Results and Discussion 
Brucella mutants in the BvrR/BvrS two-component regulatory system are pleiotropic [3]-[6].
Whole-genome microarray analysis was made to determine the effect of the mutation in BvrR/BvrS in the gene expression pattern of Brucella.
B. abortus RNA was obtained from three independent cultures of each wild type and bvrR mutant cells grown in the same conditions.
To confirm the reproducibility of the gene expression data, the array experiment was composed of six slides (three for each type of cells) yielding six measurements per gene, representing three biological replicates (since each gene is present twice on each slide).
The ORFeome-based Brucella whole-genome DNA microarray used in this study has been previously validated for the analysis of gene expression under any experimental conditions [7].
The microarray experimental design was made according to the MIAME recommendations [8].
A change in gene expression was considered both statistically and biologically significant if the p-value was less than 0.01.
The statistical analysis resulted in the identification of a total of 127 genes differentially transcribed in the bvrR mutant versus the wild type.
Eighty three genes (65%) were up- and 44 (35%) were down-regulated (the complete list of differentially expressed genes in the bvrR mutant versus the wild type is show in Table S1).
Twenty three % of the differentially transcribed genes (30) encoded for hypothetical proteins.
For genes of annotated function, 59 appeared to be up regulated and 38 down regulated in the bvrR mutant.
Genes encoding proteins involved in metabolism and cellular process are among the most up regulated genes, and those encoding proteins involved in membrane transport are among the most down regulated (Figure 1).
To further validate some data generated in the microarray experiment, forty-eight differentially expressed genes were chosen to be analyzed by real-time quantitative reverse transcription-PCR (RT-PCR).
Total RNA from both Brucella strains were reverse transcribed into cDNA.
The reactions were made by triplicate from at least two independent cultures, and the cycle of threshold (Ct) was determined for each reaction.
Data were normalized by the 2-DeltaDeltaCt method [9] using the IF-1 housekeeping gene of Brucella as reference (Table 1).
Transcriptional data of forty-one (85%) of the genes selected gave identical tendency by both methods microarray and RT-PCR: 22 were up and 19 were down regulated in the bvrR mutant.
Interestingly, the level of transcription obtained by RT-PCR of the flagellar genes fliM (BAB2_0124) and motB (BAB2_1103), and the pckA gene (BAB1_2091) were the highest in the bvrR mutant.
On the other hand, exoR (BAB1_0891), omp25a (BAB1_0722), hpr-K (BAB1_2094), bvrS (BAB1_2093) and the lipoproteins (BAB1_2147, BAB1_0589, BAB1_0358) were among the less expressed genes (Table 1).
These results confirmed a good correlation between microarray and RT-PCR data, thus validating the model.
Next, we will focus on the genes differentially expressed in the bvrR mutant (a complete representation of the differentially expressed genes is show in Figure 2).
Cell envelope modulation It is well know that the transcription of omp25a y omp22 genes is under the control of the BvrR/BvrS system, and that bvrR/bvrS mutants have increased amount of underacylated lipid A species in the LPS [2], [3].
In addition, proteomic analysis of Brucella outer membrane fragments demonstrated that the expression of several OMPs, lipoproteins and chaperones was altered in these mutants [6].
These observations led to the hypothesis that the BvrR/BvrS system is involved in cell envelope changes required for adaptation to the intracellular environment.
Our microarray results demonstrated several genes directly involved in cell envelope or outer membrane biogenesis differentially expressed in the bvrR mutant.
As expected, these included genes that encoded OMPs like Omp25a (BAB1_0722) and Omp25d (BAB1_0115) which were down-regulated.
Other bvrR regulated genes related with cell envelope were: three lipoprotein genes (BAB1_0358; BAB1_0589; BAB1_2147), which were down-regulated; six genes for periplasmic proteins and chaperones (htpX, heat shock protein, BAB1_1821; clpA and clpB, stress response proteins, BAB1_1573 and BAB1_1868, respectively; BAB2_1107; BAB1_0505; BAB1_1022), which were all up-regulated; one gene related with LPS biosynthesis (glycosyl transferase, BAB1_1620), which was up-regulated; and five genes for fatty acids biosynthesis (fabG, ketoacyl-acyl-carrier-protein reductase, BAB1_2043; fabF, oxoacyl-acyl-carrier-protein synthase, BAB1_0872; fadD, fatty-acyl-CoA synthase, BAB1_0320; cfa, cyclopropane-fatty-acyl-phospholipid synthase, BAB1_0476; BAB1_1357).
These data confirm that BvrR/BvrS regulates bacterial envelope changes that could modify surface properties relevant for Brucella virulence [6].
