However, in the DhprK mutant an additional faster migrating band corresponding to P,EIIANtr was present. The absence of P-Ser- NPr, which is probably a poor substrate for the PEP-dependent phosphorylation, apparently allows significant phosphoryl transfer to EIIANtr. The PTS of B. melitensis is transcriptionally linked to the BvrR/S two component system The gene order around B. melitensis hprK, ptsM and ptsO is conserved in other a-proteobacteria and is as follows: (i) a transcriptional response regulator and (ii) a sensor kinase of a two- component system known to be involved in host-symbiont (chvI- exoS in S. meliloti [34]) or host-pathogen interaction (chvI-chvG in A. tumefaciens [33]; bvrR-bvrS in B. abortus [35]), (iii) hprK, (iv) ptsM, (v) ptsO, and finally (vi) sahH, which encodes an enzyme involved in the biosynthesis of methionine [36] (Fig. 5A). An additional gene called pckA that encodes PEP carboxykinase, a key enzyme of gluconeogenesis [39,40] is oriented in opposite direction to this cluster (Fig. 5A). In order to see whether this conserved organization reflects a functional link between these genes, we tried to determine whether they were transcriptionally linked. For that purpose, we performed PCR assays using cDNA of B. melitensis 16M as template (Fig. 5B). Positive and negative control experiments were performed by using as template either genomic DNA or DNase-treated RNA in the absence of reverse transcriptase, respectively. When using cDNA as template we camplified intragenic regions of each gene of the cluster (Fig. 5A and B, bars and lanes 2 to 7), confirming that these genes are expressed in cells tat have grown in rich medium to late exponential phase. PCR products were also obtained for the intragenic regions of ptsP and ptsN (Fig. 5B, lanes 8 and 9, respectively), and for the neibhouring pckA gene (Fig. 5A and B, bar and lanes 1). The use of appropriate primers and cDNA as template also allowed the amplification of intergenic regions (lanes 11 to 15 in Fig. 5B), demonstrating that the following pairs of genes are co-transcribed: bvrR-bvrS, bvrS-hprK, hprK-ptsM, ptsM-ptsO and ptsO-sahH (Fig. 5B). As expected we could not amplify by RT- PCR the intergenic region between pckA and bvrR, two genes Brucella PTS oriented in opposite directions (Fig. 5A and B, bar and lanes 10). In conclusion, we demonstrated that the B. melitensis pts genes and hprK are expressed during vegetative growth and that hprK, ptsM and ptsO can be co-transcribed with bvrR/S and sahH. DptsP and DptsO mutants barely produce VirB5 and VirB10 Knowing that transpositional PTS mutants of B. melitensis are attenuated [19,20,21] and having demonstrated a transcriptional link with several pts genes and the genes for the BvrR/S two component system, which regulates major virulence determinants [35], we wanted to investigate a possible link between PTS and virulence by constructing deletion mutants of the corresponding genes. Since hprK, ptsM and ptsO are probably organized in an operon with bvrR, bvrS and sahH, we chose to construct the mutants by allelic replacement using the non-polar cassette aphA4 as previously described [46]. Mutants were obtained for the ptsP, ptsO, ptsN and hprK genes. Despite numerous attempts, we were not able to delete ptsM. VirB is a major virulence factor of Brucella spp. composed of twelve subunits encoded in the virB operon [47,48,49,50], that is induced in response to nutrient availability [46,51,52] and controlled by (p)ppGpp, a bacterial alarmone that mediates global physiological control in response to starvation [46]. Since PTS proteins are also involved in global regulation in response to nutrient supply (for review see [1]), we examined the role of pts genes and hprK in the control of virB expression. Western blot analyses using anti-VirB5 and anti-VirB10 antisera [53] were performed to determine the relative amounts of VirB5 and VirB10 proteins in DptsP, DptsO, DptsN and DhprK mutants compared to the wild-type strain B. melitensis 16M. Crude extracts of bacteria grown in 2YT to late exponential – early stationary phase (OD600 of 0.8–1.2) were prepared and analyzed by Western blot. The DptsN and DhprK mutants produced VirB5 and VirB10 in amounts similar to those of the wild-type strain (Fig. 6A). However, no or very little VirB5 and VirB10 were detected in extracts prepared from DptsP and DptsO mutants, suggesting that EINtr and NPr are required for production or stability of several B. melitensis VirB Figure 5. Transcriptional link between pts genes and the genes encoding the two-component system BvrR/BvrS. (A) Schematic representation of the genomic region encoding hprK, ptsM and ptsO in B. melitensis 16M. The regions amplified (1–15) by RT-PCR are indicated and the primers are listed in Table S2. (B) Agarose gel of the RT-PCR amplified products. For each primer pair, three lanes are shown: a, positive control using B. melitensis16M genomic DNA as template; b, RT-PCR; and c, a negative control using RNA as template (without RT). Identical results were obtained in several independent experiments. doi:10.1371/journal.pone.0012679.g005 PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e12679