Department of Biochemistry, 11937 US Highway at 271, The University of Texas Health Center at Tyler, Tyler, TX 75708-3154, USA1
Author for correspondence: Murty V. V. S. Madiraju. Tel: +1 903 877 2877. Fax: +1 903 877 5969. e-mail: murty{at}uthct.edu
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ABSTRACT |
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Keywords: dnaA, oriC, Mycobacterium avium, replication initiation
Abbreviations: Km, kanamycin; OAD, oleic acidalbumindextrose
The GenBank accession number for the sequence reported in this paper is U19185.
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INTRODUCTION |
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The currently accepted model is that replication in eubacteria is initiated when the DnaA initiator protein binds to DnaA boxes (9 nt DnaA protein recognition sequences) located in the origin of replication (oriC; Bramhill & Kornberg, 1988 ). The nucleotide sequence of these DnaA boxes is conserved and TTGTCCACA has been defined as their consensus sequence in Gram-positive bacteria with a high G+C content (see, for example, Fujita et al., 1990
; Qin et al., 1997
; Rajagopalan et al., 1995b
). The DnaA proteinbox interactions result in a cascade of events, which culminate in replication initiation and DNA synthesis (Bramhill & Kornberg, 1988
; Kornberg & Baker, 1991
). The dnaA region in eubacteria, including M. smegmatis, M. tuberculosis and Mycobacterium leprae, shows both sequence and gene order conservation. The genes are present in the order 5'-rrnpA-rpmH-dnaA-dnaN-recF-3' (Cole et al., 1998
; Qin et al., 1999
; Rajagopalan et al., 1995a
, b
; Salazar et al., 1996
). In addition it has been shown in many bacteria that either the 3', 5' or both 3' and 5' flanking regions of the dnaA gene exhibit oriC activity, i.e. the presence of these sequences renders otherwise non-replicative plasmids capable of autonomous replication. In the case of Bacillus subtilis both the 5' and 3' flanking regions act as oriC (Moriya et al., 1992
). In Pseudomonas putida and Pseudomonas aeruginosa only the 5' flanking region acts as oriC (Yee & Smith, 1990
) while in Streptomyces spp. (Zakrzewska-Czerwinksa & Schrempf, 1992
), M. smegmatis (Qin et al., 1997
; Rajagopalan et al., 1995b
; Salazar et al., 1996
), M. bovis and M. tuberculosis (Qin et al., 1999
) only the 3' flanking region functions as oriC. The genetic elements responsible for the replication process in M. avium, specifically how it is initiated and regulated, are not known. In view of the clinical significance of this organism it is important to identify and characterize the key players involved in M. avium replication initiation.
In this study we have cloned and determined the gene order of the dnaA region of M. avium and examined whether the dnaA gene flanking regions exhibit oriC activity in M. avium (Rajagopalan et al., 1995b ). We show that the gene order of the dnaA region of M. avium is similar to that reported for other bacteria, and the 3' but not the 5' flanking region of dnaA, when cloned in non-replicative plasmids, exhibited oriC activity in M. avium. Interestingly the 5' flanking region of M. avium also exhibited autonomous replication activity but only in M. bovis BCG. The implications of these results for understanding M. avium replication are discussed.
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METHODS |
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Recombinant DNA techniques.
The pYUB18-derived M. avium cosmid libraries were obtained from Drs Lisa Pascopella and Raul Barletta. All plasmids, unless otherwise stated, were derived from pUC18 and are described in Table 1. For selecting recombinants/transformants in mycobacteria, a 1·3 kb DNA fragment containing the aph gene for Km resistance was cloned into the multiple cloning site (Rajagopalan et al., 1995b
). Plasmid DNA from mycobacterial transformants was recovered into E. coli using the zirconia-based bead-beating technique (Qin et al., 1997
; Madiraju et al., 1999
). Oligonucleotides used in the study were synthesized in a Pharmacia Gene Assembler Special (Pharmacia) or were purchased from Operon Technologies. Standard molecular biology techniques such as plasmid analyses, labelling of DNA and Southern hybridization techniques were as described by Sambrook et al. (1989)
. DNA sequencing was performed either in a Pharmacia ALF DNA sequencer using the Pharmacia autocycle sequencing kit and protocol or in a LICOR DNA sequencer at McConnel Research Laboratories, San Diego, USA. Both forward and reverse sequencing primers were used as needed to determine the nucleotide sequence of both DNA strands.
