SummaryBoth a type IV secretion system and a flagellum have been described in Brucella melitensis . These two multimolecular surface appendages share several features. Their expression in bacteriological medium is growth curve dependent, both are induced intracellularly and are required for full virulence in a mouse model of infection. Here we report the identification of VjbR, a quorum sensing-related transcriptional regulator. A vjbR mutant has a downregulated expression of both virB operon and flagellar genes either during vegetative growth or during intracellular infection. In a cellular model, the vacuoles containing the vjbR mutant or a virB mutant are decorated with the same markers at similar times post infection. The vjbR mutant is also strongly attenuated in a mouse model of infection. As C 12 -homoserine lactone pheromone is known to be involved in virB repression, we postulated that VjbR is mediating this effect. In agreement with this hypothesis, we observed that, as virB operon, flagellar genes are controlled by the pheromone. All together these data support a model in which VjbR acts as a major regulator of virulence factors in Brucella .

SummaryPersistence infection is the keystone of the ruminant and human diseases called brucellosis and Malta fever, respectively, and is linked to the intracellular tropism of Brucella spp. While described as nonmotile, Brucella spp. have all the genes except the chemotactic system, necessary to assemble a functional flagellum. We undertook to determine whether these genes are expressed and are playing a role in some step of the disease process. We demonstrated that in the early log phase of a growth curve in 2YT nutrient broth, Brucella melitensis expresses genes corresponding to the basal (MS ring) and the distal (hook and filament) parts of the flagellar apparatus. Under these conditions, a polar and sheathed flagellar structure is visible by transmission electron microscopy (TEM). We evaluated the effect of mutations in flagellar genes of B. melitensis encoding various parts of the structure, MS ring, P ring, motor protein, secretion apparatus, hook and filament. None of these mutants gave a discernible phenotype as compared with the wild-type strain in cellular models of infection. In contrast, all these mutants were unable to establish a chronic infection in mice infected via the intraperitoneal route, raising the question of the biological role(s) of this flagellar appendage.

The flagellar regulon of Brucella melitensis 16M contains 31 genes clustered in three loci on the small chromosome. These genes encode a polar sheathed flagellum that is transiently expressed during vegetative growth and required for persistent infection in a mouse model. By following the expression of three flagellar genes (fliF, flgE, and fliC, corresponding to the MS ring, hook, and filament monomer, respectively), we identified a new regulator gene, ftcR (flagellar two-component regulator). Inactivation of ftcR led to a decrease in flagellar gene expression and to impaired Brucella virulence. FtcR has a two-component response regulator domain as well a DNA binding domain and is encoded in the first flagellar locus of B. melitensis. Both the ftcR sequence and its genomic context are conserved in other related ␣-proteobacteria. During vegetative growth in rich medium, ftcR expression showed a peak during the early exponential phase that paralleled fliF gene expression. VjbR, a quorum-sensing regulator of the LuxR family, was previously found to control fliF and flgE gene expression. Here, we provide some new elements suggesting that the effect of VjbR on these flagellar genes is mediated by FtcR. We found that ftcR expression is partially under the control of VjbR and that the expression in trans of ftcR in a vjbR mutant restored the production of the hook protein (FlgE). Finally, FtcR binds directly to the upstream region of the fliF gene. Therefore, our data support the role of FtcR as a flagellar master regulator in B. melitensis and perhaps in other related ␣-proteobacteria.Flagella are highly complex bacterial organelles that are usually well conserved among diverse bacterial species (17,39,46). In addition to motility, the bacterial flagellum is involved in a variety of interactions between the bacterium and its environment (e.g., adhesion, biofilm formation, secretion, and modulation of the host reponse) (7,16,20,22,30,36,42,50,53,57). Flagellar expression involves about 50 flagellar genes, distributed into three or four classes as observed in Escherichia coli (1) and Salmonella enterica serovar Typhimurium (31) or in Caulobacter crescentus (38), Pseudomonas aeruginosa (10), and Vibrio cholerae (41). Complex regulation processes control this hierarchical system, allowing for the expression of the genes of one downstream class if the genes from the upstream class have been expressed.At the top of the flagellar regulatory cascade, one or more distinct master genes encode the transcriptional regulators responsible for turning on and off flagellar synthesis, in response to environmental factors and cell cycle-related signals. In ␥-proteobacteria, this so-called class I is represented by a heterotetrameric complex, FlhD 2 C 2 (lateral flagellar systems), or a sigma 54-associated transcriptional activator of the NtrC family (polar flagellar systems) (4, 52). The situation is somewhat different in ␣-proteobacteria. In C. crescentus, a twocomponent response regulator called CtrA controls a part of the asymmetri...

BackgroundIt was recently shown that B. melitensis is flagellated. However, the flagellar structure remains poorly described.FindingsWe analyzed the structure of the polar sheathed flagellum of B. melitensis by TEM analysis and demonstrated that the Ryu staining is a good method to quickly visualize the flagellum by optical microscopy. The TEM analysis demonstrated that an extension of the outer membrane surrounds a filament ending by a club-like structure. The ΔftcR, ΔfliF, ΔflgE and ΔfliC flagellar mutants still produce an empty sheath.ConclusionsOur results demonstrate that the flagellum of B. melitensis has the characteristics of the sheathed flagella. Our results also suggest that the flagellar sheath production is not directly linked to the flagellar structure assembly and is not regulated by the FtcR master regulator.