Brucella PTS Figure 8. Detection of interaction partners for Brucella PTS proteins by Y2H assays. Interaction between NPr, HprK/P and PPa (top row). Interaction between EIIAMan-like and EIIANtr (middle row). Interaction between EIIAMan-like and SucA (bottom row). AD fusion = protein of interest fused with activating domain of Gal4; BD fusion = protein of interest fused with DNA binding domain of Gal4. The reporter used is indicated by the letter in the upper right corner of each picture: b-galactosidase activity (G); growth test without uracil (U); growth test without histidine in the presence of 40 mM of 3AT (H). ‘‘-’’ indicates an empty vector. Identical results were obtained in three independent experiments. doi:10.1371/journal.pone.0012679.g008 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 dihydrolipoa- mide succinyltransferase SucB (E2 component) and dihydrolipoa- mide 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 [1,2] 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. SucA, the E1 component of the enzymatic 2-oxoglutarate dehydrogenase complex physically interacts with the EIIAMan-like protein 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 [58]. In addition, DivIVA fused to a ‘‘bait’’ protein X can target an interacting GFP tagged ‘‘prey’’ protein Y to the pole [59]. 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- PLoS ONE | www.plosone.org 8 September 2010 | Volume 5 | Issue 9 | e12679