A novel type of DNA curvature present in a Clostridium perfringens ferredoxin gene: characterization and role in gene expression

Masato Kaji1, Osamu Matsushita2, Eiji Tamai2, Shigeru Miyata2, Yuki Taniguchi2, Seiko Shimamoto2, Seiichi Katayama3, Shushi Morita1 and Akinobu Okabe2

1 Department of Hospital Pharmacy, Kagawa Medical University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan
2 Department of Microbiology, Kagawa Medical University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan
3 Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1, Ridai-cho, Okayama 700-0005, Japan

Correspondence
Akinobu Okabe
microbio{at}kms.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This study has revealed that a Clostridium perfringens ferredoxin gene (per-fdx) possesses a novel type of DNA curvature, which is formed by five phased A-tracts extending from upstream to downstream of the -35 region. The three A-tracts upstream of the promoter and the two within the promoter are located at the positions corresponding to A-tracts present in a C. perfringens phospholipase C gene (plc) and a Clostridium pasteurianum ferredoxin gene (pas-fdx), respectively. DNA fragments of the per-fdx, pas-fdx and plc genes (nucleotide positions -69 to +1 relative to the transcription initiation site) were fused to a chloramphenicol acetyltransferase reporter gene on a plasmid, pPSV, and their in vivo promoter activities were examined by assaying the chloramphenicol acetyltransferase activity of each C. perfringens transformant. Comparison of the three constructs showed that the order of promoter activity is, in descending order, per-fdx, pas-fdx and plc. Deletion of the three upstream A-tracts of the per-fdx gene drastically decreased the promoter activity, as demonstrated previously for the plc promoter. Substitution of the most downstream A-tract decreased the promoter activities of the per-fdx and pas-fdx genes. These results indicate that not only the phased A-tracts upstream of the promoter but also those within the promoter stimulate the promoter activity, and suggest that the high activity of the per-fdx promoter is due to the combined effects of these two types of A-tracts.


Abbreviations: CAT, chloramphenicol acetyltransferase; Fdx, clostridial 2[4Fe–4S] ferredoxin; pas-fdx, C. pasteurianum ferredoxin gene; per-fdx, C. perfringens ferredoxin gene

The GenBank accession numbers for the C. perfringens and C. pasteurianum ferredoxin gene sequences discussed in this article are AP003194 and M11214, respectively.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ferredoxins, flavodoxins and rubredoxins are low-molecular-mass electron carrier proteins that function as electron acceptors for the numerous oxidation–reduction reactions carried out by members of the genus Clostridium. Clostridial 2[4Fe–4S] ferredoxin (also known as [8Fe–8S] ferredoxin) was the first isolated iron–sulfur protein (Mortenson et al., 1962). This type of ferredoxin (hereinafter abbreviated as Fdx) is present ubiquitously in many other bacteria and has been well characterized both structurally and biochemically (Meyer, 2000). It shuttles electrons between many important redox enzymes: for example, it can be reduced by pyruvate-Fdx oxidoreductase (Lovenberg et al., 1963) and NAD(P)H-Fdx oxidoreductase (Saint-Amans et al., 2001), and then reoxidized by hydrogenase, which disposes of the cellular excess of reducing equivalents by producing hydrogen (Moulis & Davasse, 1995). Clostridia grown in iron-rich media produce yields of Fdx comprising up to 2 % of the total cellular protein (Rabinowitz, 1972; Marczak et al., 1985). Considering that Fdx is a low-molecular-mass (~6000 Da) protein and that it is encoded chromosomally by a single-copy gene (fdx) (Graves et al., 1985; Graves & Rabinowitz, 1986), clostridial fdx genes appear to be transcribed at high levels. Furthermore, Fdx is replaced by flavodoxin under iron-limited conditions (Knight & Hardy, 1966; Ragsdale & Ljungdahl, 1984). Despite these characteristic features of the fdx genes, neither the transcriptional activity nor the regulation of these genes has been well characterized.