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Transformation conditions for M. smegmatis, M. bovis BCG and M. tuberculosis were as described by Qin et al. (1999 , 1997
) and Wards & Collins (1996)
. Since the oriC regions of M. bovis BCG and M. tuberculosis differ by only one nucleotide and oriC plasmids from each function in the other, the mechanism of replication initiation in M. tuberculosis and M. bovis BCG is believed to be very similar (Qin et al., 1999
). Consequently, to avoid any potential risk to the experimenters all transformation experiments involving M. tuberculosis oriC plasmids were carried out in M. bovis BCG.
oriC activity.
oriC activity was defined as the ability of dnaA region fragments from M. avium to render E. coli plasmids, which were otherwise unable to replicate, capable of autonomous replication in M. avium. The results are expressed as the total number of Kmr transformants obtained (µg input DNA)-1 (Qin et al., 1997 ; Rajagopalan et al., 1995b
). Since our results showed that the 5' and 3' flanking regions of the M. avium dnaA gene exhibited different host-specific autonomous replication activities, to distinguish between these we refer to autonomous replication activity of M. avium plasmids in M. avium hosts as oriC activity and replication in other mycobacterial hosts as autonomous replication activity.
Cloning of the dnaA region.
All plasmid manipulations were carried out in E. coli (Sambrook et al., 1989 ). MVM37, an oligonucleotide primer specific to the M. avium dnaA gene (5'-AACAAGACCCGCATCGACCGGTCGCTGGCCGAG-3'; Rajagopalan et al., 1995a
) was used to screen M. avium cosmid DNA libraries by colony hybridization (Sambrook et al., 1989
). The cosmids from the positive clones were confirmed by Southern hybridization, probing individually with DNA fragments specific to the rpmH, dnaA and dnaN genes. Based on these data pUC18-based plasmids with inserts containing the 3·0 kb rpmH and dnaA genes (pMQ434) and 2·1 kb dnaA and dnaN genes (pMQ423) and various subclones were constructed and used to determine the nucleotide sequence of both strands of the inserts (see Fig. 1
). Typically 400500 bases of sequence per sample were obtained with the Pharmacia ALF DNA sequencer and approximately 7501150 bases of sequence with the LICOR machine. Nucleotide sequence data were analysed by DNA Strider 1.3 and Gene Jockey programs. Once the nucleotide sequence was confirmed, the dnaA region fragments in both pMQ434 and pMQ423 were joined to produce a 3·9 kb DNA fragment containing the rpmH, dnaA and dnaN genes and their intergenic regions.
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RESULTS AND DISCUSSION |
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orf1 corresponds to nucleotide positions 480340 of the bottom strand. It is predicted to encode an RpmH homologue of 47 residues with a molecular mass of 5·3 kDa. Six nucleotides upstream of the predicted start codon, GTG, is AGAAGG, a sequence resembling that of a potential ribosome binding sequence. The predicted protein exhibits significant homology with the RpmH protein of other bacteria, including M. tuberculosis and M. smegmatis (data not shown). orf2 corresponds to nucleotide positions 10842610, is the largest of the three ORFs and is predicted to encode the DnaA homologue. Eight nucleotides upstream of the predicted start codon, TTG, is a potential ribosome binding sequence, AGGGAG. The predicted protein is 509 residues in length and has a molecular mass of 56·7 kDa. The deduced amino acid sequence of the M. avium dnaA gene product is 86 and 55% identical to the corresponding proteins of M. tuberculosis and M. smegmatis, respectively (data not shown). orf3 corresponds to nucleotide positions 3179end and is predicted to encode the dnaN homologue. Five nucleotides upstream of the ATG start codon is the putative ribosome binding sequence, GAAGGG. The predicted protein shows significant homology with the DnaN protein of other bacteria, including mycobacteria (data not shown).
