called the nitrogen PTS (PTSNtr) [4,5,6,7]. The phosphoryl transfer chain of this system is composed of three proteins, EINtr (encoded by ptsP), NPr (encoded by ptsO) and EIIANtr (encoded by ptsN) that are the respective paralogs of EI, HPr, and EIIA of the fructose PTS family; however, they are not associated with PTS permeases [4,5,6,7] but carry out multiple regulatory functions [8]. For example, the PTSNtr is involved in the regulation of genes related to nitrogen metabolism [9,10,11,12]. Moreover, compared to EI, the EINtr possesses an N-terminal extension homologous to the GAF N-terminal sensory domain of NifA from Azotobacter vinelandii, an activator that enhances transcription by s54- associated RNA polymerase [13]. Finally, PTSNtr might favor the utilization of organic nitrogen compounds when bacteria are exposed to multiple carbon sources [4,11,12,14] and is involved in maintaining K+ homeostasis in E. coli [15]. Brucella spp. are Gram negative intracellular pathogens belonging to the a-proteobacteria group which includes other bacteria interacting with eukaryotic hosts, such as Agrobacterium tumefaciens or Sinorhizobium meliloti [16]. They are responsible for brucellosis, a worldwide zoonosis that affects a broad range of mammals [17], and can also infect humans where it may cause Malta fever, a serious debilitating chronic disease [18]. Large-scale screens aiming at the isolation of attenuated transpositional mutants of Brucella spp. led to the identification of many genes involved in carbon and nitrogen metabolism [19,20]. Moreover, genes encoding homologues of the three components of the E. coli PTSNtr were also isolated during these screens [19,20,21]. These data suggest that carbon and nitrogen metabolism might affect the virulence of Brucella. The availability of the genome sequence of several Brucella species [22,23,24] allowed the identification of an additional PTS- related gene putatively encoding an EIIA belonging to the mannose PTS family. Moreover, a gene encoding a truncated homologue of HPr kinase/phosphorylase (HprK/P) was found in Brucella genomes [25,26,27]. In most firmicutes (Gram positive bacteria with low GC content), HprK/P catalyses the phosphor- ylation and dephosphorylation of a conserved serine residue in HPr (usually Ser46) [28,29,30]. In these bacteria, HPr phosphor- ylated on this conserved serine (P-Ser-HPr) is a central regulator of carbon metabolism mediating among others inducer exclusion and acting as a co-repressor of the catabolite control protein A (CcpA) during CCR [1,31]. Similarly to Brucella spp, other a-proteobacteria including S. meliloti [32] and A. tumefaciens (S. Poncet, A. Khemiri and J. Deutscher, unpublished) possess the predicted PTSNtr proteins, as well as an EIIAMan-like protein and HprK/P, and lack PTS permeases. It was therefore suggested [25,26,27] that Brucella PTS proteins might form a phosphoryl transfer chain exclusively dedicated to regulatory functions. Interestingly, in these three bacteria, the genes ptsO, ptsM and hprK (encoding respectively NPr, an EIIAMan-like protein and HprK/P) are localized close to genes encoding (i) a two-component system involved in virulence or symbiosis (BvrR-BvrS in Brucella spp., ChvI-ChvG in A. tumefaciens and ChvI-ExoS- in S. meliloti) [33,34,35], (ii) S-adenosyl homocys- teine hydrolase (SahH), an enzyme involved in the metabolism of methionine [36,37] and (iii) PEP carboxykinase, a key enzyme of gluconeogenesis [38,39,40]. As previously proposed by Hu and Saier [27], the conservation of this genomic locus in several a- proteobacteria suggests a functional link between PTS, HprK/P and the neighboring genes in regulating carbon/nitrogen metabolism in these organisms. In this report, we demonstrate by in vitro and in vivo experiments that the PTS proteins found in B. melitensis function in a phosphorelay. Moreover, we observed that NPr is phosphorylated Brucella PTS not only by EINtr on His-30, but also by HprK/P on a conserved serine (Ser-61). This latter phosphorylation slows the in vivo phosphotransfer to His66 in EIIANtr. We also demonstrated a transcriptional link between the PTS genes ptsO, ptsM and hprK and the two component system genes bvrR/S establishing a link between virulence and metabolism that was reinforced by the observation that both, ptsP and ptsO mutants, almost completely lost the synthesis of a type IV secretion system (T4SS). Finally by carrying out a yeast two hybrid screen against the whole B. melitensis ORFeome we identified several interaction partners of PTS proteins allowing us to propose a preliminary model of regulation for carbon and nitrogen metabolism in B. melitensis. Results The Brucella melitensis 16M genome encodes four PTS proteins and HPr kinase/phosphorylase The genome of Brucella melitensis 16M [22] contains three genes (ptsP/BMEI0190, ptsO/BMEI2031 and ptsN/BMEI1786) encod- ing homologues of the proteins composing the PTSNtr (EINtr, NPr and EIIANtr, respectively) and ptsM/BMEI2032 encoding a homologue of an EIIA of the mannose PTS family (EIIAMan- like). A homologue of HprK/P (encoded by hprK/BMEI2034) is also found in Brucella. All these genes are highly conserved in the genome of other sequenced Brucella species. Sequence analyses and multiple aligments of these five proteins were carried out and allowed the prediction of the phosphorylatable histidine or serine residues (see Figures S1 to S5). Similar to its homologues in A. tumefaciens and S. meliloti, B. melitensis EINtr contains a GAF domain resembling the sensory domain of the NifA protein of A. vinelandii [6]. The GAF domains are ubiquitous motifs present in many sensory proteins of eukaryotes and prokaryotes and are proposed to allosterically regulate catalytic activities of these proteins through the binding of small molecules [13]. When compared to HprK/Ps from firmicutes, HprK/Ps from a-proteobacteria lack about 130 N-terminal amino acids. The role of this domain is still unknown and artificially truncated L. casei HprK/P (missing the first 127 amino acids) retained kinase and phosphorylase activities and all known regulatory properties [41]. Moreover, a carboxy- terminal conserved region is present in HprK/P from firmicutes and most b-, c- and d-proteobacteria, but absent from a- proteobacteria. This region was shown to be important for the phosphorylase activity of HprK/P, suggesting that in a-proteo- bacteria HprK/P might not be able to efficiently dephosphorylate P-Ser-NPr or dephosphorylate it by a different enzyme [30,42]. Based on the sequence analyses, we propose that EINtr autopho- sphorylates on His357 in the presence of PEP and subsequently transfers its phosphoryl group to His30 of NPr, which then phosphorylates EIIANtr and the EIIAMan-like protein on His66 and His9, respectively. Moreover, we suggest that HprK/P might phosphorylate NPr on the conserved Ser61. These hypotheses were tested by carrying out in vitro phosphorylation assays with purified proteins. Phosphoryl group transfer from P,EI to the EIIAs via P,His-NPr To carry out in vitro phosphorylation assays the ptsP, ptsN, ptsM and ptsO genes as well as a mutated ptsO allele (ptsOH30A) causing a His30Ala replacement in NPr, were inserted into a His6 tag expression vector and the resulting fusion proteins were purified as described in Materials and Methods (see also Fig. 1A for the purification of NPr, EIIANtr and EIIAMan-like). We first tested the ability of EINtr to phosphorylate NPr on His30 in a PEP- dependent reaction. NPr was not phosphorylated when incubated PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12679