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Conceived and designed the experiments: JD XDB JJL. Performed the experiments: MD SP CN RC HB AM. Analyzed the data: MD CN JD XDB JJL. Wrote the paper: MD JD JJL. Helped draft the manuscript: SP XDB.
In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions.
In vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not) established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among α-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA) of 2-oxoglutarate dehydrogenase.
The B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which this regulatory PTS coordinates also C and N metabolism.
In order to successfully colonize an ecological niche, bacteria have to integrate different signals indicating environmental changes, and subsequently trigger an adequate adaptative response by modulating their cellular activities. The appropriate response to changes in nutrient availability, for example, relies on diversified mechanisms, including global regulation systems such as the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS). The PTS catalyzes the uptake and concomitant phosphorylation of carbohydrates and is composed of several proteins forming a phosphorelay transferring the phosphoryl group from PEP to the incoming sugar: (i) the general PTS proteins enzyme I (EI) and HPr are cytoplasmic components usually common to all PTS carbohydrates; (ii) the enzyme II complex is specific for one or several sugars and is generally composed of at least three domains (or distinct proteins) including the cytoplasmic EIIA and EIIB, and the membrane-crossing EIIC (sometimes also EIID) that constitutes the permease of the system , . PTS proteins are usually phosphorylated on a conserved histidine, with the exception of most EIIB components that are phosphorylated on a cysteine. Besides its function in the transport and phosphorylation of carbon sources, the PTS plays a key role in the regulation of many aspects of bacterial physiology, including carbon catabolite repression (CCR) (for reviews see , , ).
Interestingly, a paralog of the classical PTS was proposed to function as a regulatory link between carbon and nitrogen metabolism. This system was first identified in Escherichia coli and called the nitrogen PTS (PTSNtr) , , , . 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 , , ,  but carry out multiple regulatory functions . For example, the PTSNtr is involved in the regulation of genes related to nitrogen metabolism , , , . 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 σ54-associated RNA polymerase . Finally, PTSNtr might favor the utilization of organic nitrogen compounds when bacteria are exposed to multiple carbon sources , , ,  and is involved in maintaining K+ homeostasis in E. coli .
Brucella spp. are Gram negative intracellular pathogens belonging to the α-proteobacteria group which includes other bacteria interacting with eukaryotic hosts, such as Agrobacterium tumefaciens or Sinorhizobium meliloti . They are responsible for brucellosis, a worldwide zoonosis that affects a broad range of mammals , and can also infect humans where it may cause Malta fever, a serious debilitating chronic disease . 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 , . Moreover, genes encoding homologues of the three components of the E. coli PTSNtr were also isolated during these screens , , . These data suggest that carbon and nitrogen metabolism might affect the virulence of Brucella.
The availability of the genome sequence of several Brucella species , ,  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 , , . In most firmicutes (Gram positive bacteria with low GC content), HprK/P catalyses the phosphorylation and dephosphorylation of a conserved serine residue in HPr (usually Ser46) , , . In these bacteria, HPr phosphorylated 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 , .
Similarly to Brucella spp, other α-proteobacteria including S. meliloti  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 , ,  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) , , , (ii) S-adenosyl homocysteine hydrolase (SahH), an enzyme involved in the metabolism of methionine ,  and (iii) PEP carboxykinase, a key enzyme of gluconeogenesis , , . As previously proposed by Hu and Saier , the conservation of this genomic locus in several α-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 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.
The genome of Brucella melitensis 16M  contains three genes (ptsP/BMEI0190, ptsO/BMEI2031 and ptsN/BMEI1786) encoding 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 . 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 . When compared to HprK/Ps from firmicutes, HprK/Ps from α-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 . Moreover, a carboxy-terminal conserved region is present in HprK/P from firmicutes and most β-, γ- and δ-proteobacteria, but absent from α-proteobacteria. This region was shown to be important for the phosphorylase activity of HprK/P, suggesting that in α-proteobacteria HprK/P might not be able to efficiently dephosphorylate P-Ser-NPr or dephosphorylate it by a different enzyme , .
Based on the sequence analyses, we propose that EINtr autophosphorylates 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.
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 with [32P]PEP (Fig. 1B, lane 3). In agreement with our prediction (Fig. S4), the additional presence of EI allowed the phosphorylation of wild-type NPr (Fig. 1B, lane 4) and NPrS61A (data not shown), but not of NPrH30A (Fig. 1B, lane 2). We subsequently tested whether B. melitensis EIIANtr and the EIIAMan-like protein were phosphorylated by P~His-NPr. Incubation of EIIANtr or the EIIAMan-like protein with [32P]PEP (data not shown) or [32P]PEP and EINtr (Fig. 1B, lanes 5 and 6) did not allow their phosphorylation. By contrast, EIIANtr and the EIIAMan-like protein were phosphorylated by [32P]PEP in the presence of both general PTS proteins EINtr and NPr, establishing that after its own phosphorylation on His30 by EINtr, P~His-NPr is able to transfer its phosphoryl group to EIIANtr and the EIIAMan-like protein (Fig. 1B, lanes 7 and 8). Purification of the EIIAMan-like protein provided always two distinct forms migrating to slightly different positions on SDS polyacrylamide gels (Fig. 1A), which apparently became both phosphorylated by P~His-NPr. The reason for the appearance of two EIIAMan-like forms is not known, but a similar observation has been reported for EIIABMan from Streptococcus salivarius, which was also isolated in two distinct forms migrating to slightly different positions on SDS polyacrylamide gels .
