Brucella PTS Figure 7. Dpts mutants mutants display colony size heterogeneity on solid medium. (A) Colony size heterogeneity displayed by the mutants DptsP, DptsO, DptsN and DhprK in comparison to the wild-type strain. Late log phase cultures were diluted and plated on 2YT medium and grown at 37uC for 8 to 10 days. (B) Complementation of the colony size heterogeneity phenotype for the DptsP and DptsO mutants. Wild-type, DptsP and DptsO strains (carrying the empty vector pMR10-cat; see Table S1) and the complemented mutants DptsP/ptsP and DptsO/ptsO (carrying vectors pRH001-ptsP and -ptsO, respectively; see Table S1) were plated on 2YT supplemented with 20 mg/ml of chloramphenicol as described in (A) and grown at 37uC for 8 to 10 days. (C) Growth of Dpts and DhprK mutants in 2YT liquid cultures in comparison to the wild-type strain. Overnight 2YT cultures of the five strains were back diluted to an OD600 (optical density at 600 nm) of 0.05, and growth was monitored by measuring the OD600 at diffent time intervals. Identical results were obtained in three independent experiments. When re-isolated on new plates small and large colonies give always the same size of colonies (except for some ‘‘suppressors’’ that appeared in the small phenotype background (see arrowhead in figure 7A). doi:10.1371/journal.pone.0012679.g007 Yeast two hybrid assays reveal oligomerization of EINtr, the EIIAMan-like protein and HprK/P, and interaction between NPr and HprK/P Since in B. melitensis 16M two pts genes, hprK and the two- component system genes bvrR/bvrS are co-transcribed and functionally linked we wanted to test if there existed any physical interactions between PTS components, HprK/P and the BvrR/S proteins. A yeast two hybrid (Y2H) interaction matrix of 64 interactions was performed with the four PTS proteins, HprK/P, BvrR and BvrS fused to the Gal4 DNA binding domain (BD) and tested against the same proteins fused to the Gal4 activating domain (AD). Each BD and AD fusion was also tested against Gal4-AD and Gal4-BD alone. The previously evidenced interac- tion between BvrR and BvrS [54] was used as a positive control. The results presented in Fig. 8 and S6 show that two PTS proteins (EINtr, EIIAMan-like) and HprK/P interacted with themselve, suggesting that these proteins form oligomers similar to some well- studied EI, EIIAMan and HprK/P homologues [55,56,57]. Additionally, a bidirectional interaction was evidenced between NPr and HprK/P (Fig. 8 and S6) confirming the results of the in vitro phosphorylation test. No interaction was observed between other PTS proteins that were shown to phosphorylate each other in vitro. Finally, an interaction between BvrR and BvrS could be demonstrated (Fig. S6), but no interaction was detected between any of the PTS proteins or HprK/P and the two-component partners. A yeast two-hybrid screen against the B. melitensis ORFeome reveals interaction partners of the EIIAMan-like protein and NPr Having demonstrated that the incomplete PTS of B. melitensis is functional and knowing that PTS-dependent regulations are mediated either by allosteric interaction or by direct phosphory- lation of target proteins [1], we performed a Y2H screen to detect interaction partners of the EIIAMan-like protein and NPr of B. melitensis 16M to get some preliminary clues about the functional role of this PTS. Briefly, these PTS proteins were fused to the Gal4 DNA binding domain and used as baits to identify interaction partners in an « ORFeomic » library. Two clones provided a positive signal with at least two of the three reporter genes and the PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e12679