Inhibition of chromosome replication in Mycobacterium smegmatis: effect of the rpmH–dnaA promoter region

Leiria Salazar1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In a previous study a functional mycobacterial origin of replication, oriC, was isolated on a plasmid. However, it was found that origin function was inhibited by the presence of the adjacent dnaA gene or its regulatory region, so that plasmids containing both of these regions next to the origin did not yield transformants. This inhibition could be due either to overexpression of dnaA on a plasmid being toxic, the transcription of dnaA into the downstream origin topologically inhibiting its function, or to the DnaA boxes upstream of dnaA somehow interacting with the DnaA boxes in the origin to prevent its function. To distinguish between these possibilities, plasmids were constructed lacking different parts of the dnaA gene: the promoter, the DnaA boxes, or both. Additionally, the putative dnaA promoter region was replaced by mycobacterial sequences that exhibit weaker or null promoter activity. The results indicate that the rpmH–dnaA promoter region, but not the DnaA boxes, is the principal cause of the incompatibility observed and suggest that this region could be playing a role in the inhibition of chromosome replication.

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


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
 
The genus Mycobacterium comprises a wide variety of organisms ranging from rapid-growing saprophytes or opportunists such as Mycobacterium smegmatis and M. fortuitum, which duplicate in 2–3 h, to slow-growing pathogens like M. tuberculosis and M. bovis, which have a duplication time of around 18–24 h.

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 oriC–DnaA complexes containing 20–40 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 (ars’s) 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 ars’s 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 dnaA–dnaN 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 (rpmH–dnaA 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 rpmH–dnaA 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 rpmH–dnaA promoter with the dnaA gene was cloned in trans, a stronger incompatibility was observed.


   METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strain, plasmids and media.
E. coli XL-1 Blue (Stratagene) was used for routine cloning and plasmid amplification. M. smegmatis high-frequency electroporation strain mc2155 was grown in Middlebrook 7H9 or 7H10 broth supplemented with 0·5% (v/v) glycerol and 10% OADC. Tween 80 (0·05%) was added to liquid media. Aliquots of 0·1–0·5 µg plasmid DNA were used in electroporation experiments as described previously (Salazar et al., 1996 ). The transformation efficiencies for each plasmid are shown as the number of transformants per µg DNA, based on the mean from at least four independent electroporations. Colony transformants were counted after incubation at 37 °C for 72–120 h. As control, plasmids pOM11 and pIJ963 were electroporated. Carbenicillin (Cb; 50 µg ml-1), kanamycin (Km; 25 µg ml-1) and hygromycin (Hyg; 50 µg ml-1) were added to the culture media as appropriate.

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). BamHI–KpnI 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 HindIII–EcoRI region, containing PdnaA (-35 and -10 sequences), from pOS239 and pOS239K, respectively. Similarly, pOS247 was constructed by deletion of the 113 bp EcoRI–NcoI region (containing DnaA boxes) from pOS239. pOS248 was constructed by deletion of PdnaA and the DnaA boxes (383 bp HindIII–NcoI 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 rnpA–rpmH 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 ({Omega} fragment composed of a streptomycin–spectinomycin 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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Table 1. Plasmids used in this study

 

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Table 2. Primers and amplified fragments used

 
Transcriptional fusion to GFP and measurement of the fluorescence.
The shuttle plasmid pFPV27 (Valdivia et al., 1996 ) was used for cloning fragments fused to the gfp gene. The PCR reaction was carried out using the primers LS60B (5'-AAGGATCCGAAGGTCCGCTTGCCCTT-3') and SM15B (5'-GCGGATCCGGACGATTACCCCCTTTGAG-3'), or LS60B and SM17E (5'-CTCGAATTCGCCCCGTGCT-3') and DNA from the plasmids pOS239, pOS246, pOS249, pOS250, pOS251, pOS252 and pOS253 as substrate. The resulting fragments containing the upstream dnaA region, or only the promoter region, were cloned into pFPV27 (BamHI or BamHI/EcoRI site, respectively). The direction of the inserts was determined by digestion with restriction endonucleases and sequencing. M. smegmatis mc2155 cells bearing the transcriptional fusion to gfp were grown in 7H9 medium containing 25 µg kanamycin ml-1 at 37 °C. Fluorescence and culture density (OD640, Molecular Devices Spectra Max 340) were determined from aliquots of the cultures taken at early, mid-exponential and stationary phase. GFP fluorescence was quantified by fluorometry in a Spectrafluor Tecan (Microplate Reader).