The truncated HprK/P possibly adds an additional dimension of regulation to the B. melitensis PTS by phosphorylating NPr on a conserved serine residue (Ser61; see Fig S4). We therefore tested the ability of HprK/P to phosphorylate NPr on Ser61. As for the pts genes, we cloned the hprK-coding sequence in an expression vector and purified the His6-tagged fusion protein. As shown in Fig. 2A, HprK/P phosphorylated wild type NPr and NPrH30A in an ATP-dependent way, whereas NPrS61A was not phosphorylated.
HprK/Ps of firmicutes possess antagonistic kinase (HPr phosphorylation) and phosphorylase (P-Ser-HPr dephosphorylation) activities, which are regulated by intracellular concentrations of inorganic phosphate (Pi) and glycolytic intermediates, such as fructose-1,6-bisphosphate (FBP) , , . Indeed, the ATP-dependent kinase activity of HprK/P from B. subtilis is stimulated by FBP, but inhibited by Pi, which is also one of the substrates in the phosphorylase reaction. Moreover, in addition to ATP, HprK/P can also use pyrophosphate (PPi), the product of the HprK/P-catalyzed phosphorylase reaction, as phosphate donor . The effect of increasing concentrations of FBP on ATP- and PPi-dependent kinase activities of B. melitensis HprK/P was tested. With both phosphoryl group donors, HprK/P was active as a kinase in the absence of FBP. Moreover, FBP has no stimulatory effect on the kinase activity in the absence of Pi (data not shown). Nevertheless, in the presence of 0.5 mM Pi, increasing concentrations of FBP (up to 10 mM) enhanced the ATP-dependent kinase activity, whereas under the same conditions almost no stimulatory effect was observed on the PPi-dependent activity (Fig. 2B). Similar results have been reported for L. casei HprK/P . Kinase activity assays were also carried out in the presence of increasing concentrations of Pi. As observed for all studied Gram-positive HprK/Ps, the addition of Pi resulted in an inhibition of both the ATP- and PPi-dependent kinase activities of B. melitensis HprK/P (Fig. 2C).
We tested whether B. melitensis HprK/P also exhibits Pi-requiring phosphorylase activity. Even at high Pi concentrations (25 mM), P~Ser-NPr was barely dephosphorylated by B. melitensis HprK/P (Fig. 3). In HprK/P of firmicutes, Pi binds to the same site as PPi and the β-phosphate of ATP, which is thought to be responsible for the inhibition of the ATP- and PPi-dependent kinase functions. However, although B. melitensis HprK/P seems to bind Pi, because its ATP- and PPi-dependent kinase activities are inhibited by Pi (Fig. 2C), B. melitensis HprK/P failed to promote efficient P~Ser-NPr dephosphorylation. This seems to be the case for HprK/P from other proteobacteria, such as A. tumefaciens (I. Mijakovic, A. Khemiri and J. Deutscher, unpublished) and Neisseria meningitidis (S. Poncet, M.-K. Taha, M. Larribe and J. Deutscher, unpublished).
To ascertain that the PTS phosphorylation cascade is also functional in vivo we used Western blots in order to demonstrate the presence of P~EIIANtr in B. melitensis crude extracts. This was possible because we demonstrated with purified proteins that EIIANtr and P~EIIANtr can be separated on non-denaturing polyacrylamide gels, with P~EIIANtr migrating significantly faster than EIIANtr (data not shown). Extracts were prepared from the wild-type strain and the ΔptsP, ΔptsO and ΔhprK mutants grown in rich medium to exponential phase (OD600=0.8). and aliquots containing 60 µg of protein were loaded on a non-denaturing polyacrylamide gel. EIIANtr and P~EIIANtr were separated by electrophoresis and detected by Western blotting with anti-EIIANtr polyclonal antibodies. Under the conditions employed, only the slower migrating EIIANtr band could be detected in extracts of the wild-type strain and the ΔptsP and ΔptsO mutants (Fig. 4). However, in the ΔhprK 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 gene order around B. melitensis hprK, ptsM and ptsO is conserved in other α-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 ) or host-pathogen interaction (chvI-chvG in A. tumefaciens ; bvrR-bvrS in B. abortus ), (iii) hprK, (iv) ptsM, (v) ptsO, and finally (vi) sahH, which encodes an enzyme involved in the biosynthesis of methionine  (Fig. 5A). An additional gene called pckA that encodes PEP carboxykinase, a key enzyme of gluconeogenesis ,  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 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.
Knowing that transpositional PTS mutants of B. melitensis are attenuated , ,  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 , 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 . 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 , , , , that is induced in response to nutrient availability , ,  and controlled by (p)ppGpp, a bacterial alarmone that mediates global physiological control in response to starvation . Since PTS proteins are also involved in global regulation in response to nutrient supply (for review see ), 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  were performed to determine the relative amounts of VirB5 and VirB10 proteins in ΔptsP, ΔptsO, ΔptsN and ΔhprK 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 ΔptsN and ΔhprK 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 ΔptsP and ΔptsO mutants, suggesting that EINtr and NPr are required for production or stability of several B. melitensis VirB subunits (Fig. 6A). Complementation of the ΔptsO mutant with wild-type ptsO constitutively expressed from a low copy plasmid fully restored VirB10 (Fig. 6B) and VirB5 production (data not shown). For unknown reasons, plasmid-encoded ptsP did not restore VirB5 or VirB10 production in the ΔptsP mutant, although the plasmid was functional because it complemented the “small colony” phenotype of the ΔptsP mutant (see Fig. 7B). Knowing that EINtr is strictly required for P~His-NPr formation we tried to complement the ΔptsO mutant with the mutant alleles ptsOH30A and ptsOS61A. Constitutive expression of ptsO and ptsOS61A in ΔptsO restored VirB10 production, whereas the ΔptsO/ptsOH30A strain failed to produce VirB10 (Fig. 6B). This confirms that P~His-NPr is needed for VirB 10 synthesis and consequently that EINtr is also required.