Clostridium perfringens is a commensal bacterium that causes a variety of diseases ranging from a mild food-borne diarrhoeal disease to fulminant and fatal infections such as dysentery and enterotoxaemia in animals and gas gangrene in humans (Hatheway, 1990; Songer, 1996). This organism exhibits volatile fermentation and grows very rapidly, as its doubling time is less than 10 min under optimal conditions (Morris, 1991). Since Fdx plays a central role in clostridial fermentation, the productivity of Fdx may also be associated with the proliferation of clostridia and their pathogenicity. To characterize the expression of Fdx in C. perfringens, we have cloned the C. perfringens ferredoxin gene (per-fdx) based on the nucleotide sequence deposited in the GenBank database (Shimizu et al., 2002). Inspection of the per-fdx sequence revealed that it possesses five phased A-tracts located from nucleotide positions -63 to -18 relative to the putative transcription initiation site.

Previously, we demonstrated that three phased A-tracts located immediately upstream of a promoter of the C. perfringens phospholipase C gene (plc) form an intrinsic DNA curvature (Toyonaga et al., 1992; Matsushita, C. et al., 1996), and that they stimulate plc promoter activity through interaction with the {alpha}-subunit C-terminal domain of RNA polymerase in a low-temperature-dependent manner (Katayama et al., 1999, 2001). The five phased A-tracts of the per-fdx gene, which we describe here for the first time, consist of three upstream A-tracts located at almost the same positions as the ones in the plc gene, and two downstream A-tracts located within the promoter. We also found two phased A-tracts corresponding to the latter ones in the Clostridium pasteurianum ferredoxin gene (pas-fdx). Promoters of prokaryotes, especially those of mesophilic bacteria, are, in general, preceded by a DNA curvature (Gabrielian et al., 1999–2000; Bolshoy & Nevo, 2000; Pedersen et al., 2000), and promoter upstream phased A-tracts have been well documented as a paradigm illustrating the role of the promoter upstream DNA curvature (Pérez-Martín et al., 1994; Ohyama, 2001). However, phased A-tracts within the promoter region have not been reported. This may or may not represent a special element affecting the promoter architecture and function.

The present study was aimed at characterizing the five phased A-tracts of per-fdx and the two phased A-tracts of pas-fdx, and defining the roles of these A-tracts in gene expression. We constructed derivatives of a chloramphenicol acetyltransferase (CAT) reporter plasmid, pPSV (Matsushita, C. et al., 1994), of which the promoterless CAT gene (catP) was transcriptionally fused to the region from -69 to +1 of per-fdx and its deletion or substitution derivatives. By comparing the CAT activities of transformants carrying the deletion or substitution constructs, we examined the role of the phased A-tracts in promoter activity. We also compared the promoter activities of the CAT reporter constructs, in which catP was transcriptionally fused to the same regions of the per-fdx, pas-fdx and plc genes. The results presented here indicate that the promoter activity is stimulated by the two phased A-tracts within the promoter, but that it is most strongly stimulated by the five phased A-tracts. We also discuss a possible mechanism underlying the stimulation of promoter activity by these two types of phased A-tracts.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmids, bacterial strains and culture conditions.
pPSV, an Escherichia coliC. perfringens shuttle vector containing a promoterless catP gene (Matsushita, C. et al., 1994), was used as the CAT reporter plasmid in this study. pT7Blue T-vector (Novagen) was used to clone the per-fdx gene. The bacterial strains used in this study were C. perfringens 13 (Mahony & Moore, 1976), C. pasteurianum JCM 1408T (ATCC 6013T) and E. coli NovaBlue (Novagen). C. perfringens was grown anaerobically in tryptone/yeast extract/glucose (TYG) broth at 37 °C as described previously (Kaji et al., 1999). C. perfringens transformants were grown in TYG broth containing 20 µg erythromycin ml-1. E. coli transformants were grown on Luria–Bertani agar plates supplemented with 100 µg erythromycin ml-1 or 100 µg ampicillin ml-1.

