Characterization of the functional replication origin of Mycobacterium tuberculosis

Characterization of the functional replication origin of Mycobacterium tuberculosis

Gene 233 (1999) 121–130 www.elsevier.com/locate/gene Characterization of the functional replication origin of Mycobacterium tuberculosis Ming-Hui Qi...

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Gene 233 (1999) 121–130

www.elsevier.com/locate/gene

Characterization of the functional replication origin of Mycobacterium tuberculosis Ming-Hui Qin, Murty V.V.S. Madiraju, Malini Rajagopalan * Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, TX 75708-3154, USA Received 18 December 1998; received in revised form 8 March 1999; accepted 8 April 1999; Received by J. Wild

Abstract The gene order in the 5 kb Mycobacterium tuberculosis dnaA region is rnpA, rpmH, dnaA, dnaN and recF. We show that M. tuberculosis DNA fragment containing the dnaA–dnaN intergenic region functioned as oriC, i.e., allowed autonomous replication to otherwise nonreplicative plasmids, in M. tuberculosis H37Ra (H37Ra), avirulent strain of M. tuberculosis, and in Mycobacterium bovis BCG (BCG), a closely related, slowly growing mycobacterial strain. Removal of Escherichia coli plasmid replication origin (ColE1) from the M. tuberculosis oriC plasmids did not abolish their ability to function as oriC, confirming that the autonomous replication activity of these plasmids is due to the presence of the DNA fragment containing the dnaA–dnaN intergenic region. Deletion analyses revealed that the minimal oriC DNA fragment is 814 bp. The copy number of M. tuberculosis oriC plasmids containing ColE1 ori relative to chromosomal oriC is one and the 5∞ flanking region of minimal oriC contains features that support stable autonomous replication. The M. tuberculosis oriC did not function in rapidly growing mycobacterial species such as M. smegmatis. M. smegmatis oriC functioned only in M. fortuitum, but not in any of the slowly growing mycobacterial species such as M. tuberculosis and BCG. Together these data suggest that the replication initiation mechanisms in the slowly growing Mycobacteria are similar and probably different from those in the rapidly growing Mycobacteria and vice versa. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Autonomous replication; BCG; Mycobacteria; Mycobacterium bovis; oriC

1. Introduction Mycobacterium tuberculosis, the causative agent for tuberculosis, is a slow grower with a doubling time of 22–24 h. The genus Mycobacterium also includes several other slowly growing pathogenic species, such as M. bovis with a doubling time of 22–24 h and M. avium with a doubling time of 10–16 h and rapidly growing nonpathogenic members such as M. smegmatis and M. fortuitum with a doubling time of 2–3 h ( Wheeler and Ratledge, 1994). Replication of the bacterial chromosome leading to subsequent multiplication of the pathogen inside the

Abbreviations: Ap, ampicillin; BCG, Mycobacterium bovis BCG; Enase, restriction endonuclease; H37Ra, Mycobacterium tuberculosis H37Ra; Km, kanamycin; LB, Luria–Bertani (medium); MCS, multiple cloning site(s); nt, nucleotide(s); oligo, oligonucleotide; ori, origin(s) of DNA replication; oriC, chromosomal origin of DNA replication; PCR, polymerase chain reaction; R, resistant/resistance. * Corresponding author. Tel.: 903-877-7731; fax: 903-877-5969. E-mail address: [email protected] (M. Rajagopalan)

host is the first step in the onset of infection. Replication is initiated when DnaA, the initiator protein binds at a unique site on the chromosome called oriC which triggers a cascade of downstream events (Bramhill and Kornberg, 1988a, 1988b; Kornberg and Baker, 1991). These include opening of the double-stranded oriC region to allow entry of DnaB protein followed by unwinding of the double-stranded DNA, priming of DNA synthesis and the formation of replication forks. The structural organization and the nt sequence features of oriC appear to be conserved in eubacteria ( Kornberg and Baker, 1991). The oriC from one bacterial species has been shown to be functional, i.e., support autonomous replication in other members of the same genus ( Yee and Smith, 1990; Kornberg and Baker, 1991). We and others previously reported that either the 3.5 kb dnaA region containing the rpmH, dnaA and dnaN genes and their intergenic regions (Rajagopalan et al., 1995b) or just the dnaA–dnaN intergenic region alone (Salazar et al., 1996; Qin et al., 1997) of M. smegmatis functioned as oriC in M. smegmatis, i.e.

