Laboratorio de Genética Molecular, Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020A, Venezuela1
Tel: +58 2 504 1653. Fax: +58 2 504 1382. e-mail: lsalazar{at}pasteur.ivic.ve
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ABSTRACT |
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Keywords: oriC/replication, dnaA promoter, incompatibility, Mycobacterium
Abbreviations: FIS, factor for inversion stimulation; GFP, green fluorescent protein; IHF, integration host factor protein; OADC, oleic acid albumin dextrose complex
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INTRODUCTION |
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Tuberculosis (TB) remains a major, global public health problem, particularly in low-income countries. In 1993, the World Bank estimated that the disease accounts for more than 25% of avoidable adult deaths in developing countries. Moreover, the global number of TB cases is expected to continue to increase (Dolin et al., 1994 ). Better application of current diagnostic, treatment and prevention strategies could lead to a gradual decrease in the disease, but eliminating TB will require new tools and a better understanding of the metabolism of the mycobacteria. An improved knowledge of the replication process would aid in finding susceptible factors for controlling the mycobacterial cell cycle.
Initiation of DNA replication is a global process, which in bacteria, plasmids and some viruses, involves the binding of initiator proteins to repetitive sequences at the origin of replication, oriC. The oriC structure, as well as the basic gene order and the primary structures of the encoded proteins in its vicinity, are remarkably conserved among eubacteria. The most important element leading to initiation of bacterial chromosome replication is the DnaA protein. The binding of DnaA to DNA-binding sites (DnaA boxes) has been demonstrated for Escherichia coli (Crook et al., 1993 ), Bacillus subtilis (Krause et al., 1997
) and Streptomyces (Jakimowicz et al., 1998
). The DnaA protein binds to DnaA boxes in the E. coli oriC region, leading to formation of regularly shaped oriCDnaA complexes containing 2040 DnaA monomers (Bramhill & Kornberg, 1988
). The formation of this complex might involve other proteins, such as FIS and IHF (Roth et al., 1994
), and lead to DnaA-facilitated strand opening of a region containing three AT-rich 13-mer sequences, preceding the formation of the primosome (Baker et al., 1987
; Sekimizu et al., 1988
; Marszalek & Kaguni, 1994
). In vivo studies indicate that the DnaA protein also controls transcriptional events in the oriC region. This could lead to a strongly increased negative supercoiling of the 13-mer region up to the time when the cell will be ready to initiate replication and thus facilitate strand opening (Asai et al., 1992
).
Autonomous replicating sequences (arss) 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 arss contain either the region upstream of dnaA or a DnaA box region downstream of rnpA. In contrast, in Gram-positive bacteria oriC always includes the intergenic dnaAdnaN region, although DnaA boxes exist in two noncoding regions upstream and downstream of the dnaA gene. An exception is B. subtilis, where both upstream and downstream DnaA box regions are required in cis for initiation of replication, suggesting that in this bacterium, the initiation of replication involves formation of a loop between the upstream and downstream DnaA boxes, mediated by the DnaA protein (Moriya et al., 1992
, 1999
).
Salazar et al. (1996) reported that with oriC plasmids of M. smegmatis, containing both upstream and downstream DnaA box regions in cis, transformants could not be obtained in this bacterium, even at a low copy number. Since this incompatibility was partially relieved when a small deletion that inactivated the dnaA gene was introduced, or when the upstream dnaA region and 5' end of the dnaA gene were deleted, it was suggested that the incompatibility could be due to overproduction of the DnaA protein.
In this work, a series of oriC plasmids of M. smegmatis was constructed in which the upstream dnaA region (rpmHdnaA promoter and DnaA boxes) was deleted or exchanged with sequences that expressed a different promoter activity (weak or null) or by sequences that did not contain a consensus DnaA box motif. A short sequence (270 bp) containing the strong rpmHdnaA promoter was found to exert an inhibitory effect on oriC activity. The three DnaA boxes upstream of dnaA exhibited a limited effect on ori activity. When the rpmHdnaA promoter with the dnaA gene was cloned in trans, a stronger incompatibility was observed.
