials: A
authors.lastname: Osorio
authors.forename: Georges
authors.initials: G
authors.lastname: Dreyfus
authors.forename: Laura
authors.initials: L
authors.lastname: Camarena
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019295:Computational Biology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D005407:Flagella / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D050939:Gene Fusion; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D008954:Models, Biological; D008969:Molecular Sequence Data; D009068:Movement; D016296:Mutagenesis; D009154:Mutation; D016366:Open Reading Frames; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54 / Q000502:physiology; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D051877:RNA Polymerase Sigma 54