File S1 - Systematic Dissection of the <i>Agrobacterium</i> Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation

Contains: Information S1; Figure S1. Complementation analysis of identified mutant impaired in Hcp secretion. (A) Complementation test of the identified mutants deficient in Hcp secretion. The wild-type C58 and various mutants alone or harboring the vector pRL662 (V) or complemented plasmid (C) were analyzed for Hcp secretion. (B) Hcp secretion analysis of fha and tssC41 revertants. Total (T) and secreted (S) proteins isolated from wild-type C58 and various strains grown in AB-MES (pH 5.5) for 6 h at 25°C were separated by 12% Glycine-SDS-PAGE and examined by western blot analysis. The secreted proteins were collected from 1 ml of culture medium after removal of bacterial cells by centrifugation and were concentrated by TCA precipitation [44]. The non-secreted protein ActC was an internal control. The proteins analyzed and sizes of molecular weight standards are indicated on the left and right, respectively. Figure S2. Whole-cell ELISA and Hcp secretion assay. (A) ActC signals were significantly increased from wild-type C58 with lysozyme treatment. A. tumefaciens wild-type C58 and ΔactCBA grown in AB-MES (pH 5.5) for 6 h at 25°C were collected, and intact cells were treated with lysozyme (Lysozyme) or without lysozyme (None) and used for ELISA with various antibodies. (B) AopB surface localization is independent of T6SS. A. tumefaciens wild-type C58, ΔaopB, ΔtssC41, and ΔtssB grown in AB-MES (pH 5.5) for 6 h at 25°C were collected, and intact cells were used for ELISA with various antibodies. The strains used and proteins analyzed are indicated on the right and below, respectively. The Y-axis indicates the OD450 value representing the signal intensity of reaction to specific antibody. Data are mean±SD of triplicate samples. (C) AopB does not significantly affect secretion of Hcp. Total (T) and secreted (S) proteins isolated from wild-type C58 and two ΔaopB strains grown in AB-MES (pH 5.5) for 6 h at 25°C were separated by 12% Glycine-SDS-PAGE and examined by western blot analysis with specific antibodies. The secreted proteins were collected from 1 ml of culture medium after removal of bacterial cells by centrifugation and were concentrated by TCA precipitation [44]. The non-secreted protein ActC was an internal control. The proteins analyzed and sizes of molecular weight standards are indicated on the left and right, respectively. Figure S3. Biochemical fractionation of sheath components and exoproteins. Equal volumes of total proteins (T), periplasmic fraction (P), proteins from Spheroplast (Sp), soluble cytoplasmic fraction (S), and insoluble fraction (IS) isolated from A. tumefaciens wild-type C58 were separated by 10% or 12% Glycine-SDS-PAGE followed by western blot analysis. Proteins analyzed with antibodies against specific proteins are indicated on the left and sizes of molecular weight standards are indicated on the right, and with arrows when necessary. The quality of biochemical fractionation was monitored by TssM used as insoluble protein markers, ActC as a periplasmic protein marker [37], [46], and Fha as a cytoplasmic protein marker. Figure S4. Strep-tagged TssB has full function in mediating Hcp secretion. The ΔtssB mutant harboring the vector pRL662 (V) or complemented plasmid (C) or complemented plasmid with C-terminal Strep tag (C-Strep) were analyzed for Hcp secretion. Total (T) and secreted (S) proteins isolated from wild-type C58 and various strains grown in AB-MES (pH 5.5) for 6 h at 25°C were separated by 12% Glycine-SDS-PAGE and examined by western blot analysis. The non-secreted soluble protein ActC was an internal control. The proteins analyzed and sizes of molecular weight standards are indicated on the left and right, respectively. Figure S5. Commassie blue staining of co-purified fractions from E. coli. (A) Co-purification of Hcp (pTrc-Hcp) with Vector (pET22b only), TssC41-His (pET22b-TssC41-His), TssB-His (pET22b-TssB-His), or TssB plus TssC41-His (pET22b-TssB-TssC41-His) from E. coli BL21 (DE3). (B) Co-purification of Atu4347 (pTrc-Atu4347) with Vector (pET22b only), TssC41-His (pET22b-TssC41-His), TssB-His (pET22b-TssB-His), or TssB plus TssC41-His (pET22b-TssB-TssC41-His) from E. coli BL21 (DE3). (C) Co-purification of VgrG-1 (pTrc-VgrG-1) with Vector (pET22b only), TssC41-His (pET22b-TssC41-His), TssB-His (pET22b-TssB-His), or TssB plus TssC41-His (pET22b-TssB-TssC41-His) from E. coli BL21 (DE3). (D) Co-purification of ExoR-Strep (pTrc-ExoR-Strep) with Vector (pET22b only), TssC41-His (pET22b-TssC41-His), TssB-His (pET22b-TssB-His), or TssB plus TssC41-His (pET22b-TssB-TssC41-His) from E. coli BL21 (DE3). Proteins were induced by IPTG and the soluble protein extracts were passed through Ni-NTA His binding resins to purify His-tagged proteins and their interacting proteins. The fractions of load (L), and elution (E) were analyzed by western blot analysis of Hcp, VgrG-1, Atu4347, TssB, or TssC. Two TssB-specific protein bands are detected when expressed in E. coli, which suggests that a truncated TssB (*) may be formed by proteolysis due to the overexpression in E. coli. The proteins analyzed and sizes of molecular weight standards are indicated on the right and left, respectively, and with arrows when necessary. All the samples were also analyzed by SDS-PAGE followed by Coomassie blue staining; the positions of putative TssC41-His, TssB-His, and TssB proteins are indicated by arrows. Table S1. Bacterial strains and plasmids. Table S2. Primers used in this study. Table S3. Characteristics of proteins encoded by the imp cluster. (PDF)