[34,38]. Genetic studies indicate that FtsW is required for productive FtsZ ring formation and that FtsW possibly functions to promote and stabilize Z-rings in mycobacteria [34]. More recently, FipA, a FtsZ interacting protein has been shown to be required for sustenance of cell division in M. tuberculosis under oxidative stress conditions [43]. The results presented in this study attest that M. tuberculosis ClpX is a bona fide interaction partner of FtsZ and potentially acts as a negative regulator of Z-ring assembly and cell division. Thus, ClpX joins the list of proteins that regulate Z-ring assembly and cell division in the human pathogen M. tuberculosis. As reviewed, the modes of action of ClpX or ClpXP in cell division control are distinctly different in different bacterial species. C. crescentus ClpXP indirectly regulates cell division by degrading the CtrA regulator thereby modulating the expression of cell division genes [11,44,45]. B. subtilis ClpX inhibits FtsZ polymer- ization activity in vitro and Z-ring assembly in vivo, independent of the ClpXP protease activity [24,25]. The physical interaction between the ClpX and FtsZ proteins in B. subtilis, however, was not investigated in these studies. ClpX is an abundant protein in B. subtilis, with 8,400 molecules per cell [46]. Thus, it is likely that ClpX operates at stoichiometric levels in affecting FtsZ activities in B. subtilis. While this work was in progress, two recent reports indicated contrasting roles for E. coli ClpX in cell division. One study concluded that ClpXP operates catalytically to degrade FtsZ polymers and monomers, thereby modulates the equilibrium between free and polymeric FtsZ required for Z-ring assembly in an ATP-dependent manner [19]. The other study reported that E. coli ClpX directly modulates FtsZ polymer dynamics by disassem- bling FtsZ polymers in an ATP-independent manner [20] and that ClpXP protease activity contributes to a minor role in cell division regulation. Therefore, our studies showing a physical interaction between ClpX and FtsZ (Figs. 3–5), the similar intracellular FtsZ levels in cells producing altered levels of ClpX (supplementary Fig. S7), the modulation of FtsZ polymerization in vitro and Z-ring assembly in vivo (Figs. 2 and 7), combined with the finding that the intracellular ratio of ClpX to FtsZ is 1:2 (Fig. 6A), are consistent with the notion that in M. tuberculosis ClpX interacts with FtsZ and possibly interferes with Z-ring assembly by sequestering the available intracellular pools of FtsZ. While this reasoning assumes that overproduction of ClpX in vivo could lead to cell division arrest and extensive filamenation, in reality this might not happen (see below). For example, our results showed that a 6-fold overproduction of ClpX caused ,50% increase in cell length. While this can be considered modest, it is noteworthy that a 2-fold increase in ClpX in B. subtilis resulted in only a ,20% increase in cell length [25]. ClpX is the substrate recognition part of the ClpXP protease complex. Thus, increased levels of ClpX could promote its interaction not only with FtsZ but also with its other substrates. For example, the ClpXP complex in E. coli is known to associate with ,60 proteins [23]. Therefore, while the in vitro results suggested that FtsZ and ClpX interact stoichiometrically, the extent of in vivo filamentation would likely depend on the ClpX ‘available’ for FtsZ (cell division) inhibition. The observed modulation of Z-ring assembly upon ClpX overproduction was carried out in M. tuberculosis background WT for clpP1 and clpP2. Thus, our data also suggest that ClpX mediated modulation of FtsZ assembly is independent of the presumptive M. tuberculosis ClpXP protease activity as in B. subtilis and possibly in other bacteria. Our studies with FtsZ mutant proteins, FtsZG103S, defective in binding to GTP and, hence, defective in polymerization [28], FtsZD210G, defective in GTP hydrolysis and, hence, deficient in exhibiting polymer dynamics [47], FtsZD374-76A and FtsZDC21 M. tuberculosis ClpX and FtsZ which are defective for interactions with FtsW [34,38], and possibly other proteins, indicate that M. tuberculosis ClpX interacts with FtsZ independent of FtsZ oligomeric state or associations with other proteins. Parallel data showing that ClpX interferes with FtsZ polymerization activity in the presence of the non- hydrolysable GTP analog, GMPCPP [31], reduces the amount of FtsZ polymers formed even when added to preformed FtsZ polymers (Fig. S3) and the colocalization of ClpX-CFP with FtsZ- YFP at midcell Z-rings (Fig. 8) are in partial support of the above conclusions. Together, these results are consistent with a model in which M. tuberculosis ClpX interacts with FtsZ independent of its oligomeric state or associations with other proteins and sequesters FtsZ from engaging in its activities. This line of thinking suggests that ClpX, like SulA of E. coli, inhibits the FtsZ polymerization by a sequestration mechanism [31]. However, unlike ClpX, the E. coli SulA protein does not inhibit FtsZ polymerization in the absence of GTP hydrolysis. The E. coli MinC also requires GTP hydrolysis activity of FtsZ for inhibition of polymer assembly. Since MinC does not affect the GTPase activity of FtsZ it is thought that MinC requires dynamic FtsZ polymers to exert its effect [30]. Our studies raise questions as to when the consequences of FtsZ and ClpX interactions lead to modulation of Z-ring assembly in M. tuberculosis, especially since overproduction of ClpX led to only modest changes in cell length and growth rate (Fig. 6). To answer this question, it is important to recognize that both M. tuberculosis ClpX and FtsZ are relatively abundant proteins and are present at an intracellular ratio of 1:2. ClpX is a component of the ClpXP protease and could be interacting with other hitherto unrecog- nized interaction partners, similar to the situation reported in E. coli [23]. Thus, we expect that under normal growth conditions ClpX interactions with FtsZ would not lead to interference of Z- ring assembly in M. tuberculosis. Limited ,6-fold overproduction of ClpX (Fig. 6B inset), however, could impair the optimal intracellular ratio between ClpX and FtsZ. This could in turn interfere with FtsZ activities and affect Z-ring assembly. It is important to note in this regard that ClpX expression is upregulated during intracellular growth and upon exposure to antibiotic stress, the same growth conditions where the intracel- lular levels of FtsZ are not affected [26]. FtsZ ring assembly is modulated by these conditions, however, and cell division is delayed [26]. The expression of clpX is likely modulated in response to various environmental cues and the induced ClpX possibly functions as a chaperone and as part of the ClpXP protease. We propose that under some circumstances such as growth of M. tuberculosis in macrophages and upon exposure to antibiotic cephalexin, ClpX also modulates FtsZ-ring assembly such that the pathogen can cope with the stress of survival. M. tuberculosis Z-ring assembly, as in E. coli and B. subtilis [48,49], is a dynamic process, and the subunits in the FtsZ ring undergo constant turnover [47]. Studies have also indicated that ,30% of the FtsZ protein pool is in the Z-ring, while the remainder is likely to be present as short polymers and monomers [49]. Therefore, it is likely that in M. tuberculosis, ClpX-bound FtsZ monomers are incompatible for polymerization or exchange with the subunits in FtsZ polymers or protofilaments [32,47,50]. This, in turn, could limit subunit exchange and prevent productive Z-ring assembly. Although further studies are required to address these issues in detail, our data suggest that ClpX could be one of the factors regulating M. tuberculosis Z-ring assembly during intracellular growth. We found approximately three times more colocalization of ClpX and FtsZ at the cell poles compared to midcell sites (Fig. 8). However, our data do not rule out a possibility that colocalization of ClpX and FtsZ could also be due to their respective interactions PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11058