ClpX interaction with mutant FtsZ proteins As reviewed, FtsZ binds GTP and exhibits GTP-dependent polymerization and GTP hydrolysis activities [28,32]. FtsZ also interacts with FtsW in vitro [38] and in vivo [34]. The amino acid residues in FtsZ important for these activities have been identified. For example, FtsZG103S is defective in binding to GTP; hence, is also defective in GTP hydrolysis. FtsZD210G, on the other hand, is proficient in GTP binding, but is defective in GTP hydrolysis [28]. FtsZD374-76A is defective in its interactions with FtsW [34]. FtsZDC21 lacks 21 amino acids from the C- terminus, a region believed to be critical for interactions of FtsZ with other cell division proteins [38,39,40,41]. To investigate if ClpX interacts with FtsZ mutants defective in the above-described activities, we purified FtsZG103S, FtsZD210G, FtsZD374-76A and FtsZDC21 recombinant proteins and investigated their interaction using pull-down and BACTH assays [34,35,38]. Pull-down assays with mixtures of equimolar amounts of His-ClpX and various FtsZ mutant proteins followed by immunoblotting confirmed the presence of FtsZ in fractions containing ClpX (Fig. 5A). The BACTH assay confirmed these data (Fig. 5B, 5C). ClpX also inhibited the assembly of FtsZDC21 (Fig. 5D). These data suggest that ClpX is capable of interacting with monomeric or polymeric FtsZ and that the C-terminal region of FtsZ is not required for this interaction. M. tuberculosis ClpX and FtsZ Overexpression of clpX inhibits Z-ring assembly and reduces M. tuberculosis viability Saturated transposon mutagenesis studies have indicated that clpX is an essential gene, like the ftsH protease [42]. While it is still possible that clpX is not essential in M. tuberculosis, saturation transposon screens are comprehensive. We would like to note that our innumerable attempts to make a knock-out ftsH, another protease shown by transposon mutagenesis to be essential, by homologous recombination were unsuccessful (Chauhan and Rajagopalan, unpublished data). Therefore, to evaluate the consequences of the interaction of ClpX with FtsZ in vivo, we created M. tuberculosis strains expressing altered levels of ClpX from the inducible tetracycline promoter and characterized them with respect to growth, viability and Z-ring structures. Quantitative immunoblotting determined the intracellular levels of ClpX monomers in exponential phase cultures of WT M. tuberculosis to be ,14,000 molecules per cell (Fig. 6A, see legend for details). Under the same conditions, FtsZ levels were previously determined to be ,30,000 molecules per cell (12), and this makes the approximate ratio of ClpX (monomeric) to FtsZ 1:2. Immunoblot- ting also revealed that the ClpX levels were elevated by ,six-fold in the presence of anhydrotetracycline in the sense clpX strain (pRD23, Table S2; inset in Fig. 6B). The low level of the ClpX signal in the antisense strain (asClpX) made it difficult to accurately determine Figure 5. ClpX interacts with various FtsZ mutants. (A) Pull-down assay. Immunoblots of His-ClpX and various tag-free FtsZ mutant proteins copurified on Ni-NTA resin. Equimolar amounts of ClpX and various FtsZ mutant proteins were used for the pull-down assay. Load, wash and proteins eluted with 0.3 M imidazole were resolved on a 10% NuPAGE gel, transferred to a PVDF membrane and probed with a-ClpXBS and a-FtsZ antibodies. (i) FtsZG103S, (ii) FtsZD210G and (iii) FtsZD374-76A. (B) Bacterial two-hybrid analyses of ClpX-FtsZ mutant protein interactions. Experimental details are as in Fig. 4A. Interactions of ClpX-FtsZDC21, ClpX-FtsZG103S, and ClpX-FtsZD210G on MacConkey agar plates containing 1% maltose. Red indicates strong interaction. (C) Quantitative analysis of BACTH interactions: Extent of interactions was measured by b-galactosidase activity as described under Fig. 4A. Mean 6 SD from three experiments are shown. (D) ClpX inhibits polymerization of FtsZDC21. The effect of ClpX on FtsZDC21 assembly was monitored by right angle light scatter assay. ClpX (2 mM) was mixed with FtsZDC21 (5. 4 mM) in the presence of GTP at a final concentration 1 mM. Note both the extent and the rate of FtsZDC21polymerization was decreased in the presence of ClpX. doi:10.1371/journal.pone.0011058.g005 PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11058