Yeast two hybrid assay Haplo¨ıd yeast Mav103 and Mav203 [92] 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 b- galactosidase coloration assays. For all Y2H assays used in this study, except for the interaction test between PTS proteins and BvrR and BvrS, b-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 30uC. The filter was then placed in liquid nitrogen to lyse the cells, transferred on a new plate containing two Whatman papers saturated with b-galactosidase assay solution (for each plate 5 ml of Z-buffer, 120 ml of 4% X-gal and 13 ml of 100% b- mercaptoethanol), and finally incubated at 37uC. In the case of interaction tests between PTS proteins and BvrR or BvrS, b- galactosidase coloration tests were performed using an overlay plate assay as described in [93]. Y2H screen against the ORFeome of B. melitensis 16M Briefly, entry vectors pDONR201 of the ORFeome [89] 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 [91] using LR. Pools of pVV213 were used to transform the haplo¨ıd 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. Diplo¨ıds were selected using SD-LW medium. As a first screen for selecting interactions, an overnight culture of the diplo¨ıds was grown in SD- HLW medium at 30uC 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) [94]. 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 b-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. DivIVA interaction test 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). Brucella PTS 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 mg/ml) until the OD600 reached 0.1. Arabinose (10 mM) induction was performed during three hours before the microscopic observation. Rabbit immunization In order to produce monospecific polyclonal antisera against EIIANtr, rabbits were immunized with the purified protein (50 mg 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. Detection of in vivo phosphorylated EIIANtr Wild-type strain and DhprK, DptsP and DptsO 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 phosphor- ylation 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 contain- ing 60 mg 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). PLoS ONE | www.plosone.org 13 September 2010 | Volume 5 | Issue 9 | e12679