title: Systematic targeted mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators in the control of virulence.
abstract: In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest.
authors.affiliation: Unité de Recherche en Biologie Moléculaire, Facultés Universitaires Notre Dame de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium.
authors.forename: Valérie
authors.initials: V
authors.lastname: Haine
authors.forename: Audrey
authors.initials: A
authors.lastname: Sinon
authors.forename: Frédéric
authors.initials: F
authors.lastname: Van Steen
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Rousseau
authors.forename: Marie
authors.initials: M
authors.lastname: Dozot
authors.forename: Pascal
authors.initials: P
authors.lastname: Lestrate
authors.forename: Christophe
authors.initials: C
authors.lastname: Lambert
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Letesson
authors.forename: Xavier
authors.initials: X
authors.lastname: De Bolle
mesh_terms: D000818:Animals; D017347:Brucella melitensis / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D002460:Cell Line; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D018390:Gene Targeting; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016296:Mutagenesis; D012097:Repressor Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D014774:Virulence
chemical_list: D012097:Repressor Proteins; D014157:Transcription Factors