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METHODS |
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The plasmids used in this study are listed in Table 1. The ori plasmids pOS239, pOS242 and pOS245 were described previously (Salazar et al., 1996
). For the construction of pOS239K and pOS242K, the aph gene (Kmr) from pYUB53 (Jacobs et al., 1996) (1·3 kb fragment) was cloned in the SalI site of pUC19 (pUC19Kmr). BamHIKpnI fragment from pOS239 and SmaI fragment from pOS242, 3·3 kb and 2·65 kb, respectively, were cloned in pUC19Kmr. pOS246 and pOS246K were constructed by deletion (cut, blunted and religated) of the 270 bp HindIIIEcoRI region, containing PdnaA (-35 and -10 sequences), from pOS239 and pOS239K, respectively. Similarly, pOS247 was constructed by deletion of the 113 bp EcoRINcoI region (containing DnaA boxes) from pOS239. pOS248 was constructed by deletion of PdnaA and the DnaA boxes (383 bp HindIIINcoI fragment) from pOS239. For the construction of pOS249, the fragment containing the promoter region of a sigma factor of M. smegmatis was amplified by PCR (fragment A, Table 2
) and cloned into the HindIII/EcoRI site of pOS239. In a similar way, pOS250 was constructed by the cloning of a PCR fragment containing the promoter of the gyrBA gene of M. smegmatis (fragment B, Table 2
) into pOS239. The promoter of the dnaA gene and/or DnaA boxes region upstream of dnaA were replaced by sequences that did not exhibit promoter activity nor contained consensus sequences for DnaA boxes, by cloning PCR amplification fragments containing the rnpArpmH genes of M. tuberculosis into pOS239 (fragments C, D and E, respectively; Table 2
). In the PCR reactions, DNA of the cosmid pIV101 for M. smegmatis and pIV305 for M. tuberculosis (Salazar et al., 1996
) was used as substrate. To create pOS254, pJEM15 (Timm et al., 1994
) was digested with ClaI to release the transcriptional termination sequence (
fragment composed of a streptomycinspectinomycin resistance gene flanked by short inverted repeats containing tT4), and pOS239 was linearized with the endonuclease MluI. Both fragments were treated with Klenow DNA polymerase and deoxynucleoside triphosphates, purified from 1% agarose gels and ligated. The resulting plasmid carries an oriC with a transcription stop signal precisely 123 nucleotides from the stop of the coding sequence (TGA) of the dnaA gene, between the AT-rich region and the first DnaA box on oriC of M. smegmatis. To screen for deletion or replacement oriC mutants, DNA from transformants was amplified with specific primers flanking the upstream dnaA region, LS60 (5'-TGGAAGGTCCGGTTGCCCTTG-3') and SM15 (5'-GGACGATTACCCCCTTTGAGG-3'), and the fragments obtained were analysed by sequencing. To clone the dnaA gene in trans, the dnaA gene including its upstream region was amplified by PCR using specific primers LS60P (5'-TTCAGCTGGAAGGTCCGCTTGCC-3') and SM2P (5'-CTCAGCTGTCAGCGTTTGGCGCGCTGGC-3') and DNA from plasmid pOS239 as template. The PCR product was digested with PvuII and cloned in the ScaI site of pOS245. Similar constructions were made using DNA from pOS249 and pOS250 as template.
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Other molecular techniques.
Digestions, ligations, filling of protruding ends and plasmid DNA isolation were performed according to standard procedures (Ausubel et al., 1995 ). Amplified fragments were sequenced with USB Sequenase 2.0 (USB, Amersham) and [35S]dATP
S or with a dye terminator cycle sequencing kit and an ABI377 sequencer (PE Biosystem), using the appropriate primers. PCR amplification reactions were carried out with Taq DNA polymerase (Perkin Elmer), according to the manufacturers recommendations.
Determination of plasmid copy number.
M. smegmatis mc2155 cells bearing oriC plasmids were grown overnight at 37 °C to mid-exponential phase, in 7H9 medium containing 50 µg hygromycin ml-1. Total DNA was then prepared as described by Ausubel et al. (1995) , linearized by digestion with PvuII and separated by 1% agarose gel electrophoresis. Southern blot analysis was performed with [
32P]dCTP-labelled oriC from M. smegmatis as probe. The oriC probe was obtained by PCR amplification using the primers SM10 (5'-GCCCCTTCGATAATCCCCGCA-3') and SM11 (5'-CACGCTCGGCGGCTGTGGATA-3') and pIV101 cosmid as template. Hybridization was at 65 °C, overnight, and final wash conditions were 65 °C for 30 min with 0·1xSSC and 0·1% SDS. DNA bands were scanned and analysed using ALPHAEASE software (Alpha Innotech). The copy number was determined as the ratio between the chromosomal and plasmid band intensities after hybridization.
