Characterization of the oriC region of Mycobacterium smegmatis

Abstract

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.The dnaA gene flanking region in many bacteria is the origin of chromosomal DNA replication, or oriC region (10,26,27). The oriC region is generally AT nucleotide rich, containing repeats of AT-rich nucleotide sequences varying in length from 13 (e.g., Escherichia coli and pseudomonads) to 16 (e.g., Bacillus subtilis) together with several 9-nucleotide DnaA protein recognition sequences called DnaA boxes (10,17,26,27). Biochemical studies with purified proteins and the oriC region of E. coli revealed that the first step in the initiation of DNA replication involved binding of 20 or more DnaA molecules to the DnaA boxes (3). This step is presumed to be followed by opening of the double-stranded oriC DNA at 13-mer AT-rich cluster sites for the entry of DnaC-directed DnaB protein.Additional events at the oriC region then result in unwinding of double-stranded DNA, priming of DNA synthesis, and formation of replication forks. Genetic studies with mutant DnaA proteins and mutant origin regions further clarified the role(s) of the DnaA protein and the mechanism of replication initiation in E. coli (10, 15).The mycobacteria exhibit varied growth rates, with the doubling times ranging from 2 to 3 h (Mycobacterium smegmatis and M. fortuitum) to 10 to 12 h (M. avium-intracellulare complex) to 22 to 24 h (M. tuberculosis and M. bovis BCG) (29). The genetic and biochemical aspects of replication, especially initiation, in mycobacteria are not well understood. As a first step toward identifying the key playe...

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

“…1). (ii) Despite the fact that the oriC region from M. smegmatis contained seven DnaA box motifs, while the region upstream of dnaA contains only three, the ars plasmids are highly stable and did not insert into chromosomes ( Fig. 5 ; Qin et al, 1997 ;Salazar et al, 1996). Nevertheless, the DnaA box region upstream of the dnaA gene might be playing some regulatory role.…”

“…Qin et al (1997) reported for ars plasmids of M. smegmatis a copy number of two. In this study, variations in the copy number of the ars plasmids of M. smegmatis were found (Fig.…”

“…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.…”

Mycobacterium tuberculosis DnaA initiator protein: purification and DNA-binding requirements

“…of the fact that the DNA fragments containing the dnaA-dnaN intergenic region from M. avium, M. leprae, M. smegmatis and M. tuberculosis were shown to promote autonomous replication [8][9][10][11][12], little is known about interactions between the two key elements of the initiation of mycobacterial chromosome, oriC and DnaA protein. In contrast with E. coli, the structure of mycobacterial oriC region is more complex; the oriC is longer than the one in E. coli and possesses numerous 'non-perfect' DnaA boxes.…”

“…In contrast with E. coli, the structure of mycobacterial oriC region is more complex; the oriC is longer than the one in E. coli and possesses numerous 'non-perfect' DnaA boxes. Each of the 13 DnaA boxes located within the M. tuberculosis oriC [12] region differs at least by one base from the perfect E. coli DnaA box (TTATCCACA). Thus it is important to understand more of the initiation of DNA replication in these slow-growing organisms.…”