Mycobacterium smegmatis dnaA region and autonomous replication activity

Abstract

Two key elements that are thought to be required for replication initiation in eubacteria are the DnaA protein, a trans-acting factor, and the replication origin, a cis-acting element. As a first step in studying the replication initiation process in mycobacteria, we have isolated a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that contains the dnaA gene. Nucleotide sequence analysis of this region revealed homologies with the rpmH gene, which codes for the ribosomal protein L34, the dnaA gene, which codes for the replication initiator protein DnaA, and the 5 end of the dnaN gene, which codes for the beta subunit of DNA polymerase III. Further, we provide evidence that when cloned into pUC18, a plasmid that is nonreplicative in M. smegmatis, the DNA fragment containing the dnaA gene and its flanking regions rendered the former capable of autonomous replication in M. smegmatis. We suggest that the M. smegmatis chromosomal origin of replication is located within the 4-kb DNA fragment.The genus Mycobacterium includes both rapid growers (e.g., Mycobacterium smegmatis and M. fortuitum) and slow growers (e.g., M. tuberculosis, M. bovis BCG, and M. leprae). The doubling time for rapid growers is approximately 3 to 4 h, compared with 20 to 24 h for slow growers. Some of the slow growers, such as M. tuberculosis and M. leprae, are major human pathogens. M. tuberculosis is a well-recognized opportunistic infectious agent for immunocompromised patients (24), and recent years have seen the emergence of M. tuberculosis strains that are resistant to one or more traditionally used antimycobacterial drugs. Our inability to control mycobacterial infections is due to a limited understanding of the basic metabolic processes, such as DNA replication and recombination, in these pathogens (33,36). An improved understanding of the key steps involved in the replication process of both rapid growers and slow growers would enable one to identify metabolic targets against which new generation drugs could be directed (2).Initiation of replication is believed to occur when DnaA, the initiator protein, interacts with the replication origin called the replicon or the replicator (15,17). Detailed genetic studies carried out in Escherichia coli (15,17) and limited studies carried out in Bacillus subtilis (9,(20)(21)(22) revealed that DnaA protein is required for replication initiation. DnaA protein initiates replication by selectively binding to repetitive units of nine-nucleotide DnaA protein recognition sequences, present in the origin of replication, called the DnaA boxes. This process triggers a cascade of events which result in replication initiation (4,15,17). In many bacteria, the dnaA gene is flanked by the rpmH and rnpA genes on the 5Ј side and the dnaN, recF, and gyrB genes on the 3Ј side (15,22,28). Exceptions to this gene order have been found in Rhizobium meliloti, Caulobacter crescentus, and Synechocystis sp. (16,25,38). Comparative analyses of the amino acid sequences of DnaA proteins from different bacteria have re...

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

“…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.…”

“…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.…”

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

“…The consensus sequence for Mycobacterium DnaA box is TTG/CT-CCACA [8]. Within the M. tuberculosis oriC, there is only one DnaA box with a single mismatch from the consensus sequence (DnaA box no.…”

“…Comparison of DnaA boxes from several Mycobacteria oriC regions allowed the consensus sequence to be determined: TT(G/C)-TCCACA (named a 'perfect' DnaA box) [8]. It corresponds to the Streptomyces consensus sequence for DnaA box (TTGTCCACA) [20].…”

“…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.…”