In order to confirm the impact of the ptsP mutations on T4SS expression we carried out a transcriptional analysis of the whole virB operon with the wild-type strain and the ptsP mutant. Indeed, the expression level of the individual virB genes was more than thirty times lower in the ptsP mutant than in the wild-type strain (Fig. 6C)
When plated on 2YT rich medium, the ΔptsP, ΔptsO, ΔptsN and, to a lesser extent, the ΔhprK mutant displayed a heterogeneity in colony size compared to the wild-type strain B. melitensis 16M (Fig. 7A). Small colonies were detected only 8 to 10 days after inoculation, whereas the larger colonies were visible after 3 to 4 days as usually observed for the wild-type strain. To ensure that the colony size heterogeneity of pts and hprK mutants resulted from the deletion of the corresponding genes, we first carried out a complete typing of these strains confirming that they all derived from B. melitensis 16M wild-type, exhibited a smooth phenotype, and were not contaminated with other strains (data not shown). Next, we complemented the two mutants with the most marked phenotype (ΔptsP and ΔptsO) by constitutively expressing wild-type copies of the ptsP and ptsO genes in the corresponding mutants. As shown in Fig. 7B, the complemented strains ΔptsP/ptsP and ΔptsO/ptsO displayed bigger colonies than the ΔptsP and ΔptsO mutants transformed with the empty vector-pMR10cat. Their colonies resembled those of the wild-type strain carrying the empty vector-pMR10cat.
Finally, we measured growth of the four mutants when cultivated in liquid 2YT medium (Fig. 7C). No differences were observed between growth of the mutants and the wild-type strain, suggesting that the growth heterogeneity observed on solid medium might not result from the composition of the medium, but rather from parameters that distinguish liquid and solid cultures, such as oxygen supply, nutrient or water availability.
Since in B. melitensis 16M two pts genes, hprK and the two-component system genes bvrR/bvrS are co-transcribed and functionally linked we wanted to test if there existed any physical interactions between PTS components, HprK/P and the BvrR/S proteins. A yeast two hybrid (Y2H) interaction matrix of 64 interactions was performed with the four PTS proteins, HprK/P, BvrR and BvrS fused to the Gal4 DNA binding domain (BD) and tested against the same proteins fused to the Gal4 activating domain (AD). Each BD and AD fusion was also tested against Gal4-AD and Gal4-BD alone. The previously evidenced interaction between BvrR and BvrS  was used as a positive control. The results presented in Fig. 8 and S6 show that two PTS proteins (EINtr, EIIAMan-like) and HprK/P interacted with themselve, suggesting that these proteins form oligomers similar to some well-studied EI, EIIAMan and HprK/P homologues , , . Additionally, a bidirectional interaction was evidenced between NPr and HprK/P (Fig. 8 and S6) confirming the results of the in vitro phosphorylation test. No interaction was observed between other PTS proteins that were shown to phosphorylate each other in vitro. Finally, an interaction between BvrR and BvrS could be demonstrated (Fig. S6), but no interaction was detected between any of the PTS proteins or HprK/P and the two-component partners.
Having demonstrated that the incomplete PTS of B. melitensis is functional and knowing that PTS-dependent regulations are mediated either by allosteric interaction or by direct phosphorylation of target proteins , we performed a Y2H screen to detect interaction partners of the EIIAMan-like protein and NPr of B. melitensis 16M to get some preliminary clues about the functional role of this PTS. Briefly, these PTS proteins were fused to the Gal4 DNA binding domain and used as baits to identify interaction partners in an « ORFeomic » library. Two clones provided a positive signal with at least two of the three reporter genes and the corresponding proteins were identified as Ppa and SucA by sequencing the inserts in the pVV213 vector. NPr interacts with the inorganic pyrophosphatase PPa (BMEI0076) and the EIIAMan-like protein with the E1 component (SucA) (BMEI0140) of 2-oxoglutarate dehydrogenase.
In order to validate these interactions, the ORFeome entry clones for ptsO (NPr), ppa, hprK, sucA and ptsM (EIIAMan-like) were checked by sequencing and the coding sequences were subcloned in the Y2H vectors pVV212 and pVV213. Three interaction matrices were designed and the interactions between NPr and Ppa and the EIIAMan-like protein and SucA were confirmed (Fig. 8). In addition, a new interaction between PPa and HprK was established. SucA is the E1 component of the 2-oxoglutarate dehydrogenase complex, which contains also the dihydrolipoamide succinyltransferase SucB (E2 component) and dihydrolipoamide dehydrogenase (E3 component) and plays a crucial role in the TCA cycle by converting 2-oxoglutarate to succinyl-CoA and CO2. Knowing that the PTSNtr presumably links regulation of carbon and nitrogen metabolism ,  and that 2-oxoglutarate is at the cross-road between the TCA cycle and nitrogen assimilation, we tried to confirm the interaction between the EIIAMan-like protein and SucA by another independent method.
DivIVA is attracted to and remains at cell poles not only in its native organism, B. subtilis, but also in E. coli and other bacteria . In addition, DivIVA fused to a “bait” protein X can target an interacting GFP tagged “prey” protein Y to the pole . To confirm the interaction between the EIIAMan-like protein and SucA we fused DivIVA to SucA and the EIIAMan-like protein to GFP.
After arabinose induction, strain DH10B[pSKoriTcat-pBad-divIVA-gfp] synthesizing DivIVA-GFP exhibits fluorescens mainly at the cell poles (positive control; data not shown), whereas DH10B[pMR10kan-ptsM-gfp] producing EIIAMan-GFP (with or without arabinose induction) was uniformly fluorescent (Fig. 9B; negative control). DH10B bearing the two plasmids (pSKoriTcat-pBad-divIVA-sucA and pMR10kan-ptsM-gfp) showed a bipolar fluorescence pattern only when arabinose was present (Fig. 9C). This illustrates that SucA was targeted to the pole by DivIVA and able to recruit the EIIAMan-GFP fusion at the same location, thus confirming the interaction detected in the Y2H experiments.
Since PTS permeases are lacking in α-proteobacteria, their soluble PTS proteins are not involved in carbohydrate transport and phosphorylation, but probably participate only in a regulatory phosphorelay (Fig. 10) , , . In this paper, we present the first extensive biochemical and genetic characterization of the PTS components in an organism that lacks PTS permeases. A few studies on similar systems have previously been carried out, but were either limited to biochemical studies of PTS protein phosphorylation  or to genetic studies of mutants . First we established that in B. melitensis the phosphoryl group transfer from PEP to the EIIAs is fully functional. Second, several pieces of evidence allow us to propose a link between the PTS and the virulence of B. melitensis. Finally, we report a connection between the PTS and systems likely to maintain the N/C balance. These three points are discussed in detail hereunder.
B. melitensis EINtr autophosphorylates with PEP and transfers the phosphoryl group to the conserved His30 of NPr before it is passed on to either EIIANtr or the EIIAMan-like protein (Fig. 10). EINtr probably senses the PEP availability (PEP/pyruvate ratio), that is translated into relative levels of phosphorylated vs. non-phosphorylated forms of NPr and EIIAs . In addition to His30, NPr is also phosphorylated on a serine residue. Similar to firmicutes, B. melitensis possesses an HprK/P using ATP or PPi as phosphoryl donor. Only phosphorylation of NPr with ATP is stimulated by FBP (Fig. 2B). Identical observations were made for L. casei HprK/P . However, Brucella spp lack a 6-phosphofructokinase and consequently hexose catabolism does not occur via the Embden-Meyerhof-Parnas pathway, but is redirected through the pentose phosphate and perhaps the Entner-Doudorof pathway. Accordingly, the only pathway that is expected to produce FBP is gluconeogenesis . We therefore propose that, in contrast to firmicutes, the FBP signal sensed by B. melitensis HprK/P reflects gluconeogenetic instead of glycolytic activity. High gluconeogenetic flux will probably activate HprK/P (via FBP) (Fig. 2B), which in turn will slow the PEP-dependent phosphoryl transfer from P-Ser-NPr to the EIIAs (Fig. 4) , , .
Interestingly, an inorganic pyrophosphatase named PPa interacts with NPr and HprK/P in Y2H tests (Fig. 8). PPi serves as substrate for the kinase reaction and is formed during P-Ser-HPr dephosphorylation . Hydrolysis of PPi by PPa not only lowers the PPi concentration, but also produces inorganic phosphate (Pi), which inhibits both, ATP- and PPi-dependent kinase activities of B. melitensis HprK/P (Fig. 2C). Elevated PPa activity might therefore reduce phosphorylation on Ser61 of NPr. Similarly, Mijakovic  proposed that the B. subtilis pyrophosphatase YvoE (the yvoE gene is located in the hprK operon) indirectly decreases the kinase activity of HprK/P, and stimulates P-Ser-HPr dephosphorylation by HprK/P. The physical interaction of PPa with NPr and HprK/P might allow efficient regulation of HprK/P activity in B. melitensis. Alternatively, a link might exist between the PTS and the ppGpp production/degradation system (also called stringent response) as was demonstrated in E. coli . NPr might affect the PPa-catalyzed conversion of PPi to Pi and thus modulate the PPi-producing ppGpp degrading activity of Rsh (RelA/SpoT homologue).
Purified B. melitensis HprK/P barely dephosphorylated P-Ser-NPr under in vitro conditions (Fig. 3). Similar observations were made for HprK/P from Treponema denticola , M. pneumoniae , 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 α-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) , , . In M. pneumoniae and possibly other bacteria, dephosphorylation of P-Ser-HPr seems to be catalyzed by a protein phosphatase of the PP2C family .
It seems that PTS-mediated carbon source utilization can affect host-bacteria interactions , . B. melitensis pts mutants were previously shown to be attenuated , ,  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 α-proteobacteria the pts genes are located downstream from the two-component system genes essential for infection or symbiosis , , . In addition, a recent transcriptome analysis with B. abortus showed that hprK (BAB1_2094) was downregulated in the bvrR:Tn5 mutant . 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 , , . 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 . 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 ,  and HutC, a transcriptional repressor of the histidine utilization (hut) genes .
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 ΔptsO, ΔptsP and to a lesser extent the ΔptsN and ΔhprK 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 . 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 , , , , . 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 .
In many bacteria the PTS is linked to carbon catabolite repression (CCR). Interestingly, in the α-proteobacterium S. meliloti HprK/P regulates succinate-mediated CCR . In Brucella, erythritol is the most favoured carbon source and is able to inhibit glucose incorporation , but to our knownledge the underlying mechanism is not known and diauxic growth has not been reported. CcpA as well as Crp  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 , , , , , , . 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 . 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 . 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:
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  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 , ; 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 and99).