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rendered nonreplicative plasmids capable of autonomous replication in M. smegmatis. Further, the 5∞ flanking region of the dnaA–dnaN intergenic region of M. smegmatis was shown to promote its oriC activity (Qin et al., 1997). The dnaA gene region of M. tuberculosis and M. leprae have been cloned and sequenced (Rajagopalan et al., 1995a; Salazar et al., 1996). The dnaA region of M. tuberculosis, however, did not show autonomous replication activity in the two mycobacterial species tested, namely, M. smegmatis and BCG (Salazar et al., 1996). Availability of functional ori of M. tuberculosis is essential to evaluate how its replication initiation is regulated. More importantly, it would allow us to address whether or not M. tuberculosis oriC functions in M. smegmatis or vice versa, and whether there are any differences in the oriC activities of the two organisms. In this study, we first set out to clone the oriC of M. tuberculosis, characterize its autonomous replication activity in both the presence and absence of E. coli plasmid ori and, finally, evaluate whether M. tuberculosis oriC functions in other mycobacterial species. We show that a 814 bp DNA fragment containing the dnaA–dnaN intergenic region of M. tuberculosis is the minimal oriC and does not function in any of the rapidly growing mycobacteria.

2. Materials and methods 2.1. Bacterial strains, plasmids, media and antibiotics The bacterial strains and plasmids used are listed in Table 1. E. coli strains were grown in LB broth, whereas mycobacterial strains were grown in 7H9 broth supplemented with OADC (Rajagopalan et al., 1995b). For some experiments BCG and H37Ra were grown on 1% Ogawa egg slants (Goto et al., 1991). E. coli transformants were selected on LB agar plates containing Ap, Km or both and mycobacterial transformants were selected on 7H10 agar plates containing Km (Sambrook et al., 1989; Rajagopalan et al., 1995b). Km and Ap were used at a final concentration of 50 mg/ml. 2.2. Recombinant DNA techniques Chromosomal DNA from BCG was prepared as described by Pelicic et al. (1997). DNA amplification, labeling of DNA with radioactive nucleotides, transfer of DNA to nitrocellulose membranes, Southern hybridization and DNA sequencing were carried out as described (Sambrook et al., 1989; Qin et al., 1997). DNA amplification products were either digested with appropriate restriction enzymes or kinased, and then gel purified and cloned into various plasmid vectors (Sambrook et al., 1989). The transformation conditions for M. smegmatis, M. fortuitum, H37Ra and BCG were

as described (Goto et al., 1991; Qin et al., 1997). Approximately 0.5–1.0 mg of DNA was used for each transformation. Appropriate dilutions of the transformation mix were spread on antibiotic plates, incubated at 37°C for up to 1 week for M. smegmatis and M. fortuitum and for 3–4 weeks for BCG and H37Ra. Transformation of BCG and H37Ra with pMV206, E. coli–Mycobacterium shuttle plasmid produced approx. 1×104 transformants per mg DNA. Isolation and analyses of plasmid DNA from E. coli and mycobacterial transformants were as described (Qin et al., 1997). The oriC activity was defined as the ability of dnaA region fragments from M. tuberculosis H37Rv, BCG or M. smegmatis mc2155 to render E. coli plasmids capable of autonomous replication in the respective mycobacterial hosts and is expressed as the total number of KmR transformants obtained per mg of input DNA ( Rajagopalan et al., 1995b; Qin et al., 1997). 2.2.1. GenBank accession numbers The nt sequence of the 5 kb dnaA region of M. tuberculosis and the 897 bp fragment containing the dnaA–dnaN intergenic region of BCG were deposited in GenBank under the accession numbers U38891 and U75298, respectively. 2.3. Construction of M. tuberculosis and BCG oriC plasmids Cloning of the 5 kb dnaA region of M. tuberculosis and the 3.5 kb dnaA region of M. smegmatis was as described (Rajagopalan et al., 1995a,b). Various fragments from the dnaA region of M. tuberculosis, BCG and M. smegmatis were generated either by PCR or by restriction digestion followed by gel purification. Purified DNA fragments were cloned into pZErO-2.1 plasmids (see Table 1) and subsequently tested for oriC activity. The following combination of oligos was used to derive the DNA fragments cloned in respective oriC plasmids: Q49 (5∞-TGTGCGTGAGCTCACCGA) and Q61 (5∞-GCCGAGACCTCGTAGTCGAA) for pMQ56 and pMQ57; Mty51 (5∞-GGGGAATTCTTAAAAAAACTTCTC ) and Mty53 (5∞-GGGCCCGGGCCTGGCTGG CAGATT ) for pMQ361; Mty52 (5∞-GGGAATATTGGCTGTGAGTGTCGC ) and Mty53 for pMQ363; Mty52 and Q63 (5∞-CGGAATTCTGCGCCCTTTCAC ) for pMQ375; Mty52 and Q64 (5∞ -CGGAATTCGTCAAGTCGG) for pMQ377; Q37 (5∞-GGAATTCTGCAGGGCCTGGCTGGCA GATT ) and Q65 (5∞-CGGAATTCCCGGCGGTTCCG) for pMQ379; Q37 and Q66 (5∞-CGGAATTCCACGCCTCATCCCC ) for pMQ381 and MVM 78 (5∞-CGATCGGATGGTCCGCACT ) and MVM80 (5∞- AGCTCGAAGCACTCCGACCACATCACG) for pMR50. The boldface letters indicate the EcoRI, SmaI, SspI and PstI recognition sites.