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RESULTS |
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To determine if oriC function is negatively regulated by the transcription of dnaA, two different approaches were adopted, which avoided the topological effect but left dnaA expression intact. First, the insertion of the transcription terminator tT4 at the end of the dnaA gene, which prevents dnaA transcripts from entering and traversing oriC, did not release the inhibitory effect (pOS254; Table 3). On the other hand, while trying to establish a system in which the dnaA gene was supplied in trans, PdnaAdnaA gene was cloned into the ScaI site of pOS245, disrupting the ampicillin-resistance gene (Fig. 2a
). Unexpectedly, on several occasions, the restriction analysis of the plasmids isolated from transformed colonies of E. coli revealed a deletion instead of the expected lengthening. The same results were also observed when PsigM or PgyrB drove dnaA expression (Fig. 2b
), or when the dnaA gene was cloned in another site on the plasmid (data not shown).
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Phenotype of M. smegmatis colonies containing oriC plasmids and copy number determination
M. smegmatis mc2155 colonies bearing the oriC plasmids were smaller than those carrying the control plasmid, pOM11 (Fig. 4), indicating some incompatibility towards the chromosome. Additionally, differences in colony morphology were found between strains carrying the oriC plasmids. When only PdnaA was modified, the resulting colonies were smaller than when an additional modification in the DnaA boxes region was introduced (compare strains with plasmids pOS253 and pOS246).
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DISCUSSION |
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Some additional results were found to be inconsistent with the hypothesis that the direct interaction between the plasmid-borne and chromosomal DnaA boxes, mediated by DnaA protein, could be the cause of the strong incompatibility between the plasmid and chromosomal oriCs, as has been suggested to occur in B. subtilis (Moriya et al., 1992 ) and S. lividians (Zakrzewska-Czerwinska et al., 1992
). (i) No significant variations in the transformation efficiency were observed when the ars contained the DnaA boxes upstream of dnaA (compare plasmids pOS246 and pOS248 in Fig. 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. First, an atypical morphology of colonies was found when M. smegmatis carried oriC plasmids. In cases in which PdnaA and DnaA box regions had been modified simultaneously (plasmid pOS253), the cells generated large colonies similar to the control (Fig. 4
). On the other hand, deletion of the DnaA boxes increased the plasmid copy number (Fig. 5
). The effect of the DnaA boxes upstream of dnaA on replication could be indirect, via repression of transcription from the dnaA promoter, as deletion of the DnaA box causes an increase in the dnaAGFP activity (Fig. 3
). In E. coli, deletion of the DnaA box localized between dnaA1P and dnaA2P causes an increase in expression from dnaA1P and a decrease in the activity of promoter 2P (Atlung et al., 1985
; Polaczek & Wright, 1990
).
The possibility that oriC function is negatively regulated in cis by the transcripts initiated from upstream of dnaA that enter and traverse oriC was discarded (plasmid pOS254). However, topological effects exerted by dnaA gene expression were not demonstrated, because a strong incompatibility in E. coli was found when this gene was supplied in trans, regardless of whether a strong or weak promoter drove the transcription of the dnaA gene. The significance of this result is not clear but the dnaA and rpmH promoters of M. smegmatis and M. tuberculosis are very well expressed in E. coli (data not shown). These genes show a good consensus 70 promoter sequence (L. Salazar and others, unpublished data). It is possible that transcription from upstream of dnaA interfered with replication initiation from the origin of replication of E. coli on the plasmid. Or alternatively, an intra-molecular interaction may have occurred between both DnaA boxes on the plasmid mediated by DnaA protein. Moriya et al. (1988)
observed that in the transformant colonies obtained when Region B of B. subtilis was cloned into a low-copy-number plasmid, the DNA plasmid isolated showed drastic changes in the structure of the inserted B region. The B region of B. subtilis is equivalent to the DnaA box region upstream of dnaA in M. smegmatis.
In spite of this, the incompatibility was overcome when PdnaA was deleted. The introduction of the weak promoter led to a transformation frequency 26 times higher (Table 2), suggesting that some transcription from upstream of dnaA contributed to replication of the oriC plasmid, perhaps because the amount of DnaA protein is a limiting factor (Moriya et al., 1990
; von Meyenburg & Hansen, 1987
). However, excess of DnaA seems to be toxic. The dnaA promoter of mycobacteria is relatively strong in relation to those governing other essential genes and dnaA transcription is coupled with growth (L. Salazar and others, unpublished; Fig. 3
). In B. subtilis (Ogasawara et al., 1991
) the presence of an intact dnaA gene inhibited growth, and the cloning of the dnaA gene of S. lividians, using high- or mediumhigh-copy-numbers vectors, led to very poor growth and loss of the inserts (Zakrzewska-Czerwinska et al., 1994
).