Gene cloning and plasmid construction.
The 660 bp fragment extending from nucleotide positions -343 to +317 relative to the transcription initiation site of per-fdx was PCR-amplified using chromosomal DNA from C. perfringens 13 as template. Oligonucleotides complementary to the 5' and 3' ends of the fragment were used as primers (Table 1). The PCR product was cloned into pT7Blue T-vector. The resulting plasmid, pFDX, was transformed into E. coli NovaBlue. pFDX DNA and chromosomal DNA from C. pasteurianum JCM 1408T and C. perfringens 13 were used as templates for PCR amplification of the per-fdx, pas-fdx and plc gene fragments, respectively. The DNA fragments spanning nucleotide positions -69 to +1 of the per-fdx, pas-fdx and plc genes were PCR-amplified using forward and reverse primers, which were complementary to the relevant positions, and contained BamHI and KpnI overhangs at their 5' ends, respectively. (Table 1). DNA fragments with deletions or nucleotide substitutions in the phased A-tracts of the per-fdx and pas-fdx genes were generated by PCR amplification using appropriate primers (Table 1). The PCR products were digested with restriction enzymes and then ligated into the multiple cloning site of pT7Blue T-vector. The BamHI–KpnI fragments were ligated into the multiple cloning site of pPSV for fusion to the catP gene. The nucleotide sequences of the insert DNA were confirmed to be identical with those deposited in the GenBank database: the accession numbers for the per-fdx, pas-fdx and plc genes are AP003194, M11214 and D32127, respectively.


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Table 1. Primers used to amplify DNA fragments

 
DNA manipulations and sequencing.
Restriction endonucleases, the Klenow enzyme and a DNA ligation kit were purchased from Takara Shuzo (Kyoto, Japan). All recombinant DNA procedures were carried out as described by Sambrook et al. (1989). Nucleotide sequencing was performed as described previously (Matsushita, O. et al., 1998).

Analysis of curved DNA.
The presence of DNA curvature and the degree of DNA bending were predicted by means of in silico analysis with the CURVATURE program (Shpigelman et al., 1993). The bend centre of the five phased A-tracts in the per-fdx gene was determined experimentally as follows. Eleven different 160 or 161 bp DNA fragments were PCR-amplified using pFDX DNA and the synthetic oligonucleotides listed in Table 1 as the template DNA and sets of primers, respectively. The gel-mobility assay for determination of the bend centre was performed as described previously (Matsushita, C. et al., 1996). Briefly, DNA fragments were electrophoresed on a 10 % polyacrylamide gel at 6 V cm-1, the temperature being kept constant at 4±0·5 or 55±0·5 °C. A 100 bp DNA ladder (New England Biolabs) was used as the non-curved molecular mass marker. The gel migration anomaly is presented in terms of RL, which is defined as the ratio of the apparent to the true fragment length.

Northern blot and primer extension analyses of the per-fdx gene.
Preparation of total RNA from C. perfringens cells, Northern blot analysis and primer extension were carried out as described previously (Fujinaga et al., 1999). In brief, C. perfringens was grown at 37 °C to the mid-exponential phase of growth (OD600=0·8), then total RNA was prepared by the SDS/phenol method. For Northern blot analysis, 2 µg of RNA were hybridized at 55 °C with a DNA probe (fragment 11, Table 1), which had been PCR-amplified and labelled with digoxigenin-11-dUTP (Roche Diagnostics). Primer extension analysis was performed for determination of the transcriptional initiation site of the per-fdx gene. A 30 nt primer, which was complementary to the sequence from nucleotide positions +78 to +107 of the per-fdx gene, was 5' end-labelled with [{gamma}-32P]dATP [4·5 kCi mmol-1 (166·5 TBq mmol-1); ICN Biochemicals] and then hybridized with total RNA. The hybrids were extended with reverse transcriptase (Superscript RT; Life Technologies) and the extension products were electrophoresed on a sequencing gel.