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M.-H. Qin et al. / Gene 233 (1999) 121–130 Table 1 Strains and plasmids used in this study Strain or plasmid Strains Top 10F ∞ M. bovis BCG S10 M. fortuitum ATCC 6841 M. smegmatis mc2 155 M. tuberculosis H37Ra Palsmids pZErOTM-2.1 pUC18 pUC18 derived pMR42 pRZ30 pMQ21 pRZ35 pMQ391 pMQ392 pRZErO -2.1 derived pMQ212 pMQ215 pMQ224 pMQ56 pMQ219 pMQ218 pMQ361 pMQ363 pMQ375 pMQ377 pMQ379 pMQ381 pMQ57 pMQ50

Genotype of description

Source or reference

F ∞{laclqtetR} mcrA D(mrr-hsdRMS-mrcBC )ø80lacZ DM15 DlacX74 deoR rec A1 araD139 D(ara-leu)7697 galU galK rpsL endA1 nupG Transformation proficient strain, kanamycin sensitive Transformation proficient strain, kanamycin sensitive Transformation proficient strain, kanamycin sensitive Transformation proficient strain, kanamycin sensitive

Invitrogen

E. coli plasmid vector, KmR E. coli plasmid vector, ApR

Invitrogen Laboratory stock

pUC18 plasmid containing aph gene, KmR, ApR

Rajagopalan et al. (1995b) This work

A 953 bp PCR fragment (nt 2719–3412a) from M. smegmatis genomic DNA cloned in SmaI site of pUC18; 1.3 kb aph fragment was inserted at EcoRI site, KmR, ApR 5 kb BamHI fragment containing the dnaA region of M. tuberculosis in BamHI site of pUC18, ApR A 897 bp PCR fragment (nt 2719–3616b) from M. tuberculosis H37Rv genomic DNA cloned in SmaI site of pUC18; 1.3 kb aph fragment cloned at EcoRI site, KmR ApR 2.4 kb DNA fragment amplified from pRZ35 using primers Q61A and Q62, digested with BglII and ligated, KmR 2.4 kb PCR amplified product from pRZ35 was digested with PvuII and ligated, KmR 5 kb BamHI fragment from the pMQ21 cloned in same sites of pZErO-2.1, KmR A 3 kb BamHI–EcoRI fragment of pMQ21 cloned in same sites of pZErO-2.1, KmR 1 kb ApaI deletion of pMQ212 followed by self-ligation, KmR A 897 bp PCR fragment (nt 2719–3616b) from M. tuberculosis genomic DNA cloned in pZErO-2.1, KmR SacII–BamHI deletion of pMQ56 followed by self-ligation, KmR A 1.4 kb HincII fragment of pMQ21 cloned in pZErO-2.1, KmR EcoRI+SmaI digested PCR fragment (615 bp, nt 2918–3532b) from pMQ21 cloned in pZErO-2.1, KmR SspI+SmaI digested PCR fragment (571 bp, nt 2962–3532b) from pMQ21 cloned in pZErO-2.1, KmR) SspI+EcoRI digested fragment (459 bp nt 2963–3421b) from pMQ21 cloned in EcoRV–EcoRI sites of pZErO-2.1, KmR SspI+EcoRI digested fragment (501 bp nt 2963–3463b) from pMQ21 cloned in EcoRV–EcoRI sites of pZErO-2.1, KmR PstI+EcoRI digested PCR fragment (523 bp, nt 3010–3532b) from pMQ21 cloned in same sites of pZErO-2.1, KmR PstI+EcoRI digested PCR fragment (523 bp nt 3031–3532b) from pMQ21 cloned in same sites of pZErO-2.1, KmR A 897 bp PCR fragment (nt 1–897c) from BCG genomic DNA cloned in pZErO-2.1, KmR A 5 kb PCR fragment (corresponding to the dnaA region on pMQ212) from BCG genomic DNA cloned in pZErO-2.1, KmR

Goto et al. (1991) Laboratory collection W.R. Jacobs, Jr. ATCC

Rajagopalan et al. (1995a) This work This work This work This This This This

work work work work

This work This work This work This work This work This work This work This work This work This work

a Nucleotide numbering was based on dnaA region sequence of M. smegmatis (Rajagopalan et al., 1995a). b Nucleotide numbering was based on dnaA region sequence of M. tuberculosis (GenBank accession No. U38891). c Nucleotide numbering was based on dnaA region sequence of M. bovis ( U75298).

