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Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor-regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na(+)/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.

The genome of Brucella melitensis contains genes coding for the sigma factors RpoD, RpoN, RpoH1, RpoH2, RpoE1 and RpoE2. Previously published data show that B. melitensis is flagellated and that an rpoE1 mutant overexpresses the flagellar protein FlgE. In this study, we demonstrate that mutation of rpoE1 causes an overexpression of the flagellar genes fliF, flgE, fliC, flaF and flbT, correlating with the production of a longer filament and thereby demonstrating that RpoE1 acts as a flagellar repressor. Moreover, mutation of rpoE1 increases the promoter activity of the flagellar master regulator ftcR, suggesting that RpoE1 acts upstream of ftcR. Together, these data show that RpoE1 represses the flagellar synthesis and filament length in B. melitensis.

It was recently demonstrated that the pathogen Brucella melitensis produces a polar sheathed flagellum under the control of the master regulator FtcR. However, the regulatory mechanism controlling the flagellar assembly remains unknown. In this work, we investigate the flagellar hierarchy of B. melitensis as well as the flagellin FliC regulation. We show that a mutation in fliF or flgE (coding for the basal body structure and the hook, respectively) does not affect FliC synthesis, suggesting that production of FliC does not depend on the flagellar assembly. We demonstrate that FlbT is a FliC activator since inactivation of flbT causes a decrease in fliC expression by using a fliC-lacZ translational reporter construct. Moreover, the quantitative real-time PCR and Western blot analysis show a marked decrease in fliC mRNA and FliC protein level, respectively. Conversely, the B. melitensis wild-type strain overexpressing flaF fails to produce FliC, suggesting an opposite function. Interestingly, the expression of the flbT gene in an ftcR or an flbT mutant restores FliC production, demonstrating that FlbT plays a regulatory checkpoint role in FliC synthesis. This mechanism could be conserved in the Rhizobiales since complementation of an flbT or an ftcR mutant with flbT from Sinorhizobium meliloti restores FliC synthesis.

Some Brucella rough mutants cause cytotoxicity that resembles oncosis and necrosis in macrophages. This cytotoxicity requires the type IV secretion system (T4SS). In rough mutants, the cell-surface O antigen is shortened and the T4SS structure is thus exposed on the surface. Cytotoxicity effector proteins can therefore be more easily secreted. This enhanced secretion of effector proteins might cause the increased levels of cytotoxicity observed. However, whether this cytotoxicity is unique to the rough mutant and is mediated by overexpression of the T4SS has not been definitively determined. To test this, in the present study, a virB inactivation mutant (BMDeltavirB) and an overexpression strain (BM-VIR) of a smooth Brucella melitensis strain (BM) were constructed and their cytotoxicity for macrophages and intracellular survival capability were analysed and compared. Cytotoxicity was detected in macrophages infected with higher concentrations of strains BM or BM-VIR, but not in those infected with BMDeltavirB. The quorum sensing signal molecule N-dodecanoyl-dl-homoserine lactone (C(12)-HSL), a molecule that can inhibit expression of virB, inhibited the cytotoxicity of BM and BM-VIR, but not of BMDeltavirB. These results indicated that overexpression of virB is responsible for Brucella cytotoxicity in macrophages. Transcription analysis showed that virB is regulated in a cell-density-dependent manner both in in vitro culture and during macrophage infection. When compared with BM, BM-VIR showed a reduced survival capacity in macrophages and mice, but both strains demonstrated similar resistance to in vitro stress conditions designed to simulate intracellular environments. Taken together, the cytotoxicity of Brucella for macrophages is probably mediated by increased secretion of effector proteins that results from overexpression of virB or an increase in the number of bacterial cells. The observation that both inactivation and overexpression of virB are detrimental for Brucella intracellular survival also indicated that the expression of virB is tightly regulated in a cell-density-dependent manner.