Brucella PTS Figure 6. Synthesis of VirB proteins in DhprK and Dpts mutants. (A) Detection of VirB10 (top) and VirB5 (bottom) by Western blot analysis in the wild-type (Bm16M), DptsP, DptsO, DptsN and DhprK strains following growth in 2YT to late-exponential phase. An established negative control (DrshBm) was included in the anti-VirB5 Western blot analysis [46] (B) Western blot analysis of VirB10 with Bm16M (+pMR10cat), DptsO (+pMR10cat) and the complemented strain DptsO/ptsO, DptsO/ptsOH30A, and DptsO/ptsOS61A. Identical results were obtained in a second independent experiment. (C) Transcription analysis of virB gene expression in wild-type and ptsP mutant. The values presented by the bars correspond to the ratio of normalized and averaged microarray data (n = 263) obtained for 10 virB ORFs in the ptsP mutant and the wild-type strain grown under the same conditions. BMEI0575 is a control ORF whose expression is not modulated whatever the strain considered. doi:10.1371/journal.pone.0012679.g006 subunits (Fig. 6A). Complementation of the DptsO mutant with wild-type ptsO constitutively expressed from a low copy plasmid fully restored VirB10 (Fig. 6B) and VirB5 production (data not shown). For unknown reasons, plasmid-encoded ptsP did not restore VirB5 or VirB10 production in the DptsP mutant, although the plasmid was functional because it complemented the ‘‘small colony’’ phenotype of the DptsP mutant (see Fig. 7B). Knowing that EINtr is strictly required for P,His-NPr formation we tried to complement the DptsO mutant with the mutant alleles ptsOH30A and ptsOS61A. Constitutive expression of ptsO and ptsOS61A in DptsO restored VirB10 production, whereas the DptsO/ptsOH30A strain failed to produce VirB10 (Fig. 6B). This confirms that P,His-NPr is needed for VirB 10 synthesis and consequently that EINtr is also required. In order to confirm the impact of the ptsP mutations on T4SS expression we carried out a transcriptional analysis of the whole virB operon with the wild-type strain and the ptsP mutant. Indeed, the expression level of the individual virB genes was more than thirty times lower in the ptsP mutant than in the wild-type strain (Fig. 6C) Colony size heterogeneity of pts and hprK deletion mutants plated on rich medium When plated on 2YT rich medium, the DptsP, DptsO, DptsN and, to a lesser extent, the DhprK mutant displayed a heterogeneity in colony size compared to the wild-type strain B. melitensis 16M (Fig. 7A). Small colonies were detected only 8 to 10 days after inoculation, whereas the larger colonies were visible after 3 to 4 days as usually observed for the wild-type strain. To ensure that the colony size heterogeneity of pts and hprK mutants resulted from the deletion of the corresponding genes, we first carried out a complete typing of these strains confirming that they all derived from B. melitensis 16M wild-type, exhibited a smooth phenotype, and were not contaminated with other strains (data not shown). Next, we complemented the two mutants with the most marked phenotype (DptsP and DptsO) by constitutively expressing wild-type copies of the ptsP and ptsO genes in the corresponding mutants. As shown in Fig. 7B, the complemented strains DptsP/ptsP and DptsO/ ptsO displayed bigger colonies than the DptsP and DptsO mutants transformed with the empty vector-pMR10cat. Their colonies resembled those of the wild-type strain carrying the empty vector- pMR10cat. Finally, we measured growth of the four mutants when cultivated in liquid 2YT medium (Fig. 7C). No differences were observed between growth of the mutants and the wild-type strain, suggesting that the growth heterogeneity observed on solid medium might not result from the composition of the medium, but rather from parameters that distinguish liquid and solid cultures, such as oxygen supply, nutrient or water availability. PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e12679