2.4. Construction of M. tuberculosis oriC plasmids lacking E. coli plasmid ori To construct M. tuberculosis plasmids lacking ColE1 ori, we chose pRZ35, a pUC18-derived plasmid containing the 897 bp oriC region and the 1.3 kb aph gene (for KmR) located in MCS ( Table 1). From this, a 3 kb fragment containing the oriC of M. tuberculosis and the aph gene was amplified using oligos Q61A (5∞GAAGATCTCCCCGCGCGTTGG) and Q62 (5∞-G-

AAGATCTCATGA GCGGATAC ), digested with BglII, and self-ligated. The resulting plasmid, pMQ391 was directly used to transform BCG. The 3.0 kb PCR product was also digested with PvuII enzyme. This released a 468 bp fragment downstream and 21 bp sequence upstream of the plasmid lacZ gene. The resulting 2.5 kb fragment was purified, self-ligated and used to transform BCG. The plasmid was designated as pMQ392. Since pMQ391 and pMQ392 plasmids lack the E. coli plasmid ori, they could not be amplified and

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propagated in E. coli. Further, as mycobacterial cells are not susceptible to alkali-SDS lysis, for analysis of pMQ391 and pMQ392 plasmids from BCG cells, total DNA was isolated from the propagated cultures of BCG containing these oriC plasmids and examined by Southern hybridization. 2.5. Stability and copy number of oriC plasmids 2.5.1. Stability experiments BCG containing representative oriC plasmids, pMQ56, pMQ212, pMQ215 and pMQ392, were inoculated into 7H9 broth lacking Km. An aliquot of the culture after appropriate dilution was plated on 7H10 agar plates with and without Km, and the plates were incubated at 37°C. The remaining broth was incubated standing at 37°C in tissue culture flasks. Aliquots of cultures were removed after 3, 6, 10 and 15 days of incubation, diluted with 7H9 broth and plated on 7H10 agar plates with and without Km. Colonies from both series of plates were counted and the ratio of colonies obtained in the presence and absence of Km was determined for each set. Plasmid DNA was recovered from colonies grown on both series of plates for subsequent analyses. 2.5.2. Copy number determination Total DNA from BCG containing different oriC plasmids was prepared following the protocol of Pelicic et al. (1997). Unless otherwise specified, BCG cells containing all oriC plasmids were colony purified and propagated prior to total DNA isolation. Appropriate amounts of DNA were digested with restriction enzymes, gel electrophoresed, transferred to nitrocellulose membranes and probed with oriC-specific DNA fragment in a Southern hybridization experiment and visualized by autoradiography. The copy number of oriC plasmids relative to chromosomal oriC was determined by comparing the relative intensities of the chromosomal oriC bands with that of the plasmid oriC by densitometric scans of autoradiographs in a Bio-Image Densitometer (Millipore Corporation) and Phosphor Imager.

3. Results and discussion

of the dnaA region). These data are consistent with the other published reports (Salazar et al., 1996; Cole et al., 1998). To test whether the plasmids containing the M. tuberculosis dnaA–dnaN intergenic region replicate autonomously, H37Ra and BCG cells were transformed with pMQ219 (see Table 1, Fig. 1). H37Ra is an avirulent derivative of M. tuberculosis H37Rv, whereas BCG is an attenuated vaccine strain of M. bovis. KmR colonies of H37Ra and BCG were obtained with pMQ219 similar to those obtained with pMV206, an E. coli– Mycobacterium shuttle vector ( Fig. 1). Neither H37Ra and BCG strains alone nor these strains transformed with the control plasmid, pZErO-2.1, grew on 7H10 Km plates. Analyses of the plasmid DNA recovered from the KmR transformants of BCG and H37Ra by restriction digestion ( Fig. 2) and Southern hybridization (data not shown) indicated that sequence changes, if any, in pMQ219 plasmids following electroporation in the two mycobacterial strains were minimal or none ( Fig. 2; compare lanes 1 and 5 with lanes 2–4 and 6–8, respectively). These data further indicated that pMQ219 plasmids maintained as extrachromosomal elements in H37Ra, and in BCG, a closely related mycobacterial species. The corresponding DNA fragment containing the dnaA–dnaN intergenic region from BCG when cloned in pZErO-2.1 vectors also exhibited autonomous replication activity in BCG and H37Ra and maintained as stable extrachromosomal elements (data not shown, see pMQ57 in Table 1). Both M. tuberculosis and BCG DNA fragments containing the dnaA–dnaN intergenic region when cloned in modified pUC18 plasmids containing gene for KmR also exhibited autonomous replication activity in respective hosts (data not shown). Since these DNA fragments, irrespective of the cloning vectors used, exhibited autonomous replication activity, we will henceforth refer to this region as the oriC of M. tuberculosis. The nt sequence of the DNA fragment containing the dnaA–dnaN intergenic region of BCG was found to be essentially identical to that of M. tuberculosis counterpart, with only a single base difference (see Fig. 3A, G instead of A at nt position 629, GenBank accession No. U75298). Since oriC activity and the nt sequence of the oriC region from M. tuberculosis and BCG were similar, all subsequent transformation experiments were carried out in BCG.