We therefore propose a model in which EINtr senses the metabolic status of the cell via the PEP/pyruvate ratio . The existence of a GAF domain in EINtr provides a link between GAF-sensed signals (α-ketoglutarate ,  or other ligands ) and PTS phosphoryl transfer. The signals (EI phosophorylation state and HprK/P activity) are transmitted to the EIIAMan-like protein, which in turn regulates the activity of 2-oxoglutarate dehydrogenase (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 probably 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 , , , 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 α-proteobacteria possessing homologues of the PTS regulatory proteins and the crucial two component system encoded by genes arranged in a strictly conserved genomic context.
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'éthique en experimentation animale approval N° FUNDP08/106).
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 concentrations when appropriate: nalidixic acid, 25 µg/ml; kanamycin, 50 µg/ml; chloramphenicol, 20 µg/ml; ampicillin, 100 µg/ml; gentamycin, 50 µg/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 37°C 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 37°C.
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 37°C with constant shaking. The experiment was carried out twice.
For the construction of overexpression plasmids, the B. melitensis ORFeome entry vectors  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.
The published genome sequence predicts a ptsO gene starting with a GUG codon and lacking a ribosome binding site (RBS) . We therefore assumed that NPr might be 4 amino acids longer and that the gene starts with an ATG preceded by a RBS. Accordingly, a new pDONR201 entry vector (pDONR201-ptsO) bearing a longer version of the ptsO gene was constructed. Briefly, the B. melitensis 16M ptsO CDS (BMEI2031) was amplified by PCR with genomic DNA with and the Gateway™ primers GWnprF and GWnprR (Table S2) and cloned in the entry vector pDONR201 (Invitrogen Life-technologies) as previously described . Directed mutagenesis of ptsO was performed with the QuickChange™ Site Directed Mutagenesis kit (Stratagene) using plasmid pDONR201-ptsO as a template. Primers used to obtain the ptsOH30A and ptsOS61A alleles are listed in Table S2. The correct sequence of all PCR products was confirmed by DNA sequencing. Plasmids pQE30-ptsO, -ptsOH30A and -ptsOS61A encoding (His)6-NPr and its two mutant forms, were obtained by amplification of the corresponding allele using oligonucleotides SP65 and SP66, and plasmids pDONR201-ptsO, -ptsOH30A and -ptsOS61A respectively, as templates. The PCR products were digested with BamHI and KpnI and cloned into pQE30 digested with the same enzymes.
The E. coli NM522 (Stratagene) transformants (Table S1) harboring the various pQE30-derivatives were grown in 500 ml of LB medium supplemented with 100 µg/ml ampicillin to an OD600 of 0.7. The synthesis of (His)6-fusion proteins was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside and growth was continued for 3 hours at 37°C. Protein extracts were prepared and loaded on a 1 ml Ni-NTA column (Qiagen); purification was carried out under native conditions by following the recommendations of the manufacturer. (His)6-tagged EINtr, NPr, NPrH30A, NPrS61A, EIIANtr, EIIAMan-like and HprK/P were recovered as soluble proteins.
[32P]PEP was synthesized by following the PEP-pyruvate isotope exchange method in the presence of pyruvate kinase and [γ-32P]ATP . Transfer of the phosphoryl group from [32P]PEP via EINtr, NPr or NPrH30A to EIIANtr or EIIAMan-like was tested at 37°C in 30 µl reaction mixtures containing 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 10 µM [32P]PEP, 1.5 µg of EINtr, 3 µg of NPr or NPrH30A, 4.5 µg of EIIANtr or EIIAMan-like. Samples were incubated for 20 min at 37°C and reactions were stopped by addition of SDS sample buffer. Proteins were separated by electrophoresis on 0.1% SDS-15% polyacrylamide gels, which were subsequently dried and exposed overnight to a storage phosphor screen (STORM).
ATP-dependent NPr phosphorylation assays were performed in 50 µl reaction mixtures containing 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 25 µM [γ-32P]ATP or [32P]PPi and varying amounts of either FBP or potassium phosphate. The assay mixtures were incubated for 20 min at 37°C and the reaction was stopped by addition of SDS sample buffer. Proteins were separated on 0.1% SDS-15% polyacrylamide gels. After electrophoresis, gels were boiled for 10 min in 0.5 N HCl, dried and exposed overnight to a storage phosphor screen.
For P-Ser-NPr dephosphorylation assays, P-Ser-NPr was obtained by incubating B. melitensis (His)6-NPr with (His)6-HprK/P for 30 min at 37°C in 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 5 mM ATP and 25 mM FBP. HprK/P was subsequently inactivated by keeping the reaction mixture for 10 min at 65°C. P-Ser-NPr was then loaded on a PD-10 column (GE Healthcare), eluted with 20 mM NH4HCO3 to eliminate ATP, and lyophilized. Dephosphorylation assays were carried out in reaction mixtures containing 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 3 µg of P-Ser-NPr, 0.45 µg of HprK/P and various concentrations of potassium phosphate. The assay mixtures were incubated for 30 min at 37°C before HprK/P was heat-inactivated for 10 min at 65°C. The different forms of NPr were separated by electrophoresis on non-denaturing 12.5% polyacrylamide gels, followed by staining with Coomassie Blue.
