The published genome sequence predicts a ptsO gene starting with a GUG codon and lacking a ribosome binding site (RBS) [22]. 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 GatewayTM primers GWnprF and GWnprR (Table S2) and cloned in the entry vector pDONR201 (Invitrogen Life-technologies) as previously described [89]. Directed mutagenesis of ptsO was performed with the QuickChangeTM 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. Overexpression and purification of PTS proteins The E. coli NM522 (Stratagene) transformants (Table S1) harboring the various pQE30-derivatives were grown in 500 ml of LB medium supplemented with 100 mg/ml ampicillin to an OD600 of 0.7. The synthesis of (His)6-fusion proteins was induced with 0.1 mM isopropyl-b-D-thiogalactopyranoside and growth was continued for 3 hours at 37uC. Protein extracts were prepared and loaded on a 1 ml Ni-NTA column (Qiagen); purification was carried out under native conditions by following the recommen- dations of the manufacturer. (His)6-tagged EINtr, NPr, NPrH30A, NPrS61A, EIIANtr, EIIAMan-like and HprK/P were recovered as soluble proteins. Protein phosphorylation and dephosphorylation assays [32P]PEP was synthesized by following the PEP-pyruvate isotope exchange method in the presence of pyruvate kinase and [c-32P]ATP [90]. Transfer of the phosphoryl group from [32P]PEP via EINtr, NPr or NPrH30A to EIIANtr or EIIAMan-like was tested at 37uC in 30 ml reaction mixtures containing 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 10 mM [32P]PEP, 1.5 mg of EINtr, 3 mg of NPr or NPrH30A, 4.5 mg of EIIANtr or EIIAMan-like. Samples were incubated for 20 min at 37uC 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 ml reaction mixtures containing 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 25 mM [c-32P]ATP or [32P]PPi and varying amounts of either FBP or potassium phosphate. The assay mixtures were incubated for 20 min at 37uC and the reaction was stopped by addition of SDS sample buffer. Proteins were separated on 0.1% SDS-15% polyacrylamide gels. After electro- phoresis, 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 37uC 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 65uC. P-Ser-NPr was then loaded on a PD-10 column Brucella PTS (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 mg of P-Ser-NPr, 0.45 mg of HprK/P and various concentrations of potassium phosphate. The assay mixtures were incubated for 30 min at 37uC before HprK/P was heat- inactivated for 10 min at 65uC. The different forms of NPr were separated by electrophoresis on non-denaturing 12.5% polyacryl- amide gels, followed by staining with Coomassie Blue. RNA isolation and RT-PCR assays 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 ml 10% SDS, 20 ml proteinase K (20 mg/ml) and RNaseOUTTM (Invitrogen Life-Technologies), and incubated for 1 hour at 37uC. Total RNA was then extracted using TRIzolH 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/ml) (Invitrogen Life-Technologies) and dNTP mix (10 mM each dNTP) (Invitrogen Life-Technologies) were added to 3–4 mg of DNase-treated total RNA and the mixture was incubated at 65uC for 10 min. 5X First-Strand buffer, DTT (0.1 M) and RNaseOUTTM (Invitrogen Life-Technologies) were added to the solution, which was incubated at 25uC for 2 min. Finally, SuperScriptTM reverse transcriptase (Invitrogen Life- Technologies) was added and incubated for 10 min at 25uC and 50 min at 42uC. The enzyme was inactivated by heating to 70uC 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 ml) were then used in a PCR performed in a final volume of 30 ml 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 95uC, followed by 35 cycles of 30 sec at 95uC, 30 sec at annealing temperature (depending on the primers used), 90 sec at 72uC, and a final step of 10 min at 72uC. 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 NimbleGenTM 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 [71]. GatewayH cloning of genes of interest in Y2H vectors 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 [89] corrresponding to detected genes of interest (YFG) (Table S1) were subcloned in Y2H destination vectors pVV212 and pVV213 (Table S1) [91]. 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 [54]. PLoS ONE | www.plosone.org 12 September 2010 | Volume 5 | Issue 9 | e12679