3.1. The oriC of M. tuberculosis The nt sequence analyses of the 5 kb dnaA region of M. tuberculosis ( Rajagopalan et al., 1995a) revealed that it contained the complete coding regions for the rnpA, rpmH, dnaA and dnaN genes and the partial coding region for the recF gene ( Fig. 1, GenBank number U38891). The intergenic regions of the rpmH– dnaA and dnaA–dnaN genes were found to be 603 bp and 527 bp, respectively (see Fig. 1 for the physical map

3.2. Autonomous replication activity of the dnaA region of M. tuberculosis To evaluate the effects of sequences located upstream of M. tuberculosis oriC on its autonomous replication activity, plasmids containing different lengths of dnaA region were constructed and their abilities to exhibit oriC activity was determined. These plasmids contained either the 5 kb dnaA region (pMQ212, Fig. 1), deriva-

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Fig. 1. Localization of the oriC of M. tuberculosis H37Rv in the 5.0 kb dnaA region. Physical map of the 5.0 kb dnaA region is shown. Shaded boxes indicate the ORF of the indicated gene. Abbreviations for Enase sites: Ac, AccI; A, ApaI; B, BamHI; E, EcoRI; H, HincII; and S, SacII. The direction of transcription of each gene is indicated by an arrowhead. The names of oriC plasmids and the corresponding oriC fragments are shown on the left side. TF — the transformation efficiencies or the total number of BCG transformants obtained per mg of input oriC plasmid DNA are shown on right. Single-letter restriction enzyme codes for generating the oriC fragments are shown on either side of the DNA fragment. Q49 and Q61 are the oligonucloetide primers used to generate the cloned DNA fragment in pMQ56. A1 indicates location of 1 kb ApaI deletion in the oriC region of pMQ224 ( Table 1). Although not shown, the transformation efficiency of pMQ219 plasmid in M. tuberculosis H37Ra is comparable to that in BCG and is in the range of 1.0×104 transformants per mg in put DNA.

tives of it with a 1 kb internal deletion in the dnaA region (pMQ224, Fig. 1, see Table 1), those lacking a part of the dnaA coding region as well as its immediate 5∞ flanking regions including the rpmH–dnaA intergenic region (pMQ215, see Fig. 1) or just the 5∞ flanking region of the dnaA gene (pMQ218, see Fig. 1). All of the recombinant plasmids except pMQ218 exhibited oriC activity in BCG and the transformation efficiencies of these plasmids were found to be comparable to those obtained with pMQ219 and pMQ56 plasmids (Fig. 1). Although the 5∞- flanking region of the dnaA gene, i.e.,

the rpmH–dnaA intergenic region, like the 3∞ flanking region, is A+T rich and contains several putative DnaAboxes (GenBank entry No. U38891; see also Salazar et al., 1996), it failed to exhibit oriC activity in the absence of the 3∞ flanking region (Fig. 1, see pMQ218). Together, the above data indicated that the oriC of M. tuberculosis is located in the 3∞ flanking region of the dnaA gene and, unlike the situation with M. smegmatis (Qin et al., 1997), the 5∞ flanking region of M. tuberculosis oriC does not affect its oriC activity. Restriction digestion and Southern hybridization analyses of plasmid DNA recovered from the KmR transformants of the above oriC plasmids confirmed that the oriC plasmids were maintained as extrachromosomal elements in BCG (data not shown). A 5 kb DNA fragment from BCG corresponding to the dnaA region of M. tuberculosis (see pMQ212 and pMR50 in Table 1) also exhibited autonomous replication activity in BCG similar to that of pMQ212 (data not shown). 3.3. Deletion analyses of the M. tuberculosis oriC region

Fig. 2. Analysis of pMQ219 plasmid recovered from KmR transformants of BCG and H37Ra. Plasmid DNA was recovered from KmR BCG and H37Ra transformants into E. coli (see Section 2.2). Plasmid DNA from several independent E. coli transformants was isolated, digested with EcoRI+HindIII enzymes, gel electrophoresed and visualized by ethidium bromide staining. Lanes: M, 1.0 kb ladder; 1 and 5, input plasmid DNA of pMQ219; 2, 3 and 4, plasmid DNA recovered from BCG transformants; and 6, 7 and 8, plasmid DNA recovered from H37Ra transformants.

