SummaryPaired two-component regulatory systems consisting of a sensor kinase and a response regulator are the major means by which bacteria sense and respond to different stimuli. The role of essential response regulator, MtrA, in Mycobacterium tuberculosis proliferation is unknown. We showed that elevating the intracellular levels of MtrA prevented M. tuberculosis from multiplying in macrophages, mice lungs and spleens, but did not affect its growth in broth. Intracellular trafficking analysis revealed that a vast majority of MtrA overproducing merodiploids were associated with lysosomal associated membrane protein (LAMP-1) positive vacuoles, indicating that intracellular growth attenuation is, in part, due to an impaired ability to block phagosome-lysosome fusion. A merodiploid strain producing elevated levels of phosphorylation-defective MtrA (MtrA D53N ) was partially replicative in macrophages, but was attenuated in mice. Quantitative real-time PCR analyses revealed that expression of dna A, an essential replication gene, was sharply upregulated during intramacrophage growth in the MtrA overproducer in a phosphorylation-dependent manner. Chromatin immunoprecipitation using anti-MtrA antibodies provided direct evidence that MtrA regulator binds to dna A promoter in vivo indicating that dna A promoter is a MtrA target. Simultaneous overexpression of mtr A regulator and its cognate mtr B kinase neither inhibited growth nor sharply increased the expression levels of dna A in macrophages. We propose that proliferation of M. tuberculosis in vivo depends, in part, on the optimal ratio of phosphorylated to nonphosphorylated MtrA response regulator.

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.

SummaryOligomerization of the initiator protein, DnaA, on the origin of replication ( oriC ) is crucial for initiation of DNA replication . Studies in Escherichia coli (Gramnegative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M. tuberculosis DnaA (DnaA TB ) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli , ATPase activity of DnaA TB promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaA TB proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non-viability, presumably due to a defect in oriC -DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaA TB bound to DnaA boxes similarly with ATP or ADP. DnaA TB binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation.

Acidic phospholipids have been shown to promote dissociation of bound nucleotides from Mycobacterium tuberculosis DnaA (DnaA TB ) purified under denaturing conditions (Yamamoto et. al, Biochemical J., 363, 305-311 (2002)). In the present study, we show that a majority of DnaA TB in non-overproducing cells of M. tuberculosis is membrane associated. Estimation of phospholipid phosphorus following chloroform: methanol extraction of soluble DnaA TB purified under native conditions (nDnaA TB ) confirmed the association with phospholipids. nDnaA TB exhibited weak ATPase activity, and rapidly exchanged ATP for bound ADP in the absence of any added phospholipids. We suggest that the outcome of intracellular DnaA TB -nucleotide interactions, hence DnaA TB activity, is influenced by phospholipids. KeywordsADP; ATP; ATPase; DNA replication; mycobacteria Tuberculosis is one of the most globally prevalent infectious diseases and accounts for approximately three million deaths each year. The causative agent Mycobacterium tuberculosis, a Gram-positive bacterium, is a slow grower with approximate doubling time of 24 h. The genus Mycobacterium includes other pathogens such as M. tuberculosis, M. bovis, and M. leprae, and non-pathogens such as M. smegmatis and M. fortuitum. The doubling times of these organisms range from 2 to 3 h (M. smegmatis, M. fortuitum) to 22-24 h (M. tuberculosis, M. bovis) to 185 h (M. leprae). The genetic and biochemical factors responsible for the differences in the growth rates of various mycobacteria are largely unknown.Chromosomal DNA replication in bacteria is regulated at the initiation step, where the activity and quantity of the initiator DnaA protein is critically controlled (1-3). DNA replication in Escherichia coli is initiated by the binding of DnaA protein to the DnaA boxes in the oriC, the origin of chromosomal DNA replication, and these initial interactions result in the melting of the nearby A-T-rich region, thereby forming an open (initiation) complex (4,5). DnaA protein then recruits the DnaB helicase-DnaC protein complex to form a pre-priming complex, which allows entry of primase and establishment of the replication forks.*To whom correspondence should be addressed. Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, Tel.: +81-92-621-4991, Fax: +81-92-624-1011, yamamok@agr.kyushu-u.ac Materials and Methods Purification of M. tuberculosis DnaA ProteinCloning of M. tuberculosis dnaA was performed as described (12). Overproduction of the recombinant DnaA protein was carried out with co-producing E. coli thioredoxin (Trx) (14). For purification, bacterial pellets were collected and resuspended in Binding buffer [20 mM Tris/HCl (pH 8.0), 10 % glycerol, 500 mM NaCl, 5 mM 2-mercaptoethanol, 10 mM imidazole and 0.5 % Tween 20] and disrupted by sonication. The crude cell lysate was clarified by centrifugation, and the supernatant fraction, which contained the soluble recombinant DnaA protein, was loaded...