Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly Renata Dziedzic1, Manjot Kiran1, Przemyslaw Plocinski1, Malgorzata Ziolkiewicz2, Anna Brzostek2, Meredith Moomey1, Indumati S. Vadrevu1, Jaroslaw Dziadek2, Murty Madiraju1, Malini Rajagopalan1* 1 The University of Texas Health Science Center, Tyler Biomedical Research, Tyler, Texas, United States of America, 2 Institute for Medical Biology, Polish Academy of Sciences, Lodz, Poland Abstract FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1:2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division. Citation: Dziedzic R, Kiran M, Plocinski P, Ziolkiewicz M, Brzostek A, et al. (2010) Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly. PLoS ONE 5(7): e11058. doi:10.1371/journal.pone.0011058 Editor: David M. Ojcius, University of California Merced, United States of America Received March 9, 2010; Accepted May 14, 2010; Published July 6, 2010 Copyright: 2010 Dziedzic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funded by NIH grants RO1AI48417 (MR) and RO1AI73966, AI084734 (MM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: malini.rajagopalan@uthct.edu Introduction Mycobacterium tuberculosis, the causative agent of tuberculosis, has spread dramatically worldwide, and recent years have seen not only an increase in the number of multidrug-resistant strains but also the emergence of extensively drug-resistant M. tuberculosis [1,2]. Eradication of M. tuberculosis infection necessitates the development of novel drugs targeted to hitherto unidentified metabolic processes and pathways of the pathogen, and FtsZ catalyzed cell division is one such process. FtsZ, the homolog of eukaryotic tubulin, is a highly conserved protein and plays a central and essential role in initiation of the cell division process [3,4]. M. tuberculosis FtsZ, like its bacterial counterparts, exhibits GTP binding and hydrolysis activities and localizes to the midcell division site in the form of a Z-ring [5,6]. FtsZ-ring assembly in M. tuberculosis is delayed under several conditions relevant to its growth; two of these are growth in macrophages and exposure to cephalexin [5]. The identities of the regulators affecting Z-ring assembly and the cell division process in M. tuberculosis, however, are largely unknown. Identification and characterization of such regulators would improve our understanding of the cell division process in M. tuberculosis. Elegant genetic and cell biological studies carried out in other bacteria indicate that Z-ring assembly is regulated by the counterbalancing activities of positive and negative regulators that directly or indirectly modulate FtsZ activities (reviewed in [4]). These regulators include FtsA, ZipA, ZapA, Slm, SulA, YneA, Noc, EzrA, CrgA and ClpX; however, not all of these regulators are conserved in all bacteria (reviewed in [7]). The M. tuberculosis genome appears to lack most of the known regulators, except for the genes encoding ClpX, CrgA and a YneA-like protein, ChiZ [5,8]. We recently showed that ChiZ is a DNA damage-inducible protein that shows peptidoglycan hydrolysis activity and functions to regulate Z-ring assembly and cell division in M. tuberculosis [5]. It is unknown if ClpX serves as a potential cell division regulator in M. tuberculosis. ClpX, the substrate recognition part of the ClpXP protease, is well conserved in prokaryotes [9,10,11]. In the ClpXP complex, ClpX is present in a hexameric ring and is attached to the twin-stacked heptameric ClpP rings via the protease interface surface (reviewed [9]). ClpX belongs to the AAA ATPase family and contains the characteristic Walker A and Walker B motifs required for ATP binding and hydrolysis, respectively [12,13,14]. By itself, ClpX works as a chaperone, whereas in association with the ClpP protease, it is believed to eliminate misfolded, aggregated and dysfunctional protein targets [9]. It is interesting to note that, unlike Escherichia coli, Caulobacter crescentus and B. subtilis, the M. tuberculosis genome appears to contain one clpX and two clpP genes, designated as clpP1 and clpP2 [15]. On the other hand, the presence of clpP1 and clpP2 is common to members of Actinomycetales, including Mycobacterium spp. [16,17]. PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11058