Extraction of B. melitensis 16M total RNA was performed on cultures (40 ml) grown to late exponential growth phase in 2YT. Bacterial cells were harvested by centrifugation for 10 min at 3500 rpm, and resuspended in 100 µl 10% SDS, 20 µl proteinase K (20 mg/ml) and RNaseOUT™ (Invitrogen Life-Technologies), and incubated for 1 hour at 37°C. Total RNA was then extracted using TRIzol® reagent. Contaminating genomic DNA was digested with DNase I DNA-free (Ambion) before the enzyme was inactivated by DNase Inactivation Reagent (Ambion). Reverse transcriptions (RT) were performed as follows: random primers (200 ng/µl) (Invitrogen Life-Technologies) and dNTP mix (10 mM each dNTP) (Invitrogen Life-Technologies) were added to 3–4 µg of DNase-treated total RNA and the mixture was incubated at 65°C for 10 min. 5X First-Strand buffer, DTT (0.1 M) and RNaseOUT™ (Invitrogen Life-Technologies) were added to the solution, which was incubated at 25°C for 2 min. Finally, SuperScript™ reverse transcriptase (Invitrogen Life-Technologies) was added and incubated for 10 min at 25°C and 50 min at 42°C. The enzyme was inactivated by heating to 70°C for 15 min. To remove RNA hybridized to the cDNA, E. coli RNase H (Invitrogen Life-Technologies) was added to the RT reaction. A control reaction containing the same components but no reverse transcriptase was included to check for DNA contamination. The cDNA products (2 µl) were then used in a PCR performed in a final volume of 30 µl and containing 1.25 U of GoTaq DNA polymerase (Promega), dNTP mix (5 mM each), and 10 pmol of each primer. A PCR control in which B. melitensis 16M genomic DNA was used as template was included. The amplification consisted of one cycle of 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at annealing temperature (depending on the primers used), 90 sec at 72°C, and a final step of 10 min at 72°C. Primers used in this experiment are listed in Table S2.
Concerning the microarray data for the virB expression, RNA was reverse transcribed, labeled and hybridized by NimbleGen™ Systems, Inc using the catalogue design for B. melitensis 16M chromosomes I (NC_003317) and II (NC_003318) with 20 probes per gene (10 perfect matches and 10 mismatches). Each probe (24 mer) was replicated three times on a chip at a random position (design includes random GC probes). Duplicate samples of each strain were processed. Analysis of the data were performed “mutatis mutandis” as described previously .
For Y2H interaction tests, each protein of interest (YFP) was fused with both AD and BD domains of the transactivator Gal4. Entry vectors pDONR201 of the ORFeome  corrresponding to detected genes of interest (YFG) (Table S1) were subcloned in Y2H destination vectors pVV212 and pVV213 (Table S1) . LR recombination procedure was performed as recommended by the manufacturer (Invitrogen Life-Technologies) to fuse YFP with both Gal4-BD (in pVV212) and Gal4-AD (in pVV213) generating plasmids pVV212-YFG and pVV213-YFG .
Haploïd yeast Mav103 and Mav203  were transformed with pVV212-YFG and pVV213-YFG respectively, and selected on SD-W (tryptophan omission medium) and SD-L (leucin omission medium) respectively. Mating of two plasmid-carrying yeasts was then carried out, and SD-LW (leucin and tryptophan omission medium) was then used to select diploids containing both pVV212 and pVV213. Two growth tests can be used to detect physical interactions between proteins, i.e. (i) SD-HLW + 3-AT (medium without histidine and with 20 to 50 mM triaminotriazole (3-AT) and (ii) SD-ULW (medium without uracil). The additional lacZ reporter gene was used to detect interactions by performing β-galactosidase coloration assays. For all Y2H assays used in this study, except for the interaction test between PTS proteins and BvrR and BvrS, β-galactosidase coloration tests were performed as follow. Diploid yeasts were plated on a nitrocellulose filter laid on a yeast peptone dextrose (YPD) plate and grown overnight at 30°C. The filter was then placed in liquid nitrogen to lyse the cells, transferred on a new plate containing two Whatman papers saturated with β-galactosidase assay solution (for each plate 5 ml of Z-buffer, 120 µl of 4% X-gal and 13 µl of 100% β-mercaptoethanol), and finally incubated at 37°C. In the case of interaction tests between PTS proteins and BvrR or BvrS, β-galactosidase coloration tests were performed using an overlay plate assay as described in .
Briefly, entry vectors pDONR201 of the ORFeome  were pooled by 48 (half of a 96-wells plate) to obtain 69 pools borne in a single 96-well plate. Each pool was subcloned in the Y2H vector pVV213 in order to fuse B. melitensis proteins to the Gal4 activating domain  using LR. Pools of pVV213 were used to transform the haploïd yeast Mav203. To select interacting partners of our proteins of interest, mating was performed using the pools of Mav203 containing pVV213 plasmids and Mav103 strains containing pVV212 bearing our genes of interest. Diploïds were selected using SD-LW medium. As a first screen for selecting interactions, an overnight culture of the diploïds was grown in SD-HLW medium at 30°C under shaking, and plated on SD-HLW with 20 mM 3-AT. Five diploid controls were used for this Y2H assay containing: (i) empty pVV212 and pVV213 (negative control), (ii) a weak interaction (BD-Rb and AD-E2F), (iii) a strong interaction (BD-Fos and AD-jun), (iv) complete Gal4 with empty pVV213 and (v) a strong interaction (BD-DP and AD-E2F) . For each pool that showed growth, a maximum of four clones was cultivated in SD-HLW and plated on SD-LW (for a back-up), SD-HLW with 20 mM of 3-AT, on SD-ULW and on nitrocellulose filters placed on a YPD plate for β-galactosidase coloration tests. Clones that were positive for at least two Y2H tests were selected and PCR was carried out with primers iGAl4AD and Gal4term to amplify the inserts cloned in the pVV213-derived plasmids. Finally, the PCR products were sequenced using primer iGAl4AD to identify the putative interacting partner. Interactions between our proteins of interest and newly detected partners were confirmed as described in the Y2H assay.
