Mycobacterium tuberculosis ClpX interacts with FtsZ and interferes with FtsZ assembly.

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.

Mycobacterium tuberculosis ftsZ expression and minimal promoter activity.

Optimal levels of ftsZ gene product are shown to be required for initiation of the cell division process in Mycobacterium tuberculosis. Here, we report that the ftsZ gene expression is sharply down-regulated during starvation and hypoxia, conditions that are believed to result in growth arrest, but is restored upon dilution of cultures into fresh oxygen-rich media. Primer extension analysis identified four transcriptional start sites, designated as P1, P2, P3 and P4 at nucleotide positions -43, -101, -263, and -787, respectively, in the immediate upstream flanking region of the ftsZ initiation codon. Promoter deletion and homologous recombination experiments revealed that ftsZ expression from the 101-bp region is sufficient for M. tuberculosis viability. All promoter strains had reduced FtsZ levels compared to wild-type, although the loss of P4 severely compromised FtsZ levels during both the active and stationary phases. We propose that ftsZ expression from all promoters is required for optimal intracellular FtsZ levels and that the activities of P4 and possibly other promoters are down-regulated during growth-arrest conditions.

Mycobacterium tuberculosis ftsH expression in response to stress and viability.

FtsH is an essential membrane-bound protease that degrades integral membrane proteins as well as cytoplasmic proteins. We show that Mycobacterium tuberculosis (Mtb) ftsH expression levels are upregulated upon exposure to agents that produce reactive oxygen and nitrogen intermediates (ROI and RNI) and growth in macrophages. In partial support of this result is our observation that the Mtb merodiploid overexpressing ftsH shows increased resistance to ROI. ftsH transcript levels are downregulated during stationary phase and starvation. ftsH overexpression strain shows delayed growth and reduced viability in vitro and ex vivo. Finally, we show that the intracellular levels of FtsZ, an essential cell-division protein, are reduced in ftsH-overexpressing strain. Together, our results suggest that Mtb FtsH is a stress-response protein that promotes the pathogen's ability to deal with ROI stress and is possibly involved in the regulation of FtsZ levels.