Brucella PTS Figure 1. Purification and PEP-dependent phosphorylation of B. melitensis PTS proteins. His-tagged proteins were purified as described in Materials and Methods and analyzed on 0.1% SDS-15% polyacrylamide gels before carrying out phosphorylation experiments. (A) Electrophoretic separation of MW standards, NPr, EIIANtr and the EIIAMan-like protein on a SDS gel stained with Coomassie Blue. (B) To carry out phosphorylation experiments, samples containing 10 mM [32P]PEP and the indicated proteins were incubated for 20 min at 37uC before they were separated on a 0.1% SDS-15% polyacrylamide gel, which was dried and exposed to a storage phosphor screen (see Materials and Methods). Arrows indicate the migration positions of EINtr, EIIANtr, EIIAMan-like and NPr/NPrH30A. Preparations of the EIIAMan-like protein always gave two bands migrating to nearly identical positions on SDS polyacrylamide gels and both became phosphorylated. doi:10.1371/journal.pone.0012679.g001 with [32P]PEP (Fig. 1B, lane 3). In agreement with our prediction (Fig. S4), the additional presence of EI allowed the phosphory- lation 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 polyacryl- amide gels [43]. Serine 61 of NPr is the target of ATP-dependent phosphorylation catalyzed by HprK/P 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. HPr kinase and P-Ser-HPr phosphorylase activities of HprK/P from B. melitensis 16 M HprK/Ps of firmicutes possess antagonistic kinase (HPr phos- phorylation) 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) [28,29,44]. 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 [45]. 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 [45]. 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 b-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 PLoS ONE | www.plosone.org 3 September 2010 | Volume 5 | Issue 9 | e12679