Brucella PTS Figure 2. NPr kinase assays with B. melitensis HprK/P. (A) The NPr kinase assay was carried out with 200 ng of HprK/P and 2 mg of either wild- type NPr (WT), NPrH30A (H30A) or NPrS61A (S61A) in the presence of 25 mM [c-32P]ATP and in the absence of FBP and potassium phosphate (KPi). (B) Kinase assay with 200 ng of HprK/P and 3 mg of wild-type NPr, in the presence of 0.5 mM KPi, 25 mM [c-32P]ATP or [32P]PPi and increasing concentrations of FBP (0, 1, 2.5, 5, 10 mM, lanes 1 to 5). (C) Kinase assay with 200 ng of HprK/P and 3 mg of wild-type NPr in the presence of 25 mM [c-32P]ATP or [32P]PPi and increasing concentrations of potassium phosphate (0, 0.2, 1, 5, 25 mM, lanes 1 to 5). doi:10.1371/journal.pone.0012679.g002 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to be the case for HprK/P from other proteobacteria, such as A. tumefaciens (I. Mijakovic, A. Khemiri and J. Deutscher, unpublished) and Neisseria meningitidis (S. Poncet, M.-K. Taha, M. Larribe and J. Deutscher, unpublished). P,EIIANtr is formed in an hprK mutant, but not in pts mutants or wild-type B. melitensis To ascertain that the PTS phosphorylation cascade is also functional in vivo we used Western blots in order to demonstrate Figure 3. P-Ser-NPr dephosphorylation assay with B. melitensis HprK/P. Phosphorylase assays were carried out with 3 mg of P-Ser-NPr, 450 ng of HprK/P and increasing concentrations of KPi (2, 5, 10 and 20 mM, lanes 4 to7). Lane 1, 3 mg of NPr. Lane 2, phosphorylase assay with 100 ng HprK/P and no Pi. Lane 3, 3 mg of P-Ser-NPr. doi:10.1371/journal.pone.0012679.g003 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 DptsP, DptsO and DhprK mutants grown in rich medium to exponential phase (OD600 = 0.8). and aliquots containing 60 mg 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 DptsP and DptsO mutants (Fig. 4). Figure 4. Detection of EIIANtr and P,EIIANtr by Western blot in wild-type strain and Dpts and DhprK mutants. Extracts from the wild-type, DptsP, DptsO and DhprK strains grown in 2YT (exponential phase; OD600 about 0.8) were loaded on non-denaturing polyacryl- amide gels and subsequent electrophoresis allowed the separation of phospho and dephospho EIIANtr (independently established with purified proteins; data not shown) and the corresponding bands of the two EIIANtr forms were subsequently detected by Western blot with the anti-EIIANtr polyclonal antibody. Identical results were obtained in a second independent experiment. doi:10.1371/journal.pone.0012679.g004 PLoS ONE | www.plosone.org 4 September 2010 | Volume 5 | Issue 9 | e12679