M. tuberculosis ClpX and FtsZ Figure 3. FtsZ interacts with ClpX in vitro. (A) Visualization of FtsZ-His and tag-free FtsZ on a NuPAGE gel. Note that the mobilities of both proteins are distinct. *Digestion of His-FtsZ with thrombin to remove the polyhistidine tag often resulted in non-specific cleavage at another site in FtsZ resulting in a truncated FtsZ protein. However, this cleavage did not interfere with the interactions of FtsZ with ClpX. (B) Immunoblots of FtsZ pulled down with His-ClpX on Ni-NTA resin. Equimolar amounts of purified FtsZ and His-ClpX were mixed and loaded onto Ni-NTA resin. Following washing, bound proteins were eluted with 0.3 M imidazole and resolved on a 10% NuPAGE gel, transferred to PVDF membrane and probed with a- FtsZ or a-ClpXBS antibodies. Load, wash and elution are shown. (C) FtsZ does not copurify with FtsQDN100. FtsZ was mixed with His-FtsQDN100 and loaded on Ni-NTA resin. Proteins eluted with 0.3 M imidazole were resolved on a 10% NuPAGE gel, transferred to a PVDF membrane and probed with a-FtsZ or a-His antibodies. Load, wash and elution are shown (D) Solid-phase binding assay for FtsZ with ClpX. Wells of a microtiter plate were coated with ClpX, or BSA and incubated with various concentrations of FtsZ protein as indicated. The bound FtsZ protein was immunodetected with a-FtsZ antibodies and ELISA (AnaSpec), as described in the methods section. Mean 6 SD from three independent experiments are shown. doi:10.1371/journal.pone.0011058.g003 washing, the bound FtsZ protein was immunodetected and quantified by ELISA. As can be seen, FtsZ bound to the ClpX- coated wells but not to the wells coated with BSA (Fig. 3D). In vivo: BACTH assays reveal interactions between ClpX and FtsZ. Next, BACTH assays were performed to evaluate in vivo interactions between ClpX and FtsZ [35]. In these assays, ftsZ and clpX genes were cloned as fusions to the T25 or T18 fragments of adenylate cyclase in two separate vectors and transformed into the E. coli reporter strain, BTH101 [35]. Functional complementation of adenylate cyclase activity due to interactions between the partners leads to cAMP production and subsequent transcription of the lac reporter gene, which gives a distinct color to colonies growing on indicator agar plates. The strength of the interaction was also measured by assaying for the b-galactosidase activity. As expected, the gcn4-gcn4 positive control strain showed high b-galactosidase activity (Fig. 4A). Transformants expressing ftsZ or clpX from both vectors were also red, indicating self-interactions in these proteins (Fig. 4A). Similarly, the transformants expressing ftsZ and clpX were also red, but not when clpX or ftsZ was expressed with the control plasmid (see Fig. 4A). The amount of b-galactosidase produced with the transformants expressing ftsZ and clpX was comparable to the gcn4-gcn4 positive control and also to those expressing clpX or ftsZ from both vectors indicating that the interaction between ClpX and FtsZ is strong (Fig. 4A). These results were consistently obtained with any combination of vectors used to express the clpX and ftsZ genes (Fig. 4A). The specificity of ClpX-FtsZ interaction was indicated by the lack of interaction between ClpX and FtsQ and also between FtsZ and FtsI (see [36]) (Fig. 4A). Interactions between ClpXDN200, lacking the N-terminal Zinc-binding, ATP binding and ATP hydrolysis domains, and FtsZ appeared to be weak, as the amount of b-galactosidase produced was just above the background. These data suggest that the N-terminal region of ClpX is important for optimal interactions with FtsZ PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11058