Brucella PTS Figure 10. Model proposed for the role of the Brucella PTS in connecting C and N metabolisms. In agreement with the results of the in vitro and in vivo phosphorylation assays (Fig. 1–4), we postulate that a phosphoryl group is sequentially transferred from PEP to EINtr, NPr, and finally to the EIIAMan-like protein. By binding both an unknown ligand (possibly 2-oxoglutarate) through its GAF domain and autophosphorylating in response to the PEP/pyruvate ratio, EINtr might sense the metabolic status of the cell and communicate it to the EIIAMan-like protein that would regulate the 2-oxoglutarate dehydrogenase activity accordingly. In addition, HprK/P is expected to slow phosphorylation of the EIIAMan-like protein by hindering the phosphotransfer through the PTS in response to changes in the FBP concentration or other metabolites. Solid arrows indicate metabolic reactions, large open arrows represent phosphoryl transfer between PTS proteins, and dashed arrows show putative regulatory processes. The enzyme HprK/P was also included in this scheme since it phosphorylates NPr on a conserved serine. The names of the genes encoding B. melitensis PTS and HprK/P proteins are put between brackets. The pentose phosphate pathway, which connects glucose-6-phosphate (G6P) to glycolysis in B. melitensis, and the connection between TCA cycle and nitrogen metabolism starting from 2-oxoglutarate are indicated in grey. FBP, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate; CoA, coenzyme A; TCA cycle, tricarboxylic acid cycle; NH4+, ammonium. P,H-X and P,S-X indicate PTS enzymes phosphorylated on histidine or serine, respectively. doi:10.1371/journal.pone.0012679.g010 made for HprK/P from Treponema denticola [60], M. pneumoniae [67], N. meningitidis (S. Poncet, M.-K. Taha, M. Laribe and J. Deutscher, unpublished) and A. tumefaciens (I. Mijakovic, A. Khemiri and J. Deutscher, unpublished). In the case of a- proteobacteria, the poor phosphorylase activity of HprK/P might be due to the absence of a C-terminal conserved region required for P-Ser-HPr dephosphorylation (Fig. S5) [30,41,42]. In M. pneumoniae and possibly other bacteria, dephosphorylation of P- Ser-HPr seems to be catalyzed by a protein phosphatase of the PP2C family [67]. 2- A link between PTS and virulence of Brucella It seems that PTS-mediated carbon source utilization can affect host-bacteria interactions [61,68]. B. melitensis pts mutants were previously shown to be attenuated [19,20,21] but the underlying mechanism remained unknown. During this work two lines of evidence for a link between PTS and virulence emerged. First, we demonstrate a transcriptional link between the PTS genes hprK, ptsM and ptsO and the bvrR/S genes (Fig. 5) encoding a two component system crucial for virulence of B. melitensis. In all pathogenic or symbiotic a-proteobacteria the pts genes are located downstream from the two-component system genes essential for infection or symbiosis [33,34,35]. In addition, a recent transcrip- tome analysis with B. abortus showed that hprK (BAB1_2094) was downregulated in the bvrR:Tn5 mutant [69]. It is therefore tempting to assume that the PTS might also be involved in virulence regulation, possibly via a cross-talk between PTS proteins and the two-component system. This concept is also supported by the finding that deletion of ptsP or ptsO, but not ptsN or hprK, lowers the production of a major virulence factor, the type IV secretion system VirB, by reducing virB gene expression (Fig. 6). The expression of Brucella spp. virB has previously been shown to be controlled by nutrient availability via an unknown mechanism [46,48,51]. The Bartonella henselae BvrR/S homologues BatR/S, whose genes are also followed by hprK, ptsM and npr, have been reported to control virB expression and BatR binds to the virB promoter region [70]. It is therefore tempting to assume that the B. melitensis PTS communicates the metabolic state of the cell to the virB promoter by phosphorylating or interacting with the two component system BvrR/S. However, we cannot exclude the possibility that PTS components interact with one of the two other transcriptional regulators known to bind to the virB promoter: the quorum sensing regulator VjbR [52,71] and HutC, a transcrip- tional repressor of the histidine utilization (hut) genes [72]. PLoS ONE | www.plosone.org 10 September 2010 | Volume 5 | Issue 9 | e12679