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

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.

SUBMITTER: Dziedzic R

PROVIDER: S-EPMC2897852 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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

Dziedzic Renata R Kiran Manjot M Plocinski Przemyslaw P Ziolkiewicz Malgorzata M Brzostek Anna A Moomey Meredith M Vadrevu Indumati S IS Dziadek Jaroslaw J Madiraju Murty M Rajagopalan Malini M

PloS one 20100706 7

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 m ...[more]

PMID: 20625433

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Project description:Extracytoplasmic function ? factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-? factor. Mycobacterium tuberculosis membrane-associated anti-? factors have a small, stable RNA gene A (ssrA)-like degron for targeted proteolysis. Interaction between the unfoldase, ClpX, and a substrate with an accessible degron initiates energy-dependent proteolysis. Four anti-? factors with a mutation in the degron provided a set of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-substrate processivity. We note that a point mutation in the degron (X-Ala-Ala) leads to an order-of-magnitude difference in the dwell time of the substrate on ClpX. Differences in ClpX/anti-? interactions were correlated with changes in unfoldase activities. Green fluorescent protein (GFP) chimeras or polypeptides with a length identical to that of the anti-? factor degron also demonstrate degron-dependent variation in ClpX activities. We show that degron-dependent ClpX activity leads to differences in anti-? degradation, thereby regulating the release of free ? from the ?/anti-? complex. M. tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF ? factors.IMPORTANCE The ability of Mycobacterium tuberculosis to quickly adapt to changing environmental stimuli occurs by maintaining protein homeostasis. Extracytoplasmic function (ECF) ? factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the ? factor from the anti-? is governed by the ClpXP2P1 assembly. M. tuberculosis ECF anti-? factors have an ssrA-like degron for targeted degradation. A point mutation in the degron leads to differences in ClpX-mediated proteolysis and affects the cellular abundance of ECF ? factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affecting M. tuberculosis physiology.

Dataset Information

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

Publications

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

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