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{alpha}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 manufacturer’s 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 [{alpha}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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Contribution of the upstream dnaA region to the inhibition of replication
In an attempt to determine the nature of this inhibition, a series of oriC plasmids from pOS239 was constructed, eliminating or modifying the rpmH–dnaA promoter region (PrpmH–dnaA) and/or the DnaA box region upstream of the dnaA gene. The plasmid pOS239 carries a 3·3 kb fragment of M. smegmatis mc26, with the rnpA–dnaN region including the origin of replication. It is not able to remain as an ars in M. smegmatis mc2155 (Salazar et al., 1996 ). By cutting with restriction endonucleases and religation of pOS239, the rpmH–dnaA promoter region, the three DnaA boxes localized upstream of the dnaA gene, or both, were deleted. Similar constructions were made using the pUC19Kmr-cloning vector. All of these constructions were then transformed into M. smegmatis mc2155. Transformation frequencies were considered as an expression of the oriC activity. The results showed that the inhibitory effect observed in pOS239 was overcome, regardless of the selective marker (hygromycin or kanamycin resistance), when the PrpmH–dnaA (pOS246 and pOS246K plasmids) or PrpmH–dnaA and DnaA box region (pOS248 plasmid) were removed, but not when only the DnaA box region (pOS247 plasmid) was removed. The plasmids pOS246, pOS246K and pOS248 could efficiently transform M. smegmatis with a frequency of 0·3–0·5x104 transformants per µg DNA. In contrast, pOS247 showed a frequency of transformation close to zero (Fig. 1). On rare occasions, some transformant colonies were found with plasmids pOS239 and pOS247. These results indicate that the region that contained PrpmH–dnaA specifically causes the incompatibility observed between oriC plasmids and the chromosome of M. smegmatis.



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Fig. 1. Schematic representation of the constructs obtained from pOS239. Arrows indicate the orientation of transcription. Dotted white boxes denote the deleted regions. pOS239 and pOS242 were described previously (Salazar et al., 1996 ). , dnaA promoter region; , DnaA boxes region; , origin of replication; H, HindIII; E, EcoRI; N, NcoI. Transformation frequencies represent the mean of at least four independent assays; NT, not tested.

 
To elucidate the mechanism of replication control by PdnaA, a 270 bp HindIII–EcoRI fragment from pOS239 containing PrpmH–dnaA was replaced by sequences that exhibit a weak or null promoter activity. Assuming that a space conservation between the motifs is important in the replication function, in each case fragments having the same length (Table 2) were introduced into this region. PCR fragments containing the sigM promoter of M. smegmatis and the coding sequence of the rnpA gene of M. tuberculosis (fragments A and C, respectively) were cloned into the HindIII/EcoRI site of pOS239, resulting in plasmids pOS249 and pOS251. We have demonstrated that the expression of sigM from M. smegmatis and M. bovis BCG is very low in normal growth conditions (rich medium, 37 °C) (N. Arraíz and others, unpublished, and Fig. 3). In that work, we found that a 488 bp fragment containing the pknC–sigM intergenic region from M. smegmatis shows promoter activity. The fragment C did not show promoter activity (see below). Both of these plasmids are able to transform M. smegmatis mc2155, but pOS249 resulted in a stronger ars activity (Table 3).



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Fig. 3. Fluorescence level in M. smegmatis bearing transcriptional fusions with gfp. Transcriptional fusions were generated as described in Methods. Fluorescence was determined by spectrofluorometry. All measurements were carried out on triplicate cultures. Specific promoter activity is expressed as fluorescence intensity at 535 nm (emission filter), corrected for the fluorescence emission of untransformed cells.

 

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Table 3. Efficiency of transformation of oriC plasmids

 
Following a similar methodology and taking advantage of the fact that the DnaA boxes upstream of the dnaA gene of M. smegmatis are localized in a 113 bp EcoRI–NcoI segment, the DnaA box region was replaced with a fragment that did not show a consensus motif for DnaA binding (fragment D). The resulting plasmid pOS252, like pOS247, did not yield transformants. However, when the 383 bp fragment containing both PdnaA and DnaA box regions was exchanged with a fragment that did not contain any of these motifs (fragment E), ars activity was restored (plasmid pOS253; Table 3). These results support the hypothesis that the DnaA box region upstream of the dnaA gene is not involved in the incompatibility observed.

