Characterization of the functional replication origin of Mycobacterium tuberculosis

Qin, Ming Hui; Madiraju, Murty V. V. S.; Rajagopalan, Malini

doi:10.1016/s0378-1119(99)00148-1

Cited by 46 publications

(72 citation statements)

References 15 publications

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Section: Introductionmentioning

confidence: 99%

“…Knowledge regarding the steps of the mycobacterial cell cycle (replication, chromosome segregation and cell division) seems to be critical for understanding the mechanisms that are responsible for the transition from an active to a non-replicative persistent state (and vice versa) of pathogenic mycobacteria, particularly M. tuberculosis. While initiation of chromosome replication Qin et al, 1999;Rajagopalan et al, 1995;Zawilak et al, 2004) and cell division (FtsZ ring formation) (Chauhan et al, 2006; Dziadek et al, 2002Dziadek et al, , 2003Huang et al, 2007;Rajagopalan et al, 2005) are relatively well studied in mycobacteria, nothing is known about the segregation of chromosomes in these bacteria.Bacterial chromosome segregation has been recently found to be an active and complex process closely coupled with replication (see Bartosik & Jagura-Burdzy, 2005;Errington et al, 2005;Hayes & Barilla, 2006; Leonard et al, 2005 for reviews). In bacteria studied to date, the newly synthesized origin (oriC) regions undergo a symmetric or asymmetric segregation process; two copies of the duplicated oriC regions migrate from the cell centre toward opposite cell poles, i.e.…”

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Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis

et al. 2007

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Bacterial chromosomes (though not Escherichia coli and some other c-proteobacterial chromosomes) contain parS sequences and parAB genes encoding partitioning proteins, i.e. ParA (ATPase) and ParB (DNA-binding proteins) that are components of the segregation machinery. Here, mycobacterial parABS elements were characterized for the first time. parAB genes are not essential in Mycobacterium smegmatis; however, elimination or overexpression of ParB protein causes growth inhibition. Deletion of parB also leads to a rather severe chromosome segregation defect: up to 10 % of the cells were anucleate. Mycobacterial ParB protein uses three oriC-proximal parS sequences as targets to organize the origin region into a compact nucleoprotein complex. Formation of such a complex involves ParB-ParB interactions and is assisted by ParA protein. INTRODUCTIONTuberculosis (TB) is the world's most common disease caused by an infectious organism, Mycobacterium tuberculosis (DeAngelis & Flanagin, 2005; and see http:// www.who.int/topics/tuberculosis/en/). Due to the spread of multi-drug-resistant strains of M. tuberculosis and a synergy with HIV, the TB epidemic is growing and becoming more dangerous in both developing and industrialized countries. A unique feature of M. tuberculosis is its ability to maintain a dormant, non-replicating state for extended periods of time under unfavourable conditions. The mechanism (or mechanisms) by which this bacterium shifts to a dormant state and reverts to active growth is not well understood (Hampshire et al., 2004;Gó mez & Smith, 2000;Wayne & Hayes, 1996: Wayne & Sohaskey, 2001. Knowledge regarding the steps of the mycobacterial cell cycle (replication, chromosome segregation and cell division) seems to be critical for understanding the mechanisms that are responsible for the transition from an active to a non-replicative persistent state (and vice versa) of pathogenic mycobacteria, particularly M. tuberculosis. While initiation of chromosome replication Qin et al., 1999;Rajagopalan et al., 1995;Zawilak et al., 2004) and cell division (FtsZ ring formation) (Chauhan et al., 2006; Dziadek et al., 2002Dziadek et al., , 2003Huang et al., 2007;Rajagopalan et al., 2005) are relatively well studied in mycobacteria, nothing is known about the segregation of chromosomes in these bacteria.Bacterial chromosome segregation has been recently found to be an active and complex process closely coupled with replication (see Bartosik & Jagura-Burdzy, 2005;Errington et al., 2005;Hayes & Barilla, 2006; Leonard et al., 2005 for reviews). In bacteria studied to date, the newly synthesized origin (oriC) regions undergo a symmetric or asymmetric segregation process; two copies of the duplicated oriC regions migrate from the cell centre toward opposite cell poles, i.e. to the 1/4 and 3/4 positions (Escherichia coli, Bacillus subtilis, Vibro cholerae chromosome II), or one copy of the newly synthesized origins remains at the pole while the other copy migrates to the opposite pole Abbreviations: EMSA, electrophoretic mo...

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Section: Introductionmentioning

confidence: 99%

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confidence: 99%

Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis

et al. 2007

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“…Autonomous replicating sequences (ars's) on plasmids have been isolated from different species of bacteria, such as E. coli (Yasuda & Hirota, 1977), Pseudomonas putida, P. aeruginosa (Yee & Smith, 1990), B. subtilis (Moriya et al, 1992), Streptomyces lividans (Zakrzewska-Czerwinska & Schrempf, 1992), Spiroplasma citri (Ye et al, 1994), M. smegmatis (Salazar et al, 1996 ;Qin et al, 1997) and recently, in M. tuberculosis, M. bovis (Qin et al, 1999) and M. avium (Madiraju et al, 1999). In Gram-negative bacteria, isolated ars's contain either the region upstream of dnaA or a DnaA box region downstream of rnpA.…”

Section: Abbreviationsmentioning

confidence: 99%

Inhibition of chromosome replication in Mycobacterium smegmatis: effect of the rpmH–dnaA promoter region

Salazar

2000

Microbiology

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In a previous study a functional mycobacterial origin of replication, oriC, was isolated on a plasmid. However, it was found that origin function was inhibited by the presence of the adjacent dnaA gene or its regulatory region, so that plasmids containing both of these regions next to the origin did not yield transformants. This inhibition could be due either to overexpression of dnaA on a plasmid being toxic, the transcription of dnaA into the downstream origin topologically inhibiting its function, or to the DnaA boxes upstream of dnaA somehow interacting with the DnaA boxes in the origin to prevent its function. To distinguish between these possibilities, plasmids were constructed lacking different parts of the dnaA gene : the promoter, the DnaA boxes, or both. Additionally, the putative dnaA promoter region was replaced by mycobacterial sequences that exhibit weaker or null promoter activity. The results indicate that the rpmH-dnaA promoter region, but not the DnaA boxes, is the principal cause of the incompatibility observed and suggest that this region could be playing a role in the inhibition of chromosome replication.

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“…The key elements involved in the initiation of M. tuberculosis DNA replication, namely oriC and DnaA, have been identified (10,11). M. tuberculosis oriC is 550-bp long (10) and recombinant DnaA TB protein purified under denaturing conditions has been shown to associate with adenine nucleotides and oriC (12).…”

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confidence: 99%

“…M. tuberculosis oriC is 550-bp long (10) and recombinant DnaA TB protein purified under denaturing conditions has been shown to associate with adenine nucleotides and oriC (12). DnaA TB 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 (13).…”

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Facilitation of Dissociation Reaction of Nucleotides Bound to Mycobacterium tuberculosis DnaA

Yamamoto

Moomey

Rajagopalan

et al. 2007

Journal of Biochemistry

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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...

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Characterization of the functional replication origin of Mycobacterium tuberculosis

Cited by 46 publications

References 15 publications

Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis

Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis

Inhibition of chromosome replication in Mycobacterium smegmatis: effect of the rpmH–dnaA promoter region

Facilitation of Dissociation Reaction of Nucleotides Bound to Mycobacterium tuberculosis DnaA

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