The plasmids used for the experiments were obtained as follows. The pKD46 vector was used to amplify the pBad promoter sequence with oligonucleotide pairs (Fpbad and Rpbad) (Table S2). The pBad-PCR product was cloned into pSKoriTcat digested with EcoRV. The pZD6 vector was used to amplify the divIVA-gfp fusion with oligonucleotide pairs (FdivIVA and Rgfp) (Table S2). The divIVA-gfp PCR product was cloned into the pGEMTeasy vector. The pGEMT-divIVA-gfp vector was digested with NheI and KpnI and the fused genes were cloned into pSKoriTcat –pBad digested with the same enzymes. The plasmid pSKoriTcat –pBad-divIVA-gfp was used as positive control.
The entry vector bearing the ptsM gene (pDONR201- ptsM – Table S1) was taken from the B. melitensis ORFeome. This vector was used to amplify by PCR the ptsM gene with oligonucleotide pairs (FptsM and RptsM) (Table S2). The ptsM-PCR product was cloned into vector pSKoriTcat digested with EcoRV, giving plasmid pSKoriTcat–ptsM encoding the EIIAMan-like protein. The pZD6 vector was used to amplify the gfp gene with oligonucleotide pairs (Fgfp and Rgfp) (Table S2). The gfp-PCR product was cloned into the pGEMTeasy vector. The pGEMT-gfp vector was digested with BglII and KpnI to get gfp which was cloned into pSKoriTcat – ptsM digested with the same enzymes. The pSKoriTcat –ptsM-gfp vector was digested with HindIII and KpnI and cloned into pMR10kan digested with the same enzymes.
The pZD6 vector was used to amplify the divIVA gene with oligonucleotide pairs FdivIVA and RdivIVA (Table S2). The divIVA-PCR product was cloned into pSKoriTamp digested with EcoRV. The pSKoriTamp-divIVA vector was digested with NheI and HindIII and divIVA was cloned into pSKoriTcat-pBad digested with the same enzymes.
B. melitensis genomic DNA was used to amplify by PCR the sucA gene with oligonucleotide pairs (FsucA and RsucA) (Table S2). The sucA-PCR product was cloned into the pGEM11Zf vector. The pGEM11Zf-sucA vector was digested with HindIII and XhoI and cloned into pSKoriTcat –pBad-divIVA digested with the same enzymes. The correct sequence of all PCR products was confirmed by DNA sequencing.
The two plasmids, pSKoriTcat –pBad-divIVA-sucA and pMR10kan-ptsM-gfp, were used to co-transform E. coli DH10B competent cells. The resulting strain was cultivated in 10 ml SOB medium (tryptone 2%, yeast extract 0.5%, NaCl 0.058%, KCl 0.019% and MgCl2 0.19%) with chloramphenicol (20 µg/ml) until the OD600 reached 0.1. Arabinose (10 mM) induction was performed during three hours before the microscopic observation.
In order to produce monospecific polyclonal antisera against EIIANtr, rabbits were immunized with the purified protein (50 µg per dose), initially in the presence of complete Freund's adjuvant and on days 30 and 60 with incomplete Freund's adjuvant. Rabbits were bled 1 week after the last injection.
Wild-type strain and ΔhprK, ΔptsP and ΔptsO mutants were cultivated in 100 ml 2YT until reaching an OD600 of 0.7-0.8. Cells were harvested by centrifugation, washed and disrupted by vortexing with glass beads. Cell debris was removed by centrifugation and the supernatants were used for the phosphorylation tests. When carrying out phosphorylation experiments with purified His-tagged EIIANtr we had previously observed that EIIANtr phosphorylated with PEP, EI and NPr migrates significantly faster on non-denaturing polyacrylamide gels than unphosphorylated EIIANtr. Aliquots of the crude extracts containing 60 µg of protein were therefore loaded on a non-denaturing polyacrylamide gel and separated by electrophoresis. Proteins were transferred onto a nitrocellulose membrane, which was processed for immunodetection with a polyclonal antibody against EIIANtr and a secondary antibody coupled to horseradish peroxidase before carrying out ECL revelation (GE Healthcare).
B. melitensis 16M pts knock out mutants were obtained by gene replacement as previously described . For each pts gene, upstream and downstream regions (about 500 bp) flanking the gene were PCR amplified from B. melitensis 16M genomic DNA by using appropriate primers (Table S2). A second PCR was used to associate the two PCR products by cohesive ends. The final PCR product that carries a BglII site between the upstream and the downstream regions was inserted into the NotI site of pSKoriTcat (Table S1). The aphA4 cassette  was excised from pUC4aphA4 (Table S1) with BamHI and subsequently cloned into the BglII site to generate plasmid pSKoriTcat-Δpts (or -ΔhprK) (Table S1). These constructs were used to transform E. coli strain S17-1 and subsequently introduced into B. melitensis 16M by mating. Clones exhibiting a double recombination phenotype (Cms Kanr) were selected and their genotypes were verified by PCR and by Southern blot analysis using appropriate probes. The complementation plasmids pRH001-ptsP and -ptsO (Table S1) were constructed by using the Gateway™ technique (Invitrogen Life-Technologies). LR recombination cloning was carried out as recommended by the manufacturer (Invitrogen Life-Technologies) in order to insert selected genes in pRH001 using pDONR201-ptsP and -ptsO, -ptsOH30A and -ptsOS61A as entry vectors (Table S1). The resulting vectors pRH001-ptsP and -ptsO, -ptsOH30A and -ptsOS61A (Table S1) were transferred by mating into the ΔptsP or ΔptsO mutants to generate the complemented strains ΔptsP/ptsP, ΔptsO/ptsO, ΔptsO//ptsOH30A and ΔptsO/ptsOS61A. In parallel, pMR10cat (Table S1) was transfered into B. melitensis 16M wild-type, ΔptsP and ΔptsO strains by mating.
For VirB detection in total lysates of B. melitensis 16M and various mutants, strains were grown overnight at 37°C in 2YT complex medium and then diluted and grown at 37°C until late log phase (OD600 0.8–1.2). Aliquots of the cultures were kept for 1 hour at 80°C in order to inactivate cell functions and then adjusted to the same OD600. Following SDS-polyacrylamide gel electrophoresis and Western blot analysis, immunodetection of VirB5 and VirB10 in total lysates was performed with rabbit polyclonal anti-VirB5 and -VirB10 antisera  at respective dilutions of 1/5000 and 1/2000. Immunodetection with a monoclonal antibody anti-Omp 89  was used as loading control.
Multiple sequence alignment of N-terminal portion of enzyme INtr. The predicted PEP-dependent phosphorylated histidine of enzymes INtr regarding multiple alignment with paralogous enzymes I is marked by an asterisk and shaded, and the conserved region surrounding it is boxed. The predicted N-terminal GAF domain homologous to the NifA-sensory domain of Azotobacter vinelandii is underlined and limited by two vertical bars. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. (EInSme), Sinorhizobium meliloti, (EInAtu) Agrobacterium tumefaciens, (EInBme) Brucella melitensis and (EInEco) Escherichia coli.
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Multiple sequence alignment of enzyme IIANtr. Conserved histidine predicted to be phosphorylated by NPr in E. coli, S. meliloti, A. tumefaciens and B. melitensis is marked by an asterisk and shaded. The well-conserved region surrounding the putative phosphorylation site is boxed. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. (EInSme), Sinorhizobium meliloti, (EInAtu) Agrobacterium tumefaciens, (EInBme) Brucella melitensis and (EInEco) Escherichia coli.
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Mutiple sequence alignment of enzyme IIAMan. Conserved histidine phosphorylated by HPr in E. coli that is predicted to be phosphorylated by NPr in S. meliloti, A. tumefaciens and B. melitensis is marked by an asterisk and shaded. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. Sinorhizobium meliloti (IIAmSme), Agrobacterium tumefaciens (IIAmAtu), Brucella melitensis (IIAmBme) and domain IIA of enzyme IIABMan from Escherichia coli (IIAmEco).
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Multiple sequence alignment of NPr proteins. The conserved histidine residue phosphorylated by enzyme I on HPr from B. subtilis and E. coli, that is predicted to be phosphorylated by enzyme INtr on NPr proteins from E. coli, S. meliloti, A. tumefaciens and B. melitensis is marked by an asterisk and shaded. Similarly, the conserved serine residue phosphorylated by HprK/P on HPr protein from B. subtilis, that is predicted to be phosphorylated by HprK/P on NPr proteins from S. meliloti, A. tumefaciens and B. melitensis is marked by an asterisk and shaded. The consensus sequences surrounding these two predicted phosphorylation sites are boxed. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. Sinorhizobium meliloti (NPrSme), Agrobacterium tumefaciens (NPrAtu), Brucella melitensis (NPrBme), Escherichia coli (NPrEco) and HPr proteins from E. coli (HPrEco) and Bacillus subtilis (HPrBsu).
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Multiple sequence alignment of HprK/P proteins. The conserved Walker A motif which binds ATP, PPi and Pi in HprK/P proteins is boxed (155-GDSGVGGKS-162 in L. casei HprK/P)). The HprK/P signature sequence, whose consensus is (I,L,M)E(I,V)RG(I,L,M,V)G(I,V)(I,L,M) (residues 203 to 211 in L. casei HprK/P), is also boxed. An additional conserved region present in HprK from Gram positive bacteria and playing an important role in phosphorylase activity of the protein is underlined. This region is not conserved in HprK/P from α-proteobacteria. Shaded residues are amino acids that were shown to be required either for kinase or phosphorylase activities. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. Sinorhizobium meliloti (HprKSme), Agrobacterium tumefaciens (HprKAtu), Brucella melitensis (HprKBme) and C-terminal portion of HprK/P proteins from Lactobacillus casei (HprKLca) and Bacillus subtilis (HprKBsu).
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Interaction matrix for PTS proteins, HprK/P and the two-component system BvrS/BvrR. AD-P= protein of interest fused with the activating domain (AD) of Gal4; BD-P= protein of interest fused with the DNA binding domain (BD) of Gal4. Interactions demonstrated with one or two reporter genes (lacZ or HIS3) are shown in grey and black respectively.
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Strains and plasmids used in this study.
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List of the primers used in this study.
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We thank C. Baron for his kind gift of polyclonal anti-VirB5 and -VirB10 antisera. The work of J. Mignolet in preparing the ORFeome library pool is acknowledged. We are grateful to J-F Dierick for fruitful discussions.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by Fonds de la Recherche Fondamentale Collective (FRFC, Belgium, convention 2.4521.04), by Action de recherche concertee (ARC, No. 04/09-325 and 08/13-015) and by an agreement with Commisariat general aux Relations internationales de la Communaute française de Belgique - Fonds National pour la Recherche Scientifique - Centre National de la Recherche Scientifique (CGRI-FNRS-CNRS, reference PVB/ADK/FR/ad/2681). M. Dozot held a PhD fellowship from the Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture (FRIA). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.