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
). 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
). 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.
DnaA activity is proposed to be regulated by its binding of adenine nucleotides. DnaA protein has high affinity for ATP and ADP, and the ATP-binding form of DnaA protein (ATP-DnaA) initiates DNA replication in vitro
, whereas the ADP-binding form (ADP-DnaA) does not (6
). Membrane phospholipids are believed to play a crucial role in the regulation of DnaA activity (7
). They affect the dissociation of both ATP and ADP from DnaA and rejuvenate moribund DnaA in to an active species for the oriC
). Acidic phospholipids in a fluid membrane facilitate dissociation of ADP bound to DnaA protein in vitro
, and the resultant free form of DnaA protein is reactivated through binding to ATP, which is present at high concentrations under physiological conditions.
The key elements involved in the initiation of M. tuberculosis
DNA replication, namely oriC
and DnaA, have been identified (10
). M. tuberculosis oriC
is 550-bp long (10
) and recombinant DnaATB
protein purified under denaturing conditions has been shown to associate with adenine nucleotides and oriC
specifically recognizes DnaA boxes and dimethylsulfate footprinting revealed the presence of nine DnaA-boxes that bear little or no sequence similarity to the five DnaA boxes of E. coli oriC
). Acidic phospholipids have been shown to modulate DnaATB
interactions with adenine nucleotide and ori
C stabilizes DnaATB
-ATP interactions and promotes dissociation of DnaATB
-ADP complexes (12
). Presumably, the interaction of DnaATB
with phospholipids results in a decrease in the affinity of DnaA for ATP, oriC
or both. Other results suggest that ADP-bound form of DnaA is not competent for binding and rapid oligomerization on oriC
). Together, these results suggest that phospholipids regulate nucleotide bound state of DnaA and play important regulatory roles in M. tuberculosis oriC
). To further understand the relationships between DnaATB
and phospholipids, we purified DnaATB
as a soluble protein under native conditions and investigated its interactions with nucleotides in the absence of added phospholipids.