Localization of acidic phospholipid cardiolipin and DnaA in mycobacteria

Summary

Acidic phospholipids such as cardiolipin (CL) have been shown to modulate Mycobacterium tuberculosis (Mtb) DnaA interactions with ATP. In the present study, using nonyl acridine orange fluorescent dye we localized CL-enriched regions to midcell septa and poles of actively dividing cells. We also found that CL-enriched regions were not visualized in cells defective for septa formation as a consequence of altered FtsZ levels. Using Mtb cultures synchronized for DNA replication we show that CL localization could be used as a marker for cell division and cell cycle progression. Finally, we show that the localization pattern of the DnaA-green fluorescent fusion protein is similar to CL. Our results suggest that DnaA colocalizes with CL during cell cycle progression.

Introduction

Mycobacterium tuberculosis (Mtb) is a slow-growing pathogenic bacterium and the causative agent of the infectious disease tuberculosis. This bacterium has a doubling time of ∼24 h, and the pathogen’s efficiency as an infectious agent relies in part on its ability to shift from an active growth state to a non-replicative persistent state within the host. Mtb in this persistent state maintains metabolic activity¹ and presumably, favorable conditions resulting in reactivation of the bacterium can lead to active infection.² Such a shift between the two growth states requires tight regulation of cell cycle mechanisms; of particular importance are mechanisms governing initiation of DNA replication.

DnaA protein is the initiator of DNA replication and binds to specific nucleotide sequences within the chromosomal origin of replication or oriC.3, 4, 5 Studies with Escherichia coli DnaA revealed that it binds ATP and ADP with equal affinity, and hydrolyzes ATP. The hydrolytic product ADP associates tightly with DnaA and the ADP-bound form of DnaA is not active for oriC replication; acidic phospholipids promote dissociation of nucleotides and activation of DnaA.⁶ Binding of ATP-bound form of DnaA to the oriC results in opening of the duplex, subsequent assembly of other components and DNA synthesis.7, 8, 9, 10 The DnaA protein of Mtb is essential, has been shown to bind and hydrolyze ATP, and bind to oriC.11, 12, 13, 14, 15, 16 Mtb DnaA forms an oligomeric complex on the origin, mediated by its ATPase activity¹⁵ and this is followed by strand melting.¹⁷ Mtb DnaA protein associates with membrane lipids,¹³ and like the E. coli counterpart, its interactions with ATP and ADP are also modulated by membrane lipids.¹² However, the mechanism(s) regulating the replication initiation process in Mtb are largely unknown.

Our earlier studies using 10-nonyl acridine orange (NAO), a fluorescent dye, determined that CL-enriched regions are confined to septa and cell poles of actively growing Mycobacterium smegmatis and Mtb.¹⁸ These are nascent growth zones in mycobacteria.18, 19 Independently, we showed that lysX mutant deficient in production of lysinylated phosphatidylglycerol is elongated, defective for cell division and exhibits altered NAO staining pattern.¹⁸ We infer from these results that NAO, which stains CL, could be used as a marker for evaluating the status of cell division in mycobacteria. Given the affects of membrane phospholipids on DnaA activity, it is also assumed, but not proven that Mtb DnaA interacts with acidic phospholipids in vivo. The present study is undertaken to address two independent questions: (1) whether NAO staining could be used to mark septa and cell division status of mycobacteria; (2) whether the in vivo localization of DnaA and CL are similar.