Construction of Dpts mutants and complementation strains B. melitensis 16M pts knock out mutants were obtained by gene replacement as previously described [46]. For each pts gene, upstream and downstream regions (about 500 bp) flanking the gene were PCR amplified from B. melitensis 16M genomic DNA by using appropriate primers (Table S2). A second PCR was used to associate the two PCR products by cohesive ends. The final PCR product that carries a BglII site between the upstream and the downstream regions was inserted into the NotI site of pSKoriTcat (Table S1). The aphA4 cassette [46] was excised from pUC4aphA4 (Table S1) with BamHI and subsequently cloned into the BglII site to generate plasmid pSKoriTcat-Dpts (or -DhprK) (Table S1). These constructs were used to transform E. coli strain S17-1 and subsequently introduced into B. melitensis 16M by mating. Clones exhibiting a double recombination phenotype (Cms Kanr) were selected and their genotypes were verified by PCR and by Southern blot analysis using appropriate probes. The complementation plasmids pRH001-ptsP and -ptsO (Table S1) were constructed by using the GatewayTM technique (Invitrogen Life-Technologies). LR recombination cloning was carried out as recommended by the manufacturer (Invitrogen Life- Technologies) in order to insert selected genes in pRH001 using pDONR201-ptsP and -ptsO, -ptsOH30A and -ptsOS61A as entry vectors (Table S1). The resulting vectors pRH001-ptsP and -ptsO, -ptsOH30A and -ptsOS61A (Table S1) were transferred by mating into the DptsP or DptsO mutants to generate the complemented strains DptsP/ptsP, DptsO/ptsO, DptsO//ptsOH30A and DptsO/ptsOS61A. In parallel, pMR10cat (Table S1) was transfered into B. melitensis 16M wild-type, DptsP and DptsO strains by mating. Detection of VirB5 and VirB10 proteins by Western blot analyses For VirB detection in total lysates of B. melitensis 16M and various mutants, strains were grown overnight at 37uC in 2YT complex medium and then diluted and grown at 37uC until late log phase (OD600 0.8–1.2). Aliquots of the cultures were kept for 1 hour at 80uC in order to inactivate cell functions and then adjusted to the same OD600. Following SDS-polyacrylamide gel electrophoresis and Western blot analysis, immunodetection of VirB5 and VirB10 in total lysates was performed with rabbit polyclonal anti-VirB5 and -VirB10 antisera [53] at respective dilutions of 1/5000 and 1/2000. Immunodetection with a monoclonal antibody anti-Omp 89 [95] was used as loading control. Supporting Information Figure S1 Multiple sequence alignment of N-terminal portion of enzyme INtr. The predicted PEP-dependent phosphorylated histidine of enzymes INtr regarding multiple alignment with paralogous enzymes I is marked by an asterisk and shaded, and the conserved region surrounding it is boxed. The predicted N- terminal GAF domain homologous to the NifA-sensory domain of Azotobacter vinelandii is underlined and limited by two vertical bars. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. (EInSme), Sinorhizobium meliloti, (EInAtu) Agrobacterium tumefaciens, (EInBme) Brucella melitensis and (EInEco) Escherichia coli. Found at: doi:10.1371/journal.pone.0012679.s001 (0.86 MB TIF) Figure S2 Multiple sequence alignment of enzyme IIANtr. Conserved histidine predicted to be phosphorylated by NPr in E. coli, S. meliloti, A. tumefaciens and B. melitensis is marked by an asterisk and shaded. The well-conserved region surrounding the putative phosphorylation site is boxed. Red residues are identical Brucella PTS for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. (EInSme), Sinorhizobium meliloti, (EInAtu) Agrobacterium tumefaciens, (EInBme) Brucella melitensis and (EInEco) Escherichia coli. Found at: doi:10.1371/journal.pone.0012679.s002 (0.43 MB TIF) Figure S3 Mutiple sequence alignment of enzyme IIAMan. Conserved histidine phosphorylated by HPr in E. coli that is predicted to be phosphorylated by NPr in S. meliloti, A. tumefaciens and B. melitensis is marked by an asterisk and shaded. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. Sinorhizobium meliloti (IIAmSme), Agrobacterium tumefaciens (IIAmAtu), Brucella melitensis (IIAmBme) and domain IIA of enzyme IIABMan from Escherichia coli (IIAmEco). Found at: doi:10.1371/journal.pone.0012679.s003 (0.38 MB TIF) Figure S4 Multiple sequence alignment of NPr proteins. The conserved histidine residue phosphorylated by enzyme I on HPr from B. subtilis and E. coli, that is predicted to be phosphorylated by enzyme INtr on NPr proteins from E. coli, S. meliloti, A. tumefaciens and B. melitensis is marked by an asterisk and shaded. Similarly, the conserved serine residue phosphorylated by HprK/P on HPr protein from B. subtilis, that is predicted to be phosphorylated by HprK/P on NPr proteins from S. meliloti, A. tumefaciens and B. melitensis is marked by an asterisk and shaded. The consensus sequences surrounding these two predicted phosphorylation sites are boxed. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. Sinorhizobium meliloti (NPrSme), Agrobacterium tumefaciens (NPrAtu), Brucella melitensis (NPrBme), Escherichia coli (NPrEco) and HPr proteins from E. coli (HPrEco) and Bacillus subtilis (HPrBsu). Found at: doi:10.1371/journal.pone.0012679.s004 (0.42 MB TIF) Figure S5 Multiple sequence alignment of HprK/P proteins. The conserved Walker A motif which binds ATP, PPi and Pi in HprK/P proteins is boxed (155-GDSGVGGKS-162 in L. casei HprK/P)). The HprK/P signature sequence, whose consensus is (I,L,M)E(I,V)RG(I,L,M,V)G(I,V)(I,L,M) (residues 203 to 211 in L. casei HprK/P), is also boxed. An additional conserved region present in HprK from Gram positive bacteria and playing an important role in phosphorylase activity of the protein is underlined. This region is not conserved in HprK/P from a- proteobacteria. Shaded residues are amino acids that were shown to be required either for kinase or phosphorylase activities. Red residues are identical for the five proteins, whereas green and blue residues are strongly or weakly similar, respectively. Sinorhizobium meliloti (HprKSme), Agrobacterium tumefaciens (HprKAtu), Brucella melitensis (HprKBme) and C-terminal portion of HprK/P proteins from Lactobacillus casei (HprKLca) and Bacillus subtilis (HprKBsu). Found at: doi:10.1371/journal.pone.0012679.s005 (0.58 MB TIF) Figure S6 Interaction matrix for PTS proteins, HprK/P and the two-component system BvrS/BvrR. AD-P = protein of interest fused with the activating domain (AD) of Gal4; BD-P = protein of interest fused with the DNA binding domain (BD) of Gal4. Interactions demonstrated with one or two reporter genes (lacZ or HIS3) are shown in grey and black respectively. Found at: doi:10.1371/journal.pone.0012679.s006 (0.04 MB DOC) Table S1 Strains and plasmids used in this study. Found at: doi:10.1371/journal.pone.0012679.s007 (0.10 DOC) Table S2 List of the primers used in this study. Found at: doi:10.1371/journal.pone.0012679.s008 (0.11 DOC) MB MB PLoS ONE | www.plosone.org 14 September 2010 | Volume 5 | Issue 9 | e12679