The DNA fragment containing the dnaA–dnaN intergenic region in pMQ219 is 814 bp (Fig. 3A). This region includes the 3∞ and 5∞ ends of the dnaA and dnaN genes and their intergenic region. It contains one putative A+T rich cluster and several putative DnaA boxes with one to three mismatches to the consensus sequence TTG/CTCCACA defined for the DnaA boxes of M. smegmatis (Rajagopalan et al., 1995b). To identify the minimal DNA fragment that exhibits oriC activity and allows stable extra-chromosomal replication, plasmids containing different lengths of oriC were constructed (see Fig. 3A, B and Table 1) and their abilities to exhibit

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Fig. 3. The oriC of M. tuberculosis. (A) The nt sequence of the 814 bp oriC region of M. tuberculosis in pMQ219 plasmid is shown. Sequence in the shaded box indicates the putative A+T rich cluster. Arrows identify the position and orientation of the presumptive DnaA-boxes with one to three mismatches to the DnaA-box consensus sequence TTG/CTCCACA (Rajagopalan et al., 1995b). Asterisk in the sequence denotes the nt base (number 629) that is different in BCG oriC sequence (G instead of A). Ends of various oriC DNA fragments generated by PCR are indicated by brackets and are marked by single letter codes. Thus pMQ361 plasmid contains the DNA fragment from a to c; pMQ363 from b to c; pMQ379 from f to c; pMQ381 from g to c; pMQ377 from b to d; pMQ375 from b to e. (B) The minimal oriC of M. tuberculosis. Shaded boxes indicate the position of the presumptive DnaA-boxes. The position of the A+T rich cluster is marked. Arrowheads on top of the shaded boxes indicate the orientation of the DnaA-boxes. Open arrowheads indicate the DnaA-boxes with three mismatches to the consensus sequence ( TTG/CTCCACA), whereas closed arrowheads indicate the DnaA-boxes with one to two mismatches. The names of oriC plasmids and the corresponding oriC fragments are shown to the left and transformation efficiency of the corresponding plasmids are shown to the right. Asterisk next to the plasmid name indicates that plasmid DNA could only be recovered from the pooled primary transformants (see Section 3.5).

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oriC activity were determined. Transformation of BCG with plasmids pMQ361, pMQ363 and pMQ377, but not pMQ375, pMQ379 and pMQ381, produced several KmR colonies which were smaller than those obtained with pMQ56 and pMQ219 and appeared weeks later (Fig. 3B). Recovery of plasmid DNA was successful from the primary transformants obtained with pMQ361, pMQ363 and pMQ377, but not from their respective propagated cultures (data not shown). Southern hybridization using oriC-specific probe and PCR using plasmid-specific primers failed to indicate the presence of plasmid DNA in the total DNA preparations made from the propagated cultures of pMQ361, pMQ363 and pMQ377 (data not shown; see also Section 3.5), indicating that the above plasmids were lost during propagation of primary transformants. Some of the oriC plasmid constructs of Bacillus subtilis were also reported to be unstable (Moriya et al., 1992). Since pMQ219, unlike the pMQ361, pMQ363 and pMQ377 plasmids replicated stably, the extra 199 bp present at its 5∞ end could contain sequences that support stable autonomous replication. This region contains six putative DnaA-boxes (compare pMQ219 with pMQ361 in Fig. 3A). It is not evident whether the putative DnaAboxes or the region per se is required for stable autonomous replication of oriC plasmids. The oriC DNA fragment in pMQ363 is also missing the A+T rich cluster (compare pMQ363 with pMQ361 in Fig. 3A). While the role of A+T rich cluster in replication initiation in M. tuberculosis is not clear (compare pMQ363 with pMQ361 in Fig. 3B), deletion of A+T rich cluster from M. smegmatis oriC plasmids abolished their autonomous replication activity (Qin et al., 1997).

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somal (5 kb, see Fig. 4) and plasmid oriC (0.9 kb, Fig. 4) were noted indicating that plasmids pMQ391 and pMQ392 replicated extrachromosomally. These data also suggested that the observed autonomous replication activity of plasmids pMQ212, pMQ215, pMQ219 and pMQ56 is due to the presence of DNA fragment containing the dnaA–dnaN intergenic region and is not due to activation of any quiescent sequences in the parent E. coli plasmid, pZErO-2.1. Quantitation of the intensities of the chromosomal and plasmid oriC band by Phospor Imager indicated that the copy number of the pMQ391 and pMQ392 plasmids relative to chromosomal oriC is approx. 17. The high copy number was not due to the selective recovery of the plasmid DNA compared to the genomic DNA as experiments on copy number determination of other oriC plasmids did not show such bias (see Section 3.5). 3.5. Copy number and stability of M. tuberculosis oriC plasmids containing ColE1 ori To test whether M. tuberculosis oriC plasmids containing the ColE1 plasmid ori also maintain at high

3.4. M. tuberculosis oriC plasmids lacking the E. coli plasmid ori In the above experiments, oriC activity was determined by cloning the DNA fragments in plasmids that specifically replicate in E. coli but not in Mycobacteria and examining the ability of the recombinant plasmids to replicate in M. tuberculosis. This assay does not exclude the possibility that the cloned DNA fragments of M. tuberculosis somehow promote autonomous replication ability of the E. coli plasmids in H37Ra and BCG. To rule out this possibility, M. tuberculosis oriC plasmids lacking E. coli plasmid ori, ColE1, were constructed and the ability of the recombinant plasmids (see pMQ391 and pMQ392 in Table 1 and Section 2.4 for experimental details) to exhibit oriC activity in BCG was tested. Transformation of BCG with either of these plasmids produced several KmR colonies. To determine whether the M. tuberculosis oriC plasmids lacking ColE1 ori were replicating autonomously, total DNA prepared from the propagated cultures of three independent transformants was analyzed by Southern hybridization. Hybridization signals corresponding to both chromo-

