While no differences were observed between growth of the four mutants and the wild-type strain when cultivated in liquid 2YT medium (Fig. 7C), the DptsO, DptsP and to a lesser extent the DptsN and DhprK mutants displayed a heterogeneity in colony size compared to the wild-type strain when plated on solid 2YT rich medium (Fig.7A and C). This is reminiscent of a similar defect described for pts mutants of S. meliloti grown on solid media [61]. These small colonies resemble a phenotype called small colony variant (SCV). The presence of SCVs in pathogenic bacteria, including B. abortus, has often been associated with the persistence in the host [73,74,75,76,77]. It will be interesting to test whether pts and hprK mutants persist longer than the wild-type during mice infection, as was described for a SCV of the B. abortus vaccinal strain S19 [73]. 3- Carbon catabolite repression and the coordination of carbon and nitrogen metabolism In many bacteria the PTS is linked to carbon catabolite repression (CCR). Interestingly, in the a-proteobacterium S. meliloti HprK/P regulates succinate-mediated CCR [32]. In Brucella, erythritol is the most favoured carbon source and is able to inhibit glucose incorporation [78], but to our knownledge the underlying mechanism is not known and diauxic growth has not been reported. CcpA as well as Crp [79] and adenylate cyclase seem to be absent from Brucella spp. If general CCR exists in Brucella, it should therefore differ from the E. coli and B. subtilis CCR mechanisms. The PTSNtr proteins (EINtr, NPr and EIIANtr) have previously been suggested to provide a regulatory link between carbon and nitrogen metabolism [3,4,5,6,11,80,81]. Additionally, recent ‘‘in silico’’ analyses suggest that some of the diverse regulatory PTS functions acquired during evolution serve to assure an appropriate balance in C and N supply [82]. Key signals of C and N supply in E. coli appear to be the levels of glutamine and 2-oxoglutarate, the latter being at the crossroad between carbon and nitrogen metabolism [83]. Several results reported in our paper converge on these metabolites and prompted us to propose a model linking the PTS to the maintenance of the carbon and nitrogen balance in B. melitensis 16M: First, the EIIAMan-like protein interacts with the SucA subunit of 2-oxoglutarate dehydrogenase (Fig. 8 and 9). Second, the enzyme EINtr possesses an N-terminal GAF domain (Fig. S1). This domain is known to regulate the activity of NifA from A. vinelandii by binding 2-oxoglutarate [84,85]. Finally, three PTS genes are transcriptionaly linked to the genes encoding the two component system BvrR/S (Fig. 5). The latter finding supports a link between regulation of C and N metabolism and PTSNtr components because a proteomic study with a B. abortus bvrR mutant [86] revealed that two 2- oxoglutarate-dependent proteins are regulated by BvrR-BvrS: the first is the PII sensor protein that controls nitrogen metabolism and that was shown to bind 2-oxoglutarate [83,87]; the second is the 2-oxoglutarate dehydrogenase complex that converts 2- oxoglutarate into succinyl-CoA in the TCA cycle. This is the very same enzyme whose subunit SucA interacts with the EIIAMan-like protein (Fig. 8 and 9). We therefore propose a model in which EINtr senses the metabolic status of the cell via the PEP/pyruvate ratio [62]. The existence of a GAF domain in EINtr provides a link between GAF- sensed signals (a-ketoglutarate [84,85] or other ligands [88]) and PTS phosphoryl transfer. The signals (EI phosophorylation state and HprK/P activity) are transmitted to the EIIAMan-like protein, Brucella PTS which in turn regulates the activity of 2-oxoglutarate dehydroge- nase (Fig. 10). In this model, the dephospho EIIAMan-like protein is predicted to interact with and to inhibit 2-oxoglutarate dehydrogenase. Indeed, exclusively dephospho EIIAMan is prob- ably present in yeast during two-hybrid tests, where the EIIAMan/ SucA interaction was first detetcted. Finally, one can envisage that HprK/P might control EIIAMan-dependent regulation of 2- oxoglutarate dehydrogenase. Similar as observed for HPr from firmicutes [1,64,65], HprK/P-catalyzed phosphorylation of Ser61 of NPr probably slows phosphorylation of His30 and thus increases the amount of dephospho EIIAs (Fig. 4). It will also be interesting to test whether the N-terminal domain of EINtr is able to bind 2-oxoglutarate and whether this ligand can modulate the phosphotransfer activity of the PTS protein. It might also be worth studying the enzymatic activity of 2-oxoglutarate dehydrogenase in different mutant backgrounds. Finally, our model should be tested with other a-proteobacteria possessing homologues of the PTS regulatory proteins and the crucial two component system encoded by genes arranged in a strictly conserved genomic context. Materials and Methods Ethics statement Animal handling and experimental protocol was in accordance with European (DOCE 86/609/EEC), and National (AR25/04/ 2004) directives, and was supervised and authorized by the Ethical Committee of the University of Namur (FUNDP) (Commission d’e´thique en experimentation animale approval Nu FUNDP08/ 106). Bacterial strains and growth conditions All Brucella strains used in this study were derived from Brucella melitensis 16M NalR (Table S1) and were routinely cultivated in 2YT complex medium (10% yeast extract, 1% tryptone and 0.5% NaCl). E. coli strains (Table S1) were cultivated in Luria Bertani (LB) medium. Antibiotics were used at the following concentra- tions when appropriate: nalidixic acid, 25 mg/ml; kanamycin, 50 mg/ml; chloramphenicol, 20 mg/ml; ampicillin, 100 mg/ml; gentamycin, 50 mg/ml. To observe the colony size heterogeneity of pts and hprK mutants, overnight cultures were adjusted to an OD600 of 0.05 in 2YT complex medium and grown at 37uC with constant shaking until late log phase (OD600 of 1.0). Dilutions of these cultures were plated on 2YT agar supplemented with appropriate antibiotics and incubated for 8 to 10 days at 37uC. To evaluate growth of the pts and hprK mutants in liquid cultures, overnight cultures were diluted to an OD600 of 0.05 in 2YT complex medium and grown at 37uC with constant shaking. The experiment was carried out twice. Construction of overexpression plasmids For the construction of overexpression plasmids, the B. melitensis ORFeome entry vectors [89] bearing ptsN, ptsM and hprK (pDONR201-ptsN, ptsM and hprK, respectively – Table S1) were checked by DNA sequencing before they were used to amplify by PCR the ptsN, ptsM and hprK genes with oligonucleotide pairs SP67-SP68, SP69-SP70, and SP74-SP75 respectively (Table S2). The ptsN-, ptsM- and hprK-PCR products were digested with BamHI and KpnI and cloned into pQE30 (Table S1) digested with the same enzymes, resulting in plasmids pQE30-ptsN, -ptsM- and –hprK and encoding (His)6-EIIANtr, (His)6-EIIAMan-like and (His)6-HprK/P, respectively (Table S1). The correct sequence of all PCR products was confirmed by DNA sequencing. PLoS ONE | www.plosone.org 11 September 2010 | Volume 5 | Issue 9 | e12679