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

Content Provider	PubMed Central
Author	Haine, Valérie Sinon, Audrey Steen, Frédéric Van Rousseau, Stéphanie Marie, Dozot Pascal, Lestrate Lambert, Christophe Jean-jacques, Letesson Bolle, Xavier De
Copyright Year	2005
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.
Related Links	http://dx.doi.org/10.1128/iai.73.9.5578-5586.2005
Ending Page	5586
Page Count	9
Starting Page	5578
File Format	PDF
ISSN	00199567
e-ISSN	10985522
Journal	Infection and Immunity
Issue Number	9
Volume Number	73
Language	English
Publisher	American Society for Microbiology
Publisher Date	2005-09-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Immunology Microbiology Parasitology Infectious Diseases Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Parasitology Immunology Microbiology

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