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Infection and Immunity, September 2005, p. 5578-5586, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5578-5586.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Systematic Targeted Mutagenesis of Brucella melitensis 16M Reveals a Major Role for GntR Regulators in the Control of Virulence

Valérie Haine, Audrey Sinon, Frédéric Van Steen, Stéphanie Rousseau, Marie Dozot, Pascal Lestrate, Christophe Lambert, Jean-Jacques Letesson, and Xavier De Bolle*

Unité de Recherche en Biologie Moléculaire, University of Namur, Namur, Belgium

Received 17 December 2004/ Returned for modification 7 March 2005/ Accepted 10 May 2005

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.

* Corresponding author. Mailing address: Unité de Recherche en Biologie Moléculaire, Facultés Universitaires Notre Dame de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium. Phone: 32 81 72 44 38. Fax: 32 81 72 42 97. E-mail: xavier.debolle{at}fundp.ac.be.

Editor:V. J. DiRita

Infection and Immunity, September 2005, p. 5578-5586, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5578-5586.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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