Facilitation of Dissociation Reaction of Nucleotides Bound to Mycobacterium tuberculosis DnaA

Abstract

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...

Association of the Chromosome Replication Initiator DnaA with the Escherichia coli Inner Membrane In Vivo: Quantity and Mode of Binding

“…Later, this result was also reproduced with H. pylori and M. tuberculosis [40], [41], indicating that the phenomenon may be universal in the bacterial world. Immunolabeling of fixed cells revealed DnaA in the membrane vicinity [42].…”

“…Intracellular localization of DnaA was first investigated by fractionation of E. coli cells showing a remarkable portion of DnaA associated with the membrane [39] . Later, this result was also reproduced with H. pylori and M. tuberculosis [40] , [41] , indicating that the phenomenon may be universal in the bacterial world. Immunolabeling of fixed cells revealed DnaA in the membrane vicinity [42] .…”

Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold‐sensitive mutants

“…This situation may be analogous to that of the RIDA mechanism (Katayama et al ., 1998). M. tuberculosis DnaA is membrane associated (Yamamoto et al ., 2008) and acidic phospholipids promote dissociation of nucleotides from DnaA and possibly restore levels of active DnaA (Yamamoto et al ., 2002a; 2008). Finally, in addition to DnaA, other hitherto unidentified regulatory proteins may target both the dna A promoter and the ori C in order to regulate hyper‐initiation.…”

“…Earlier studies on DnaA‐mediated ori C replication in M. tuberculosis have revealed that both DnaA TB and ori C are essential and that DnaA TB is a DnaA box‐binding protein (Qin et al ., 1999; Dziadek et al ., 2002; Yamamoto et al ., 2002a; Zawilak et al ., 2004; Madiraju et al ., 2006). The DnaA TB protein exhibits weak ATPase activity (Yamamoto et al ., 2002a; 2008) and uses its intrinsic ATPase activity to rapidly oligomerize at ori C (Madiraju et al ., 2006). The M. tuberculosis strain producing DnaA that is defective for ATPase activity, but is proficient for binding to ATP, is inviable (Madiraju et al ., 2006).…”

Localization of acidic phospholipid cardiolipin and DnaA in mycobacteria

“…Mtb DnaA forms an oligomeric complex on the origin, mediated by its ATPase activity 15 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.…”