title: Analysis in vitro and in vivo of the transcriptional regulator CrgA of Neisseria meningitidis upon contact with target cells.
abstract: Contact between CrgA, a LysR-like regulatory protein in Neisseria meningitidis, and DNA is involved in the repression of several bacterial genes upon contact with epithelial cells. We used a defined in vitro system containing crgA promoter, purified RNA polymerase (RNAP) and purified CrgA protein to demonstrate that CrgA was directly responsible for this transcriptional repression. Interaction between the C-terminal domain of CrgA and the RNAP led to the production of short abortive transcripts, suggesting that CrgA may act by preventing RNAP from clearing the promoter. We probed the regulation by CrgA of its own production by analysing CrgA-DNA contacts during cell-bacteria interaction by assaying in vivo protection against dimethyl sulphate (DMS) methylation. Comparison of DMS footprints in vitro and in vivo suggested that CrgA repressed transcription through specific base contacts, probably in the major groove of the DNA double helix, resulting in DNA looping. Upon contact with target cells, CrgA was released from the DNA, allowing transcription of the target gene to proceed to elongation and facilitating tight control of the expression of genes regulated by CrgA.
authors.affiliation: Unité des Neisseria, Institut Pasteur, 28 Rue du Docteur Roux, 75724, Paris, Cedex 15, France.
authors.forename: Ala-Eddine
authors.initials: AE
authors.lastname: Deghmane
authors.forename: Dario
authors.initials: D
authors.lastname: Giorgini
authors.forename: Laure
authors.initials: L
authors.lastname: Maigre
authors.forename: Muhamed-Kheir
authors.initials: MK
authors.lastname: Taha
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004847:Epithelial Cells; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C406769:CrgA protein, Neisseria meningitidis; D017931:DNA Primers; D004269:DNA, Bacterial; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases