Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000

Abstract

Plasmid pAL5000 from Mycobacterium fortuitum encodes two proteins necessary for replication: RepA (307 amino acid residues) and RepB (119 residues). A single RNA species encoding these proteins was characterized, and its 5 end was defined. The proteins were expressed as maltose-binding protein fusions in Escherichia coli. The RepB protein was shown in vitro to bind specifically to a previously defined 435-bp region of pAL5000 containing the origin of replication (ori). The precise RepB binding sites were defined by DNase I footprinting experiments. RepB binds to two motifs in the ori region: a high-affinity site within its own promoter region, implying autoregulation of its expression, and a low-affinity site further upstream, presumably the origin of replication itself. The binding to the latter motif seems to occur on one DNA strand only. The high-affinity binding site contains several palindromic sequences. Gel retardation assays were performed with the different binding sites as templates, and the binding constant to each site was estimated from protein titrations. This is the first molecular dissection of mycobacterial DNA-binding proteins and their interactions with their targets.The mechanisms governing plasmid replication and maintenance in mycobacteria are little understood. Plasmids have been described in several species, e.g., Mycobacterium fortuitum, Mycobacterium scrofulaceum, Mycobacterium chelonae, and Mycobacterium avium (4,8,17,18,23,29). No plasmids have been found in the pathogen Mycobacterium tuberculosis, but it is possible to transform M. tuberculosis with plasmids from related mycobacteria, showing that the lack of plasmids is not due to an intrinsic inability to support their replication.The replicon most used in genetic manipulation of mycobacteria is the 4,837-bp M. fortuitum plasmid pAL5000 (19) (see Fig. 1) (GenBank accession number M23557). This replicon is the basis of several shuttle plasmids and cosmids used to manipulate DNA in a wide variety of mycobacteria, including the fast-growing Mycobacterium smegmatis and the slow growers Mycobacterium bovis BCG and M. tuberculosis (9,31,36,41). We have previously shown that the origin of replication (ori) of this plasmid lies within a 435-bp segment and comprises a region with a high incidence of repeated sequences (39). In addition, it has been shown that two open reading frames (ORFs) are necessary for a functional replicon (22,39). The products of these ORFs, RepA and RepB, will act in trans to activate the ori if this region is present on another plasmid (39).The ori region is also able to confer incompatibility to otherwise unrelated replicons. Whether the same sequence motifs within the region are responsible for the inc and ori functions or whether inc is physically separated from ori as in plasmid P1 (1, 5, 7) is at present unclear.The RepA protein (307 amino acid [aa]) has a high similarity to replication proteins from ColE2-related plasmids (12, 39). RepB is a small protein (119 aa) which has recently been shown to have similar...

Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

“…The RepA and RepB proteins support plasmid replication in trans. The 435bp cis element contains low-(L) and high-(H) affinity binding sites for RepB, and harbors several palindromic sequences (48,49). RepB binding to these sites causes local bending as well as a long-range polar curvature.…”

Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors

“…5b). Remarkably, in pALS000 this 15 bp sequence is positioned right between the RepB binding sites that were identified by Stolt & Stoker (1996b). We noticed that this sequence resembles the ori region of ColE2 plasmids (Hiraga et al, 1994).…”

“…The reduced plasmid stability observed in two other species probably reflects in part the involvement of host-encoded proteins in plasmid replication (initiation, termination) and partitioning. For instance, for the pAL.5000-based replicons there are indications of a requirement for mycobacterial DNA polymerase I to initiate DNA synthesis (Stolt & Stoker, 1996b).…”

A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon The EMBL accession number for the sequence determined in this work is AJ238973.

“…The N-terminal region of the pLR7 Rep is reported to contain helix-turn-helix motifs, which are characteristic features of DNA-binding regulatory proteins (Beggs et al, 1995). Among the Rep proteins of mycobacterial plasmids, only the pAL5000 RepB protein (119 residues) has been purified and its role in plasmid replication characterized (Stolt & Stoker, 1996b). This protein binds to a high-affinity site upstream of the repAB genes to regulate its own expression, and to a low-affinity site further upstream which is suggested to contain its ori site.…”