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

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.Bacteria have selected mechanisms to express only the appropriate subset of genes conferring a growth or survival advantage in a given situation (10). The expression of many bacterial genes is regulated at the initiation of transcription by regulators which, in response to specific environmental and/or cellular signals, bind at the promoter of target genes to activate or repress them (23).The availability of complete bacterial genome sequences has opened up doors to the development of new strategies to study the biology of microorganisms (59). For instance, the complete Brucella melitensis sequence (15) allowed us to conduct a systematic study of several families of regulators in this organism. Bacteria of the genus Brucella are facultative intracellular gram-negative coccobacilli that are pathogenic for domestic animals and occasionally for humans (57). Some transcriptional regulators of Brucella that have a role in the control of virulence have already been identified. Indeed, the two-component system BvrR/BvrS controls cell invasion, intracellular survival, and expression of two outer membrane proteins (25, 58). Some transcriptional regulators required for Brucella's virulence were also identified in several screens in cellular models and in BALB/c mice (19,31,35,36). Random screening for attenuated mutants may not be viewed as a comprehensive analysis that would allow the identification of all regulators involved in the virulence of the model tested. We...

NnrA Is Required for Full Virulence and Regulates Several Brucella melitensis Denitrification Genes

“…Since homology analyses support this hypothesis, we constructed mutants for NarR, NnrA, and NnrB regulators and for the catalytic subunits of the four reductases (narG, nirK, norB, and nosZ) of the denitrification process by using an integrative disruption strategy (5). Mutant genotypes were confirmed by Southern blotting as previously reported (5).…”

“…Since homology analyses support this hypothesis, we constructed mutants for NarR, NnrA, and NnrB regulators and for the catalytic subunits of the four reductases (narG, nirK, norB, and nosZ) of the denitrification process by using an integrative disruption strategy (5). Mutant genotypes were confirmed by Southern blotting as previously reported (5). We also constructed plasmidic promoterlacZ fusions using the pBBRMCS1lacZ vector (S. Léonard, unpublished data), a derivative of pBBR1MCS (9), in order to follow the activity of narK, nirK, norC, and nosR promoters in the narR, nnrA, and nnrB mutants.…”

Small Noncoding RNA AbcR1 Addressing Multiple Target mRNAs From Transcriptional Factor and Two-Component Response Regulator of Brucella melitensis

“…Transcriptional regulators and Two-component response regulators have been reported to play significant roles in the regulation of diverse biological processes, particularly in the virulence of B. melitensis (35). In the present study, the GFP reporter system was used to validate the interaction of target mRNAs with AbcR1 sRNA.…”

GntR family regulator SCO6256 is involved in antibiotic production and conditionally regulates the transcription of myo-inositol catabolic genes in Streptomyces coelicolor A3(2)

“…The GntR regulators play a fundamental role in the modulation of gene expression to respond to a variety of environmental signals. As a result, they regulate many diverse biological processes including bacterial motility (Jaques & McCarter, 2006), development (Hoskisson et al, 2006), antibiotic production (Hillerich & Westpheling, 2006), antibiotic resistance (Truong-Bolduc & Hooper, 2007), plasmid transfer (Reuther et al, 2006) and virulence (Casali et al, 2006;Haine et al, 2005).…”