Madiraju, Murty V.V.S., Ph.D.
Replication initiation and Cell cycle control in Mycobacterium tuberculosis.
We are interested in understanding the mechanisms responsible for initiation of and regulation of chromosomal DNA replication and its coordination with other events in the life cycle of Mycobacterium tuberculosis, the causative agent for tuberculosis. M. tuberculosis is also the leading cause of morbidity and mortality in AIDS patients. These problems are compounded by the emergence of strains of M. tuberculosis that are resistant to one or more antituberculous drugs. M. tuberculosis is a slow grower with an average doubling time (dt) of 24 hours. The genus mycobacteria also includes other slow growers (M. avium, ~ dt 10 hours) and rapid growers (M. smegmatis, ~ dt 2 hours). Following initial infections, M. tuberculosis frequently enters a latent or dormant state for extended periods and subsequently, under appropriate conditions or following immune suppression, revives, multiplies and causes a secondary infection. DNA replication constitutes an important step in the exit from latency. The development of novel therapeutic agents to control M. tuberculosis infections in HIV infected patients as well as other individuals is severely hindered by our limited understanding of the initiation and regulation of M. tuberculosis DNA replication and its coordination with other events in cell cycle.
Initiation of DNA replication is believed to be triggered when DnaA, the putative initiator protein, interacts with oriC or origin of replication. Although both oriC and dnaA are essential for survival, many clinical strains of M. tuberculosis appear to tolerate major deletions and IS6110 insertions in their oriC, thereby raising questions as to how these clinical strains replicate their genome. Our current research projects focuses on three specific areas: (1) Investigations of how M. tuberculosis oriC is primed for replication both during active growth and exit from latency. (2) Identification and characterization of alternate sites of origins for replication initiation, if any, used by the pathogen (3) Identification of factors/ regulators that potentially affect DnaA activity both during entry into and exit from latent state. Proteomic, genomic, surface plasmon resonance and biochemical techniques will be used to achieve these goals. Detailed knowledge on M. tuberculosis oriC activation mechanisms both during active growth and exit from latent state is an essential prerequisite for the development of drugs to inhibit the initiation of replication and thereby prevent the development of potentially lethal infections of M. tuberculosis.
We also have a general interest in understanding the RRR (replication- recombination- repair) processes in bacteria.
Selected Papers and Abstracts:
- Nair N, Dziedzic R, Greendyke R, Muniruzzaman S, Rajagopalan M, Madiraju MVVS. (2008). Synchronized replication initiation in novel Mycobacterium tuberculosis cold-sensitive dnaA mutants. (Mol Microbiol, in press DOI: 10.1111/j.1365-2958.2008.06523.x).
- Yamamoto K., Moomey M, Rajagopalan M, Madiraju MVVS. (2008). Facilitation of dissociation reaction of nucleotides bound to Mycobacterium tuberculosis DnaA. J. Biochem, 143(6): 759-764.
- Chauhan A, Lofton H, Maloney E, Moore J, Fol M, Madiraju MVVS, Rajagopalan, M. (2006). Interference of Mycobacterium tuberculosis cell division by Rv2719c, a cell wall hydrolase. Mol Micro. 62: 132-147.
- Fol M, Chauhan A, Nair N, Moomey M, Maloney E, Jagannath C, Madiraju MVVS, Rajagopalan M. (2006). Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulator. Mol Micro. 60(3): 643-647.
- Madiraju MVVS, Moomey M, Neuenschawander P, Muniruzzaman S, Yamamoto K, Grimwade JE, Rajagopalan, M. (2006). The intrinsic ATPase activity Mycobacterium tuberculosis DnaA promotes its rapid oligomerization on oriC. Mol Micro. 59: 1876-1890.
- Chauhan A, Madiraju MVVS, Fol M, Lofton H, Maloney E, Jensen R, Reyonolds RS, Rajagopalan, M. (2006). Mycobacterium tuberculosis growing in macrophages are filamentous and are deficient in FtsZ rings. J. Bacteriol. 188: 1856-1865.
- Madiraju MVVS, Rajagopalan M. (2005). DNA Replication and Cell division in Mycobacterial Molecular Microbiology. Horizon Press. Tanya Parish (Editor): p1- 28.
- Rajagopalan M, Atkinson MAL, Lofton H, Chauhan A, Madiraju MVVS. (2005). Mutations in the GTP-binding and synergy loop domains of Mycobacterium tuberculosis ftsZ compromise its function in vitro and in vivo. Biochem Biophys Res Commun. 331(4):1171-1177.
- Rajagopalan M, Maloney E, Dziadek J, Poplawska M, Lofton H, Chauhan A, Madiraju MVVS. (2005). Genetic evidence that mycobacterial FtsZ and FtsW proteins interact, and colocalize to the division site in Mycobacterium smegmatis. FEMS LETT. (250) 9-17.
- Dzaiadek J, Rutherford SA, Madiraju MV, Atkinson MAL, Rajagopalan M. (2003). Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene. Microbiology. 149: 1593-1603.
- Greendyke R, Rajagopalan M, Parish T, Madiraju MV. (2002). Conditional expression of Mycobacterium smegmatis dnaA, an essential replication gene. Microbiology; 148: 3887- 3990.
- Kantake N, Madiraju MV, Sugiyama T, Owalczykowski SC. (2002). Escherichia coli RecO protein anneals ssDNA compelxed with its cognate ssDNA-binding protein: A common step in genetic recombination. Proc. Nat. Acad. Sci. (U.S.A.). 99- 15327- 15332.
- Yamamoto K, Muniruzzaman S, Rajagopalan M, Madiraju MV. (2002). Modulation of Mycobacterium tuberculosis DnaA protein- adenine nucleotide interactions by acidic phospholipids. Bicohem. J. 363:305-311.
- Yamamoto K, Rajagopalan M, Madiraju MV. (2002) Phospholipids promote dissociation of ADP from Mycobacterium avium DnaA protein. J. Biochem. 131:219-224.
- Dziadek J, Rajagopalan M, Kurepina, Parish T, Greendke R, Kreiswirth B, Madiraju MV.(2002) Mutations in the CCGTTCACA DnaA box of Mycobacterium tuberculosis oriC that abolish replication of oriC plasmids are tolerated on the chromosome. J. Bacteriol 184:3848-3855.
- Dziadek, J, Madiraju MV, Rutherford SA, Atkinson MA, Rajagopalan M. Physiological consequences associated with the Mycobacterium tuberculosis FtsZ overproduction in mycobacterial hosts. (2002) Microbiology 148:961-971. [** equal contribution].
- Yamamoto K, Low B, Rutherford SA, Rajagopalan M, Madiraju MV. (2001) The Mycobacterium avium-intracellulare complex dnaB locus and protein intein splicing. Biochem. Biophys. Res. Comm. 280: (898-903).
- Madiraju MV, Qin MH, and Rajagopalan M. (2001). Development of a simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads. Letts. Appl Micro. 30: (38-41).
- Madiraju MV, Qin MH, Yamamoto K, Atkinson MA, Rajagopalan M. (1999). The dnaA gene region of Mycobacterium avium and the autonomous replication activities of its 5’ and 3’ flanking regions. Microbiology: 145: 2913-2921.