title: fleQ, the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma70 dependent and is downregulated by Vfr, a homolog of Escherichia coli cyclic AMP receptor protein.
abstract: The flagellar transcriptional regulator FleQ appears to be the highest-level regulator in the hierarchical regulatory cascade of flagellar biogenesis in Pseudomonas aeruginosa. Except for the posttranslational downregulation of FleQ activity by FleN, an antiactivator, not much is known about the regulation of the fleQ gene or its gene product. Some FleQ homologs in other bacterial species either are positively regulated by another regulator (e.g., CtrA, the master regulator regulating FlbD in Caulobacter crescentus) or are expressed from a sigma70-dependent promoter (e.g., FlgR of Helicobacter pylori). In this study we demonstrated that Vfr, an Escherichia coli CRP homolog known to function as an activator for various genes, including lasR, regA, and toxA, in P. aeruginosa, is capable of repressing fleQ transcription by binding to its consensus sequence in the fleQ promoter. In a DNase I footprint assay, purified Vfr protected the sequence 5'-AATTGACTAATCGTTCACATTTG-3'. When this putative Vfr binding site in the fleQ promoter was mutated, Vfr was unable to bind the fleQ promoter fragment and did not repress fleQ transcription effectively. Primer extension analysis of the fleQ transcript revealed two transcriptional start sites, t1 and t2, that map within the Vfr binding site. A putative -10 region (TAAAAT) for the t2 transcript, with a five-of-six match with the E. coli sigma70 binding consensus, overlaps with one end of the Vfr binding site. A 4-bp mutation and an 8-bp mutation in this -10 region markedly reduced the activity of the fleQ promoter. The same mutations led to the disappearance of the 203-nucleotide fleQ transcript in an in vitro transcription assay. Vfr probably represses fleQ transcription by binding to the Vfr binding site in the fleQ promoter and preventing the sigma factor from binding to the -10 region to initiate transcription.
authors.affiliation: Department of Medicine/Infectious Diseases, University of Florida, Gainesville, Florida 32610, USA.
authors.forename: Nandini
authors.initials: N
authors.lastname: Dasgupta
authors.forename: Evan P
authors.initials: EP
authors.lastname: Ferrell
authors.forename: Kristen J
authors.initials: KJ
authors.lastname: Kanack
authors.forename: Susan E H
authors.initials: SE
authors.lastname: West
authors.forename: Reuben
authors.initials: R
authors.lastname: Ramphal
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D005407:Flagella / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; C108098:FleQ protein, Pseudomonas aeruginosa; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; C091090:Vfr protein, Pseudomonas aeruginosa; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases