BvrR/BvrS and the expression of other transcriptional regulators 
Notably, seven transcriptional regulators genes were also differentially expressed in the bvrR mutant compared to parental strain: BAB1_0237, BAB1_0891 (exoR), BAB1_1397, BAB2_0118 (vjbR), BAB2_0762 (ompR), BAB2_1127 and BAB2_1152. Three of these, namely exoR, ompR and vjbR are down regulated and have been previously implicated in Brucella virulence.
B. melitensis vjbR mutant is highly attenuated in both cellular and mouse models of infection [19].
VjbR has been described as a transcriptional regulator able to activate directly the secretion system virB operon and the flagellar genes, both virulence factors associated to the surface of the bacteria [20], [21].
Moreover, it has been demonstrated that VjbR controls the synthesis of exopolysaccharides and the productions of several OMPs, some of which are also involved in virulence [22].
Interestingly, our results also showed that the expression of vjbR was also induced intracellularly (see below).
All these data suggest that VjbR, similarly to the BvrR/BvrS system, is involved in the control of outer membrane composition and virulence.
In addition, DeJong and col [20] have demonstrated that among the promoters which expression is dependent of the VjbR regulator, is the ompR gene, the regulator of the OmpR/EnvZ two component system.
E. coli OmpR/EnvZ system controls the transcription of the outer membrane porins OmpF and OmpC in response to osmolarity [23].
Moreover, a systematically transcriptome analysis of all two component regulatory systems in E. coli has demonstrated that the OmpR/EnvZ system also controls the metabolism of amino acids, flagellar synthesis and nutrient transport [24].
As we have show in this study, the expression of at least three flagellar genes (fliM, BAB2_0124/5; flgJ, BAB1_0260; motB, BAB2_1103) were increased also in the bvrR mutant (Figure 2).
The two-regulatory systems ChvG(ExoS)/ChvI in S. meliloti and A. tumefaciens posses a high level of identity with the Brucella BvrR/BvrS [25], [26].
Chaves-Olarte et al have reported that B. abortus bvrS mutant complemented with the ExoS protein recuperated the ability to invade and replicated successfully in HeLa and macrophage cells [27], suggesting that the BvrR/BvrS system is functionally interchangeable with the ExoS/ChvI system.
A. tumefaciens ChvI/ChvG system controls the expression of the Aop, an OMP homologous to Brucella Omp25a [28], and in S. meliloti, ExoS/ChvI is a key regulator of gene expression for exopolysaccharide synthesis, motility and nutrient utilization [26].
It has been described in S. meliloti that exoR gene encodes a global regulator of transcription and that ExoR interacts genetically with both ExoS and ChvI and inhibits ExoS/ChvI activity [29], [30].
Further analysis indicated that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both polysaccharides and flagellum biosynthesis [31].
In addition, the transcription of the S. meliloti lpsS gene, that encodes a sulfotransferase that modifies LPS, is dependent on the exoR gene [32].
Other authors [29] suggest that ExoR is an inhibitor of two-component signaling that may be conserved in a large number of alpha-proteobacteria.
Our results also support this hypothesis: the functional relationship between the exoR gene and the BvrR/BvrS system.
Based on all these findings, obvious comparison about the function of all these regulators in Brucella could be made.
The fact that the expression of VjbR, OmpR and ExoR was altered in the bvrR mutant demonstrated for the first time an interaction or cross-talk among these global regulators, all involved in the control of composition and structure of the cell envelope (OMPs, LPS, chaperones, flagella, em).
