No 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.

cBacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein from Mycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog in M. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [ 14 C]N-acetylglucosamine cell wall precursor. The M. smegmatis MSMEG_0023 crgA double mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA for cell wall synthesis and cell shape protein A, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria. Since its reemergence in the early 1990s, tuberculosis caused by Mycobacterium tuberculosis remains a leading cause of global morbidity and mortality. With nearly 10 million new cases reported each year and 2 billion people infected, as well as the emergence of extensively drug-resistant and completely drug-resistant strains, sustained interest in improved control measures and tuberculosis research is crucial (4, 40). These reports emphasize the importance of understanding the basic biology of the pathways needed for pathogen proliferation, namely, DNA replication and cell division, and the development of novel strategies for combating M. tuberculosis infection (27).Cell division in bacteria is accomplished by the coordinated actions of multiple proteins that form a septal ring in the middle of a dividing cell. FtsZ, a cytoskeletal protein and a GTPase, initiates the cytokinesis process by the GTP-dependent midcell assembly of a ring structure called the Z ring (2). Constriction of the Z ring, driven by the GTPase activity of FtsZ, initiates cell division. During various stages of cell division, FtsZ interacts with a number of proteins, and these interactions modulate its midcell localization, membrane tethering, biochemical activities, and influence on the assembly of other septal ring members, including some proteins involved in peptidoglycan (PG) synthesis (reviewed in reference 2). Recent data also suggest that, at least in some bacteria, FtsZ plays a role in guiding cell wall synthesis (1, 37).In the last decade, significant advancements have been made not only ...

SUMMARY 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 transcripts levels are downregulated during stationary phase and starvation. Overexpression of ftsH delayed growth and reduced the viability of Mtb 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.

SUMMARY 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.