Figure 2. ClpX inhibits the assembly of FtsZ. (A) Light scatter assay for FtsZ polymerization in the presence or absence of ClpX. Reactions containing 5.4 mM FtsZ mixed with storage buffer alone or storage buffer containing 2 mM ClpX were initiated with GTP to a final concentration of 1 mM and followed for 15 minutes. Note that both the rate and extent of FtsZ polymerization was decreased in the presence of ClpX. (B) Determination of critical concentration of FtsZ polymerization in the presence of ClpX. Reactions containing various concentrations of FtsZ and storage buffer without or with 2 mM ClpX were initiated with 1 mM GTP and sedimentation assays performed as described under Materials and Methods section. The amount of FtsZ polymerized in the absence and presence of ClpX was calculated and plotted to determine the critical concentration of polymerization (see Materials and Methods for details). These assays could not be carried out at higher concentrations of ClpX as our innumerable attempts to obtain concentrated stocks of active ClpX following the described refolding protocol were not successful (see Materials and Methods). (C) Effect of non-hydrolysable GTP analog, GMPCPP on FtsZ polymerization activity [30,31]. ClpX inhibition of FtsZ assembly was examined in the absence of GTPase activity using GMPCPP. FtsZ (2.3 mM) without or with ClpX (2 mM) was incubated in the presence of either 1 mM GMPCPP or GTP. FtsZ polymers were collected by centrifugation, supernatant (lanes marked as ‘S’) and pellet (lanes marked as ‘P’) fractions were loaded on SDS-PA and visualized by coomassie staining. doi:10.1371/journal.pone.0011058.g002 concentration of FtsZ used, the amount of FtsZ in the supernatant was the same, and the critical concentration for FtsZ polymerization was increased to ,2.4 mM (Fig. 2B). Owing to the difficulty in purifying higher than ,8 mM ClpX, these experiments could not be performed at higher concentrations of ClpX. Nonetheless, the above data indicate a stoichiometric association between the FtsZ and ClpX molecules and suggest that ClpX modulates FtsZ assembly by sequestration. M. tuberculosis ClpX and FtsZ In the above experiments, ClpX was added along with FtsZ. Next, we added ClpX to preformed FtsZ polymers (supplementary Fig. S3). Even under these conditions, unlike with the buffer control, FtsZ was present in the supernatant (supplementary Fig. S3, see reactions 4 and 5, and compare with 2 and 3). Together, these experiments indicate that ClpX interferes with FtsZ polymerization regardless of whether it is added before the initiation of polymerization or after the formation of FtsZ polymers. GTPase activity of FtsZ is not needed for inhibition of polymerization by ClpX We next examined the inhibition of FtsZTB assembly by ClpX in the presence of GTP or GMPCPP, a non-hydrolyzable analog of GTP (Fig. 2C) [30,31]. FtsZTB polymerization occurred in the presence of GMPCPP, as with GTP. Addition of ClpX interfered with the assembly of FtsZTB in the presence GMPCPP (Fig. 2C). These data indicate that ClpX inhibition of FtsZ assembly is independent of the GTP hydrolysis activity of FtsZ. FtsZ interacts with ClpX The above data are consistent with the idea that the ClpX- mediated interference with M. tuberculosis FtsZ polymerization activity involves direct physical interaction between the two proteins in M. tuberculosis. Although M. tuberculosis FtsZ exhibits GTP binding and hydrolysis activities like other bacterial FtsZ counterparts, notable differences in these properties have been observed [28,32]. Furthermore, as reviewed, the M. tuberculosis genome lacks several identifiable homologs of known proteins of the bacterial cell division machinery [5,33,34]. Hence, we evaluated physical and functional interactions between the ClpX and FtsZ proteins of M. tuberculosis using a variety of in vitro and in vivo assays. In vitro: Pull-down assay shows that FtsZ copurifies with His-ClpX. A pull-down assay was performed to evaluate interactions between the ClpX and FtsZ proteins. Equimolar amounts of purified recombinant His-ClpX protein (molarity based on monomer concentration) and a tag-free FtsZ protein created by cleaving the polyhistidine tag were mixed and applied to Ni-NTA resin. Following washing, bound proteins were eluted with imidazole and detected by immunoblotting using anti- ClpXBS and anti-FtsZ antibodies. Both His-FtsZ and tag-free FtsZ showed distinct mobilities on NuPAGE gels (Fig. 3A). As can be seen, the elution fraction containing ClpX also contained FtsZ (Fig. 3B). Some FtsZ was also found in the flow-through and early wash fractions (data not shown), indicating that not all of the FtsZ was complexed with ClpX. In a reverse experiment, we were able to pull-down MBP-ClpX with His-FtsZ on Ni-NTA resin (data not shown). Salt concentrations of 0.2 M weakened the ClpX-FtsZ complexes and those of 0.5 M nearly abolished the interaction (Supplementary Fig. S4, panel A). ClpX-FtsZ complexes were also isolated when E. coli lysates containing the His-ClpX and FtsZ-S-tag were processed on nickel-affinity columns (Supplementary Fig. S4, panel B). Control experiments with lysates containing recombinant N-terminal FtsQ protein (amino acid 1–100; Table S2), His-FtsQN100, and FtsZ did not recover FtsZ in the fractions containing His-FtsQN100 (Fig. 3C), indicating that the observed interaction between ClpX and FtsZ is specific. In vitro: Solid-phase binding assay confirms ClpX-FtsZ interactions. Next, a solid-phase binding assay was carried out to further confirm interactions between FtsZ and ClpX. In this assay, ClpX or BSA immobilized in the wells of a microtiter plate was incubated with increasing concentrations of FtsZ. Following PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11058