Recent studies with C. crescentus and B. subtilis revealed that ClpX or ClpXP complex functions as a regulator of cell division, although the mechanisms by which it affects cell division appear to vary in the two species. Furthermore, two independent studies with E. coli ClpX led to two different conclusions (see below). In the case of Gram negative and alpha-proteobacterium C. crescentus, the ClpXP protease regulates the transcription of cell division genes via degradation of the master cell cycle regulator, CtrA [11,18]. In E. coli, another Gram negative bacterium, Camberg et al [19] reported that ClpXP regulates cell division by degrading FtsZ in an ATP-dependent manner, thereby affecting the equilibrium between monomeric and polymeric FtsZ. In contrast, single molecule analysis study with high-speed atomic force microscopy revealed that ClpX regulates dynamics of FtsZ assembly by blocking the reassembly of FtsZ in an ATP independent manner and that ClpXP protease shows only a weak protease activity against FtsZ in the presence of either GTP or GDP [20]. These results, which are inconsistent with the findings of Camberg et al [19], suggest that the contribution of FtsZ unfolding by ClpX is negligible as compared to its role in the inhibition of FtsZ assembly [20]. Nevertheless, the observed intracellular levels of ClpX and FtsZ in E. coli appear to be 600 and 15,000 molecules per cell, respectively [21,22] leading to a suggestion that ClpXP could play a catalytic role in regulating FtsZ assembly. It is pertinent to note that the proteomic studies with E. coli lysates utilizing the ClpXPtrap protocol identified FtsZEcoli as one of the ,60 proteins associated with ClpXPEcoli [23]. In B. subtilis, ClpX inhibits FtsZ assembly in vivo and interferes with the FtsZ polymerization activity in vitro indepen- dent of its ATPase activity [24,25]. Thus the above divergent results in different organisms necessitate the evaluation of the role of ClpX in cell division in other bacteria including the human pathogen, M. tuberculosis. To begin evaluating the roles of ClpX, if any, in M. tuberculosis cell division, we characterized interactions of ClpX with FtsZ in vivo and in vitro. We show that clpX expression is elevated during intracellular growth and upon cephalexin treatment, two of the conditions known to delay FtsZ ring assembly [5]. Independently, we show that ClpX functions to regulate FtsZ activity in vitro and FtsZ assembly in vivo by interacting with FtsZ and that both proteins colocalize at the cell division site. Our results are consistent with a model that ClpX activity is one of the factors responsible for the regulation of M. tuberculosis Z-ring assembly during intracellular growth and that ClpX action on FtsZ and cell division is possibly conserved in eubacteria. Results clpX expression is upregulated during intracellular growth To begin evaluating the role of ClpX in FtsZ catalyzed cell division process in M. tuberculosis, we first examined clpX expression under select growth conditions where M. tuberculosis FtsZ assembly is shown to be modulated, i.e., growth in macrophages and exposure to antibiotics [5,26]. Accordingly, we determined clpX expression relative to the housekeeping gene 16S rRNA by qRT- PCR during intramacrophage growth and upon exposure to cephalexin antibiotic (Fig. 1). Our results indicated that clpX expression was upregulated during intramacrophage growth and upon exposure to cephalexin (Fig. 1). Since M. tuberculosis cells growing in macrophages and those exposed to cephalexin antibiotic are deficient in FtsZ assembly [5,26], we examined if ClpX is involved in regulating FtsZ assembly and cell division in M. tuberculosis. M. tuberculosis ClpX and FtsZ Figure 1. clpX expression in M. tuberculosis. M. tuberculosis cells grown in Middlebrook 7H9 broth with cephalexin and lithium clavulanate (see materials and methods) or in macrophages were used to extract RNA essentially as described [5]. clpX mRNA levels were determined by quantitative real time PCR using TaqMan chemistry. Expression levels of clpX were normalized to 16S rRNA and are expressed relative to the clpX transcript levels in the broth-grown WT strain. THPI - macrophages from human acute monocytic leukemia cell line. Mean 6 SD from three independent RNA samples are shown. doi:10.1371/journal.pone.0011058.g001 ClpX inhibits the GTP-dependent FtsZ polymerization activity The 90u right angle light scatter assay is a widely used method to measure the GTP-dependent polymerization of FtsZ [27]. To examine whether ClpX interferes with FtsZ polymerization activity, purified FtsZ (5.4 mM) was incubated without or with ClpX (2 mM) at 30uC and polymerization was initiated with 1 mM GTP. Since ClpX is thought to possess ATPase activity, 1 mM ATP was included in all polymerization reactions. Consistent with our earlier data, the presence of ATP had no effect on GTP- dependent FtsZ polymerization (data not shown; [28]). In the presence of ClpX, an ,35% reduction in GTP-dependent FtsZ assembly was noted (Fig. 2A). Control experiments with RecA, an ATPase (reviewed in [29]), (Supplementary data Fig. S1) or BSA (data not shown) had no effect on FtsZ polymerization. These results suggest that M. tuberculosis ClpX interferes with FtsZ polymerization. To gain insights into the mechanism by which ClpX inhibits FtsZ assembly, increasing concentrations of FtsZ were incubated with a fixed concentration of ClpX (2 mM) and sedimentation assays performed to determine the extent of FtsZ polymerization (supplementary Fig. S2, panel B). A control experiment lacking ClpX was also performed (supplementary Fig. S2, panel A). As can be seen, in the absence of ClpX, FtsZ polymers were present in the pellet at 1 mM and beyond, (supplementary Fig. S2, panel A, compare lanes S with P). Determination of the amount of FtsZ polymer in the pellet as a function of FtsZ concentration revealed that the critical concentration for FtsZ polymerization was ,1 mM. In the presence of 2 mM ClpX, the amount of FtsZ in the pellet at all protein concentrations was decreased (supplementary Fig. S2, panel B). Furthermore, densitometric determination of the FtsZ levels in the supernatant and pellet fractions revealed that irrespective of the PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11058