Fig. 4. Copy number of M. tuberculosis oriC plasmids. Copy number of oriC plasmids was determined as described in Section 2.5. The following restriction enzymes were used to digest DNA preparations: BCG control and BCG containing the pMQ56, pMQ215, pMQ377, pMQ391 and pMQ392 with BamHI; BCG containing the pMQ212 plasmid with HindIII+EcoRI enzymes. The expected sizes of chromosomal and plasmid oriC bands are: control, 5 kb for the chromosomal oriC; pMQ212, 3 and 6 kb for chromosomal and plasmid oriC; pMQ215, 5 and 6 kb for chromosomal and plasmid oriC; pMQ56, 5 and 3.9 kb for chromosomal and plasmid oriC; pMQ377, 5 and 3.7 kb for chromosomal and plasmid oriC; pMQ391 and pMQ392, 5 and 0.9 kb for chromosomal and plasmid oriC, respectively. The primary transformants of BCG obtained with all plasmids except pMQ377 were colony purified, propagated and used to prepare total DNA. The primary transformants of BCG containing the pMQ377 plasmids were pooled and used to prepare total DNA. Separately, three independent BCG transformants containing the pMQ377 were propagated, their total DNA was isolated, digested with BamHI and processed as others ( labeled as pMQ3771).

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copy number, total DNA from propagated cultures of BCG containing the representative oriC plasmids pMQ212, pMQ215, pMQ56 and pMQ377, was analyzed by Southern hybridization (Fig. 4). Comparison of the intensities of bands corresponding to the chromosomal oriC with that of the cloned M. tuberculosis oriC by Phosphor Imager revealed that the copy number of the pMQ212, pMQ215 and pMQ56 oriC plasmids relative to chromosomal oriC is approximately one (Fig. 4). In the case of pMQ377, plasmidspecific band was detected only in the total DNA isolated from the pooled transformants (Fig. 4, lanes marked pMQ377) but not from the propagated cultures ( lane marked as pMQ3771). The copy number of pMQ377 relative to chromosomal oriC is 0.6. Plasmid copy number less than one likely indicates the loss of plasmid from some cells. Together these results indicate that the pMQ377 plasmids are not stably maintained. Similar results were also noted with the pMQ363 transformants (also see Section 3.3). Stable extra-chromosomal replication of pMQ56, pMQ212 and pMQ215 indicate that recombination events, if any, between plasmid and chromosomal oriC region with these plasmids are unlikely. The differences in the copy numbers of M. tuberculosis plasmids with and without the ColE1 ori tend to suggest that the E. coli plasmid sequences exert some interference with M. tuberculosis oriC replication (Prichard and Gover, 1981). Stability experiments indicated that in the absence of selection, at the outset up to 90% of the colony forming cells from cultures containing pMQ56 plasmids lost their plasmids whereas those of the pMQ212 and pMQ215 did not and were relatively stable (Fig. 5). The pMQ56 plasmid showed further instability and approx. 2% of the cells remained KmR after 6 days of growth in the absence of antibiotic ( Fig. 5). The number of colonies on agar plates with and without Km for plas-

Fig. 5. Stability of M. tuberculosis oriC plasmids. Stability of BCG cells containing pMQ212, pMQ215, pMQ56 or pMQ392 were determined (see Section 2.5). The percentage of KmR colonies was determined and is shown on the y-axis and number of days of culture is shown on the x-axis.

