Systematic Targeted Mutagenesis ofBrucella melitensis16M Reveals a Major Role for GntR Regulators in the Control ofVirulence
Open Access
- 1 September 2005
- journal article
- research article
- Published by American Society for Microbiology in Infection and Immunity
- Vol. 73 (9), 5578-5586
- https://doi.org/10.1128/iai.73.9.5578-5586.2005
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.This publication has 78 references indexed in Scilit:
- Generation of the Brucella melitensis ORFeome Version 1.1: Figure 1Genome Research, 2004
- Integration host factor is involved in transcriptional regulation of theBrucella abortus virBoperonMolecular Microbiology, 2004
- Intact Purine Biosynthesis Pathways Are Required for Wild-Type Virulence of Brucella abortus 2308 in the BALB/c Mouse ModelInfection and Immunity, 2004
- The rlmB Gene Is Essential for Formation of Gm2251 in 23S rRNA but Not for Ribosome Maturation in Escherichia coliJournal of Bacteriology, 2001
- Plasmid-mediated virulence genes in non-typhoidSalmonellaserovarsFEMS Microbiology Letters, 1994
- MOLECULAR BIOLOGY OF THE LysR FAMILY OF TRANSCRIPTIONAL REGULATORSAnnual Review of Microbiology, 1993
- Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium melilotiJournal of Molecular Biology, 1991
- A new family of bacterial regulatory proteinsFEMS Microbiology Letters, 1991
- A new family of bacterial regulatory proteinsFEMS Microbiology Letters, 1991
- A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative BacteriaBio/Technology, 1983