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Inorder to identify transcriptional regulators involved in virulence genecontrol inBrucella melitensis , we generated a collection of88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR,and TetR families of regulators. This collection was named LiMuR(li brary ofmu tants forr egulators). Wedeveloped a method to test several mutants simultaneously in one animalin order to identify those unable to survive. This method, called theplasmid-tagged mutagenesis method, was used to test the residualvirulence of mutants after 1 week in a mouse model of infection. Tenattenuated mutants, of which six and three belong to the GntR and LysRfamilies, respectively, were identified and individually confirmed toreplicate at lower rates in mice. Among these 10 mutants, onlygntR10 andarsR6 are attenuated in cellular models.The LiMuR also allows simple screenings to identify regulators of aparticular gene or operon. As a first example, we analyzed theexpression of thevirB operon in the LiMuR mutants. We carriedout Western blottings of whole-cell extracts to analyze the productionof VirB proteins using polyclonal antisera against VirB proteins. Fourmutants produced small amounts of VirB proteins, and one mutantoverexpressed VirB proteins compared to the wild-type strain. In thesefive mutants, reporter analysis using thevirB promoter fusedtolacZ showed that three mutants controlvirB at thetranscriptional level. The LiMuR is a resource that will providestraightforward identification of regulators involved in the control ofgenes ofinterest.

Systematic Targeted Mutagenesis ofBrucella melitensis16M Reveals a Major Role for GntR Regulators in the Control ofVirulence