The results found in the present study clearly indicate that the region upstream of the dnaA gene is playing some regulatory role in chromosome replication. They suggest that PdnaA exerts a negative effect by overexpression of DnaA protein, and DnaA boxes seem be implicated in the regulation of the copy number or stability.
Whether the dnaA regulatory region has a role in the cell cycle regulation of initiation at the origin of replication in eubacteria is still unknown. Polaczek (1998) discussed the possibility that the regulatory sequences within and surrounding the dnaA promoter region of E. coli constitute an evolutionary fossil of no significant physiological relevance, because there is not convincing experimental support that demonstrates a fine-tuned expression of the dnaA gene. However, Speck et al. (1999)
recently showed an unequivocal autoregulation of dnaA in E. coli through interaction between the DnaA protein and the dnaA promoter region. Additional functions have been assigned to the dnaA promoter region. Moriya et al. (1992)
described that in B. subtilis, the dnaA promoter region is required for autonomous replication in plasmids and that the ars activity is affected by neighbouring sequences. Removal of the promoter region of rpmH from the oriC plasmid resulted in stronger ars activity. The rpmH gene encodes the 50S ribosomal protein L34. The activity of the rpmH promoter of M. smegmatis is 1·53·5 times higher than that exhibited by the dnaA promoter (data not shown). Therefore, it is possible that the divergent transcription from rpmH and dnaA exerts topological effects, or that the strong promoter activity of rpmH will inhibit replication of the oriC plasmid. Experiments to determine the contributions of the rpmH promoter are in progress.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Atlung, T., Clausen, E. & Hansen, F. G. (1985). Autoregulation of the dnaA gene of Escherichia coli. Mol Gen Genet 200, 442-450.[Medline]
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Siedman, J. G., Smith, J. A. & Sthrul, K. (1995). Current Protocols in Molecular Biology. New York: Green Publishing Associates/Wiley-Interscience.
Baker, T. A., Funnell, B. E. & Kornberg, A. (1987). Helicase action of DnaB protein during replication from the Escherichia coli chromosomal origin in vitro. J Biol Chem 262, 6877-6885.
Bramhill, D. & Kornberg, A. (1988). A model for initiation at origins of DNA replication. Cell 54, 915-918.[Medline]
Casart, Y. (1996). Efecto del relajamiento del ADN sobre la expresión del promotor de los genes de la ADN girasa de Mycobacterium tuberculosis. Pre-doctorate thesis, IVIC, Venezuela, pp. 3640.
Crook, E., Hwanmg, D. S., Sharstad, K., Thöny, B. & Kornberg, A. (1993). E. coli minichromosome replication: regulation of initiation at oriC. Res Microbiol 142, 127-130.
Dolin, P. J., Raviglione, M. C. & Kochi, A. (1994). Global tuberculosis incidence and mortality during 19902000. Bull WHO 72, 213-220.[Medline]
Jacobs, W. R., Kalpana, G. V., Cirillo, J. D., Pascospella, L., Snapper, S. B., Udani, R. A., Jones, W., Barletta, R. G. & Bloom, B. R. (1991). Genetic systems in mycobacteria. Methods Enzymol 204, 537-555.[Medline]
Jakimowicz, D., Majka, J., Messer, W. & 8 other authors (1998). Structural elements of the Streptomyces oriC region and their interactions with DnaA protein. Microbiology 144, 12811290.[Abstract]
Krause, M., Rückert, B., Lurz, R. & Messer, W. (1997). Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein. J Mol Biol 274, 365-380.[Medline]
Madiraju, M., Qin, M. H., Yamamoto, K., Atkinson, M. & Rajagopalan, M. (1999). The dnaA gene region of Mycobacterium avium and the autonomous replication activities of its 5' and 3' flanking regions. Microbiology 145, 2913-2921.