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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Fig. 2. (a) Strategy for cloning of the dnaA gene in trans: cloning of PdnaAdnaA gene in the ScaI site of pOS245. (b) Sizes of oriC plasmids isolated from E. coli. BglII-digested DNA was electrophoresed into 1% agarose gel. Lanes: 1, 1 kb ladder; 2, pOS239; 3, pOS245; 4, PdnaAdnaA gene; 5, PsigMdnaA gene; 6, PgyrBAdnaA gene; 7, PdnaAdnaA gene. Arrows show the lengths of the restriction fragments.

 
Transcriptional fusion to gfp in plasmids
To confirm the promoter activity of the fragments used in the experiments described above, each one (fragments A–E; Table 2) was fused to the green fluorescent protein gene (gfp) on the plasmid pFVP27 (Valdivia et al., 1996 ). The results of the fluorescence measured from M. smegmatis strains carrying the different plasmids are shown in Fig. 3. The fragment containing PdnaA (plasmid pgfp87) showed promoter activity. It reached the highest level during the exponential growth phase. The promoter activity shown by PsigM (pgfp13) confirmed our previous result (N. Arraiz and others, unpublished): PsigM of M. smegmatis is a weak promoter and its activity is doubled when the cells reach the stationary phase. In some cases, with PdnaA and PsigM, promoter activity was slightly reduced by the DnaA boxes cloned downstream (plasmids pgfp85 and pgfp43, respectively). The fragments containing a portion of rnpA–rpmH gene sequences (pgfp30, pgfp434, and pgfp67) showed a fluorescence level indistinguishable from that emitted by M. smegmatis cells carrying the vector plasmid, pFVP27.

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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Fig. 4. Photographs of M. smegmatis colonies bearing oriC plasmids: (a) pOM11, (b) pOS210, (c) pOS246, (d) pOS253. Bar, 5 mm.

 
The copy number of the oriC plasmids was measured as described in Methods. Qin et al. (1997) reported for ars plasmids of M. smegmatis a copy number of two. In this study, variations in the copy number of the ars plasmids of M. smegmatis were found (Fig. 5), depending of the modification introduced in the upstream dnaA region. When PdnaA was removed, or exchanged by a weak promoter, the copy number was close to one (plasmids pOS246 and pOS249). However, when additional modifications were introduced into the DnaA boxes region the copy number was considerably higher [plasmids pOS210 (Salazar et al., 1996 ), pOS245 and pOS253]. Additionally, in previous work (Salazar et al., 1996 ) and in this study, it was found that the oriC plasmids could transform M. smegmatis if the upstream dnaA sequence was removed. In the cases where some transformants of pOS239 or pOS252 were found, the recovery of the intact plasmid DNA from these colonies failed and insertions in the chromosome were not detected (data not shown).



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Fig. 5. Copy number of the oriC plasmids: 1, mc2155 (control); 2, pOS210; 3, pOS245; 4, pOS246; 5, pOS249; 6, pOS250; 7, pOS251; 8, pOS253. Chr indicates the chromosome band. The values represent the mean (± standard error) of at least three different measurements.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The upstream region of the dnaA gene of mycobacteria is composed of, at least, two conserved motifs: the promoter region with divergent transcription for rpmH and dnaA genes; and three highly conserved 9 bp sequences constituting the DNA-binding motif for the DnaA protein (Salazar et al., 1996 ). To understand how the upstream dnaA region regulates the replication of oriC plasmids of M. smegmatis, a large number of derivatives of pOS239 (a non-replicative plasmid that contains the oriC region) were constructed. The two recognized motifs, PdnaA and/or DnaA boxes, were modified or deleted. The incompatibility of the chromosome previously observed with pOS239 was eliminated when the PdnaA or all the upstream dnaA region was removed (Fig. 1). These results strongly suggested that it is the rpmH–dnaA promoter region, and not the DnaA boxes, which exerts the negative effect on replication.

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 dnaA–GFP 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 {sigma}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 medium–high-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 1·5–3·5 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.


   ACKNOWLEDGEMENTS
 
I thank Dr Ramakrishnan for the plasmid pFVP27 with gfp, and J. Rivas for photographic work and A. Sánchez for technical support. I am grateful to Y. Casart and P. Taylor for their critical reading of the manuscript. This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Tecnológicas Venezuela (S1–97000023) and The International Centre for Genetic Engineering and Biotechnology (CRP/VEN9702).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Asai, T., Chen, C. P., Nagata, T., Takanami, M. & Imai, M. (1992). Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication. Mol Gen Genet 231, 169-178.[Medline]

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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.[Abstract/Free Full Text]

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Received 13 March 2000; revised 25 May 2000; accepted 13 June 2000.