Regulation of carbon and nitrogen metabolism 
One of the remarkable findings observed in our microarray analysis was that several genes related with metabolism were also differentially expressed.
These included twelve genes encoding ABC transport systems (matE, BAB1_0340; dctM, BAB1_0372; fhuD, BAB1_1366; yadH, BAB1_1368; oppA, BAB1_1601; oppC, BAB2_1051; oppD, BAB2_0817; potC, BAB1_ 1624; ssuB, BAB2_0917; ugpC, BAB2_1143; BAB2_0794; BAB2_1139), ten genes related with carbohydrate, amino or fatty acids metabolism and five related with nitrogen metabolism.
Interestingly, all genes related with carbohydrate, amino or fatty acids metabolism were up-regulated in the bvrR mutant.
These included the first enzyme in gluconeogenesis (pckA, phosphoenolpyruvate carboxykinase, BAB1_2091), four genes involved in TCA cycle and pyruvate metabolism (fumB, fumarate hydratase, BAB1_0977; lpdA, dihydrolipoamide dehydrogenase, BAB2_0712; pyruvate dehydrogenase, BAB2_0032; acetyl-CoA acetyltransferase, BAB2_0443), three genes involved in amino or fatty acid metabolism (aldehyde dehydrogenases, BAB2_1130, BAB2_1114; hydroxymethylglutaryl-CoA lyase, BAB1_0017), and two genes involve in benzoate degradation (pcaC, carboxymuconolactone decarboxylase, BAB2_0597; pcaI, coenzyme A transferase, BAB2_0604).
In addition, the complete maltose transport system of Brucella, which consists in a large operon containing thirteen genes (BAB1_0236-0248) was also affected.
Ten of these genes, including malK, malG, malF, malE and an iclR regulator, were down-regulated suggesting that the complete operon was negatively regulated in the bvrR mutant.
Although it has been showed that the mutants in the BvrR/BvrS system have no obvious defects with regard to the ability to grow on standard media [4], our microarray results suggests that the BvrR/BvrS system controls elements directly involved in adjusting the Brucella metabolism to the nutrient shift expected to occur during the transit to the intracellular niche.
To determine if the BvrR/BvrS system affects the metabolism, bvrR mutant and wild type strains were grown in synthetic minimal media.
As show in Figure 3, growth of the bvrR mutant was significantly reduced in minimal media. 
Other genes differentially expressed in the bvrR mutant included denitrification genes. 
The nitrite reductase gene ( nirK, BAB2_0943) was down regulated and the nitric oxide and nitrous oxide reductases genes ( nor C, BAB2_0955; nosZ, BAB2_0928) were up regulated. 
On the other hand, two deaminases (glutaminase, BAB2_0863; guanine deaminase, BAB1_0383) were also affected.
Since Brucella is an intracellular facultative pathogen, the bacteria could use these denitrification reactions to grow under low-oxygen condition by respiration of nitrate.
Brucella may also take advantage of denitrification to cope with nitric oxide (NO) production in the macrophage during the innate response against infection.
In fact, some of these denitrification genes have been related with the virulence in mice [10], [11].
Interestingly, our experiments to study the intracellular transcriptional level of BvrR/BvrS controlled genes (see below) showed that whereas norC was induced intracellularly, nirK and nosZ were less expressed.
Taken together all these data support the proposal that one role of the BvrR/BvrS system could be neutralize the production of toxic reactive nitrogen molecules, as NO, by the host.
These results also demonstrated a connection between carbon and nitrogen metabolism and BvrR/BvrS in Brucella.
Our results also demonstrated that gene hpr-K (BAB1_2094), a member of the Brucella phosphotransferase system (PTS; BAB1_2097-2094) adjacent to the bvrR/bvrS, was down-regulated.
As mentioned before, phosphoenolpyruvate carboxykinase gene (pckA, BAB1_2091) which is located upstream of the regulatory gene bvrR and divergently expressed was up-regulated.
Comparative genome analysis revealed that in addition to the bvrR/bvrS genes, the genome structure around these genes is essentially the same for all the alpha-proteobacteria [5].
Genes encoding proteins related to the PTS, including a HPr Ser-kinase, an EIIA permease of the mannose family and a HPr homologue precede those of the two-component regulatory system.
In most of these loci, upstream of the regulatory gene the pckA is divergently expressed (Figure 2).
This gene catalyzes the reversible decarboxylation and phosphorylation of oxaloacetate to form phosphoenolpyruvate.
In alpha-proteobacteria, it has been proposed that HPr might control the phosphorylation state of the transcription regulator [12]-[14].
In this regard, Letesson and col. [15] have suggested that in Brucella the PTS could interact with the BvrS sensor kinase, which in turn phosphorylates the response regulator.
Then, the BvrR could control transcription of the pckA gene, which encodes an essential control enzyme of the gluconeogenesis and Krebs cycle.
This hypothesis could explain the observation that mutants in the regulatory gene bvrR were inhibited in minimal media (see above).
According to this, it has been demonstrated that in A. tumefaciens the pckA genes is indeed under the control of ChvG/ChvI [16] and that null mutants in S. meliloti exoS and chvI have pleiotropic growth defects and were unable to grow on several carbon sources [17].
A link between carbon and nitrogen metabolism, PTS and two-component regulatory systems have been proposed for some bacteria [18], and our microarray results strongly suggest that same relationship could be made for Brucella.