Marszalek, J. & Kaguni, J. M. (1994). DnaA protein directs the binding of DnaB protein in initiation of DNA replication in Escherichia coli. J Biol Chem 269, 4883-4890.
von Meyenburg, K. & Hansen, F. G. (1987). Regulation of chromosome replication. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp 15551577. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Moriya, S., Fukuoka, T., Ogasawara, N. & Yoshikawa, H. (1988). Regulation of initiation of the chromosomal replication by DnaA-boxes in the origin of the Bacillus subtilis chromosome. EMBO J 7, 2911-2917.[Abstract]
Moriya, S., Kato, K., Yoshikawa, H. & Ogasawara, N. (1990). Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells initiation potential. EMBO J 9, 2905-2910.[Abstract]
Moriya, S., Atlung, T., Hansen, F. G., Yoshikawa, H. & Ogasawara, N. (1992). Cloning of an autonomously replicating sequence (ars) from Bacillus subtilis chromosome. Mol Microbiol 6, 309-315.[Medline]
Moriya, S., Imai, Y., Hassan, A. K. M. & Ogasawara, N. (1999). Regulation of initiation of Bacillus subtilis chromosome replication. Plasmid 41, 17-29.[Medline]
Ogasawara, N., Moriya, S. & Yoshikawa, H. (1991). Initiation of chromosome replication: structure and function of oriC and DnaA protein in eubacteria. Res Microbiol 142, 851-859.[Medline]
Polaczek, P. (1998). Is the dnaA promoter region in Escherichia coli an evolutionary junkyard of physiologically insignificant regulatory elements? Mol Microbiol 27, 1089-1090.[Medline]
Polaczek, P. & Wright, A. (1990). Regulation of expression of the dnaA gene in Escherichia coli: role of the two promoters and the DnaA box. New Biol 2, 574-582.[Medline]
Roth, A., Urmoneit, B. & Messer, A. (1994). Functions of histone-like proteins in initiation of DNA replication at oriC of Escherichia coli. Biochimie 76, 917-923.[Medline]
Qin, M. H., Madiraju, M., Zachariah, S. & Rajagopalan, M. (1997). Characterization of the oriC region of Mycobacterium smegmatis. J Bacteriol 179, 6311-6317.[Abstract]
Qin, M. H., Madiraju, M. & Rajagopalan, M. (1999). Characterization of the functional replication origin of Mycobacterium tuberculosis. Gene 233, 121-130.[Medline]
Salazar, L., Fsihi, H., de Rossi, E., Riccardi, G., Rios, C., Cole, S. T. & Takiff, H. E. (1996). Organization of the origins of replication of the chromosome of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis. Mol Microbiol 20, 283-293.[Medline]
Sekimizu, K., Yung, B. Y. & Kornberg, A. (1988). The DnaA protein of Escherichia coli: abundant, improved purification and membrane binding. J Biol Chem 263, 7136-7140.
Speck, C., Weigel, C. & Messer, W. (1999). ATP- and ADP-DnaA protein, a molecular switch in gene regulation. EMBO J 18, 6169-6176.
Timm, J., Lim, E. M. & Gicquel, B. (1994). Escherichia colimycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series. J Bacteriol 176, 6749-6753.[Abstract]
Valdivia, R. H., Hromockyj, A. E., Monack, D., Ramakrishnan, L. & Falkow, S. (1996). Applications for the green fluorescent protein (GFP) in the study of hostpathogen interactions. Gene 173, 47-52.[Medline]
Yasuda, S. & Hirota, Y. (1977). Cloning and mapping of the replication origin of Escherichia coli. Proc Natl Acad Sci USA 74, 5458-5462.[Abstract]
Ye, F., Renaudin, J., Bové, J.-M. & Laigret, F. (1994). Cloning and sequencing of the replication origin (oriC) of the Spiroplasma citri chromosome and construction of autonomously replicating artificial plasmids. Curr Microbiol 29, 23-29.[Medline]
Yee, T. W. & Smith, D. W. (1990). Pseudomonas chromosomal replication origins: a bacterial class distinct from Escherichia coli-type origins. Proc Natl Acad Sci USA 87, 1278-1282.[Abstract]
Zakrzewska-Czerwinska, J. & Schrempf, H. (1992). Characterization of an autonomously replicating region from the Streptomyces lividians chromosome. J Bacteriol 174, 2688-2693.[Abstract]
Zakrzewska-Czerwinska, J., Nardmann, J. & Schrempf, H. (1994). Inducible transcription of the dnaA gene from Streptomyces lividians 66. Mol Gen Genet 242, 440-447.[Medline]
Received 13 March 2000;
revised 25 May 2000;
accepted 13 June 2000.