The rpmHdnaA and dnaAdnaN intergenic regions
One of the characteristic features of eubacterial oriC is the presence of several DnaA-box-like sequences. The rpmHdnaA and dnaAdnaN intergenic regions of M. avium were found to contain 14 and 16 DnaA-box-like sequences with one to three mismatches to the consensus sequence TT(G/C)TCCACA, respectively (see Fig. 2; Qin et al., 1999
, 1997
; Rajagopalan et al., 1995b
). These are clustered close to the dnaA gene itself with 10 of the 14 DnaA boxes in the rpmHdnaA intergenic region located towards the 5' end of dnaA, and 11 of the 16 DnaA boxes in the dnaAdnaN intergenic region towards its 3' end. Nucleotide sequence comparisons revealed that the M. avium rpmHdnaA intergenic region is 63 and 45% identical to that of the corresponding regions of M. tuberculosis and M. smegmatis, respectively, whereas the dnaAdnaN intergenic region is 62 and 50% homologous, respectively. Many of the designated DnaA boxes of M. avium show significant sequence homology to the corresponding position in M. tuberculosis, suggesting that the equivalent DnaA boxes in both organisms are positionally conserved (Fig. 2a
and b
). In contrast, the M. smegmatis sequence does not show DnaA boxes at many of these conserved positions (Fig. 2a
and b
). This suggests that the architectural composition of oriC of M. smegmatis differs from M. avium and M. tuberculosis (Fig. 2
).
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Autonomous replication activity of M. avium DNA fragments containing the rpmHdnaA intergenic region
We have speculated that the M. avium replication origins might have evolved from B. subtilis-like organisms (Moriya et al., 1992 ) that selectively utilize the dnaAdnaN intergenic region as their primary oriC. If this is the case the rpmHdnaA intergenic region may be a vestigial replication origin or alternatively it could be a silent replication origin whose activity can be unmasked under specific growth conditions, e.g. in the absence of specific inhibitors. To test this prediction, pMQ457 (containing the rpmHdnaA intergenic region) was transformed into both M. smegmatis and M. bovis BCG. M. bovis BCG transformed with pMQ457 produced Kmr transformants but M. smegmatis did not (data not shown; see below). The M. bovis BCG transformants appeared 2 weeks later than those with the shuttle vector pMV206. The transformation frequency was in the range of 104 (µg input DNA)-1 and was similar to that obtained with the shuttle vector pMV206. The recovered and input pMQ457 DNA showed identical restriction digestion patterns (Fig. 4
), indicating that pMQ457 was replicating in M. bovis BCG as stable extrachromosomal elements (data not shown). Reciprocal experiments using M. tuberculosis, M. bovis BCG and M. smegmatis plasmid constructs containing their rpmHdnaA intergenic region in M. avium hosts did not produce any Kmr transformants (data not shown). Since the transformation efficiency of M. avium strains is two to three orders of magnitude less than the other mycobacterial strains, it is possible that the lack of autonomous replication activity of those plasmids in M. avium may be merely due to poor transformation efficiency of M. avium or could be a property specific to M. avium. Obviously improved transformation conditions with M. avium strains would help resolve this issue.
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Overproduction of DnaA of M. avium
Since the interaction between the DnaA protein and DnaA boxes is believed to be the essential prerequisite for initiation of replication (Kornberg & Baker, 1991 ), we have begun to investigate this process in M. avium. We have overproduced M. avium DnaA in E. coli. Unfortunately, most of the DnaA was in inclusion bodies (Fig. 5
). We are pursuing a number of strategies to obtain soluble protein but until it can be obtained in a sufficient quantity in its native form it will not be possible to investigate its binding properties any further.
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ACKNOWLEDGEMENTS |
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Received 30 March 1999;
revised 1 July 1999;
accepted 5 July 1999.