FIGURE 6.

TssK1 oligomerizes and is trimeric in solution. A, TssK1 interacts with itself as shown by bacterial two-hybrid analysis. BTH101 reporter cells carrying pairs of plasmids producing the indicated T6SS proteins fused to the T18 or T25 domains of the Bordetella adenylate cyclase were spotted on X-Gal-IPTG indicator LB agar plates as described under “Experimental Procedures.” Controls include TssK1 interaction assays with TolB and Pal, two proteins that interact but unrelated to the T6SS. B, TssK1_HA co-immunoprecipitates with TssK1_FLAG. Soluble lysates from 10¹¹ E. coli K12 W3110 cells producing HA-tagged TssK1 (TssK1_HA) alone or with FLAG-tagged TssK1 (TssK1_FL) were subjected to immunoprecipitation with anti-FLAG-coupled beads. The total soluble (Tot) and the immunoprecipitated (IP) material were separated by 10%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (TssK1_FL; upper panel) and anti-HA (TssK1_HA; lower panel) monoclonal antibodies. Molecular weight markers (in kDa) are indicated on the left. C, analytical size exclusion chromatography analysis of purified TssK1 (continuous line) on a Superdex 200 column, calibrated with 43-, 75-, 158-, and 440-kDa molecular weight markers (dotted lines). The molecular weight of each marker (in kDa) is indicated on the top of the corresponding peak. The theoretical mass of the TssK1 monomer being 49.9 kDa, its elution at ∼160 kDa indicates that it behaves as a trimer in solution.