mids pMQ212 and pMQ215 at all growth periods tested were found to be similar ( Fig. 5), indicating that these plasmids were stable. Plasmid DNA could be recovered at all growth periods tested, confirming that KmR of these cultures was due to the stable extra-chromosomal replication of the oriC plasmids (data not shown). Since pMQ56 plasmids are not stable in the absence of selection, the above results suggest that the 5∞ flanking region of M. tuberculosis oriC contains appropriate partitioning signals that enable the stable maintenance of oriC plasmids. While the stability experiments also revealed that the pMQ392 plasmids are stable (Fig. 5), Southern hybridization analyses of total DNA indicated integration of the plasmid into the chromosome (data not shown). 3.6. M. tuberculosis oriC does not function in M. smegmatis and vice versa The replication initiation mechanisms in bacteria belonging to the same family or genus are thought to be similar. This inference is based in part on the finding that within the same genus (or in some cases the same family), the oriC from one species functions in another species ( Zyskind et al., 1983; Skovgaard and Hansen, 1987; Yee and Smith, 1990; Kornberg and Baker, 1991; Skarstad and Boye, 1994). Our data on demonstration of M. tuberculosis oriC activity in BCG indicate that such generalization is applicable to certain members of mycobacteria as well. However, transformation of M. smegmatis with M. tuberculosis oriC plasmids, pMQ56 and pMQ212, did not result in any KmR colonies, indicating that the M. tuberculosis oriC does not function in M. smegmatis ( Fig. 6A, B). Reciprocal experiments with M. smegmatis oriC plasmid (pRZ30) in BCG confirmed the above results (data not shown). To test whether M. smegmatis oriC functions in other rapidly growing members, e.g., M. fortuitum, we examined the ability of M. smegmatis oriC plasmid pRZ30, a pUC18-derived plasmid, to function in M. fortuitum ( Table 1). The pZErO -2.1 derived oriC plasmids could not be used in this strain, as the vector alone gave high background (data not shown). Transformation of M. fortuitum with pRZ30 (Fig. 6C ) resulted in KmR colonies with transformation efficiencies comparable to those obtained in M. smegmatis (Fig. 6D). M. smegmatis cells transformed with control plasmids did not grow on Km containing plates ( Fig. 6F, G). Plasmid DNA analyses indicated that pRZ30 maintained as extra-chromosomal elements in M. fortuitum (data not shown). These data indicate that M. smegmatis oriC functions in M. fortuitum. The genetic basis for the species specificity, i.e., lack of interspecies replication, of mycobacterial oris are not known at present. A comparative analyses of the minimal oriC of M. smegmatis (550 bp) (Qin et al., 1997)

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Fig. 6. Interspecies replication activities of M. smegmatis and M. tuberculosis oriC plasmids. M. smegmatis mc2155 and M. fortuitum ATCC6841 competent cells were transformed with the various oriC plasmids indicated below, plated on 7H10 plates containing Km, incubated at 37°C for 5– 7 days and photographed. (A) M. smegmatis transformed with pMQ56. (B) M. smegmatis transformed with pMQ212. (C ) M. fortuitum transformed with pRZ30. (D) M. smegmatis transformed with pRZ30. (E ) M. fortuitum transformed with Mycobacterium–E. coli shuttle vector pMV206. (F ) M. smegmatis transformed with pZErO -2.1. (G) M. smegmatis transformed with pMR42.

with that of the M. tuberculosis (814 bp, this study), however, identified both similarities and differences in the organization of oriC. For example, some of the DnaA-boxes are positionally conserved ( Fig. 7), while their sequences per se are not exactly the same (see

Fig. 3A; Rajagopalan et al., 1995b). The DnaA-boxes of M. tuberculosis were defined based on the consensus sequence for M. smegmatis ( TTG/CTCCACA) ( Rajagopalan et al., 1995b) and other Gram+ bacteria ( TTGTCCACA) (Moriya et al, 1985; Fujita et al., 1990;

Fig. 7. Arrangement and orientation of DnaA-boxes (e) and A+T rich cluster (L) in the oriC region of M. tuberculosis (814 bp) (this study) and M. smegmatis (531 bp) (Qin et al., 1997). The minimal oriC of M. smegmatis and M. tuberculosis were aligned using Gene Works 2.5 program. The positions of the DnaA-boxes with one to three mismatches to the consensus sequence TTG/CTCCACA and the A+T rich cluster in both sequences were identified. This information was used to create physical maps for respective oriC (see also legend to Fig. 3B). Arrowheads above the DnaA boxes indicate their orientation. A — indicates the oriC region which is 75% identical between M. tuberculosis and M. smegmatis.

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Moriya et al., 1992; Skarstad and Boye, 1994). Presumably, both the sequence organization and the oriC replication initiation process per se could be different in the two species.

4. Conclusions 1. A 814 bp fragment containing the dnaA–dnaN intergenic region of M. tuberculosis exhibits oriC activity and allows stable autonomous replication of oriC plasmids. 2. The copy number of oriC plasmids relative to chromosomal oriC is 1.0. 3. Removal of ColE1 ori confirmed that the autonomous replication ability of oriC plasmids was due to the DNA fragment containing the dnaA–dnaN intergenic region. 4. The 5∞ flanking region of oriC contains sequences that enable stable autonomous replication of oriC plasmids. 5. M. tuberculosis oriC does not function in M. smegmatis and vice versa, suggesting that the replication initiation process in the slowly growing members of mycobacteria are similar and different from those of the rapidly growing members. 6. Finally the availability of minichromosomes would help in investigating the factors that regulate replication initiation in slowly growing mycobacteria. Acknowledgement This research is supported in part by NIH grants AI37956 (M.R.) and AI41406 (M.M.). We thank Dr Mark A.L. Atkinson for helpful comments and careful reading of the manuscript and Dr H. Taniguchi for BCG S10 strain. This paper is dedicated to the memory of Mr. Vincent A. Steingrube.

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