CAT assay for promoter strength involving the CAT reporter gene.
C. perfringens cells were grown in 100 ml of TYG broth containing 20 µg erythromycin ml-1. Portions (30 ml) of the cultures were centrifuged at 6000 g at 4 °C for 10 min, when the cultures reached an OD600 value of 0·8. The cell pellets were washed once with 50 mM Tris/HCl (pH 7·8) containing 30 µM DTT, resuspended in 3 ml of the same buffer and then disrupted with a French Pressure cell at 20 000 p.s.i. (137·8 MPa) at 4 °C. The supernatants obtained upon centrifugation at 15 000 g for 10 min were stored at -80 °C until being assayed for CAT activity. CAT activity was assayed spectrophotometrically as described by Shaw (1975). Protein concentrations were determined using Bradford protein assay reagent (Bio-Rad) with BSA as the standard.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Characterization of the per-fdx gene
We cloned a region spanning nucleotide positions -343 to +317 of the per-fdx gene by amplifying the relevant region based on the sequence deposited in the genome sequence database. The transcriptional start site was identified by primer extension at a site 37 nt upstream of the translational initiation site (Figs 1a and 2). A per-fdx promoter was identified upstream of the transcription start site: the sequences of the -35 and -10 core elements match four bases of each consensus sequence (TTGACA and TATAAT), and the TG motif at nucleotide positions -14 and -15 in the extended -10 sequence is conserved (Fig. 1a). The existence of a putative rho-independent transcriptional terminator downstream of the coding region (Fig. 1a) and the detection of a 0·2 kb transcript on Northern blot analysis (data not shown) indicate that the per-fdx gene is a monocistron like the pas-fdx gene (Graves & Rabinowitz, 1986). Five phased A-tracts are present between nucleotide positions -63 and -18, which may be tentatively regarded as being divided into two groups: three A-tracts upstream of the promoter (nucleotide positions -63 to -39) and two within the promoter (nucleotide positions -32 to -18). Although promoter upstream phased A-tracts have been reported for many eukaryotic and prokaryotic promoters, phased A-tracts within the promoter region have not been reported so far. Two such novel phased A-tracts are also present in the pas-fdx gene. The locations of the phased A-tracts in the per-fdx, pas-fdx and plc genes are shown in the alignment in Fig. 1(b).



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Fig. 1. Nucleotide sequence of the per-fdx gene, and comparison of three different types of phased A-tracts. (a) Nucleotide sequence of the per-fdx gene. A putative rho-independent terminator and a stop codon are denoted by arrows and an asterisk, respectively. (b) Comparison of phased A-tracts between the per-fdx, pas-fdx and plc genes. Numbers represent nucleotide positions relative to the transcriptional start site. Phased A-tracts are dotted. The -35 and -10 promoter core hexamers and the TG motif are underlined.

 


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Fig. 2. Primer-extension mapping of the 5' end of the per-fdx mRNA. Sequencing ladders generated with oligonucleotides used for primer extension were loaded into lanes A, C, G and T, and the reaction mixture was loaded into lane R. The DNA sequence of the sense strand around the first nucleotide in the transcript (designated +1) is shown and the -10 sequence is boxed.

 
Characterization of the DNA curvature formed by the five phased A-tracts
We calculated the angle of the DNA curvature formed by the five phased A-tracts in the per-fdx gene and that formed by the two phased A-tracts in the pas-fdx gene using the CURVATURE program (Shpigelman et al., 1993), since curvatures predicted by computer analyses are fairly consistent with those determined experimentally from anomalous electrophoretic mobility. The bending angles of the phased A-tracts in the per-fdx and pas-fdx genes are predicted to be ~110° and ~20°, respectively (Fig. 3a, c). In addition to the DNA curvature formed by the five phased A-tracts, another DNA curvature, of which the angle is predicted to be ~35°, is present in a further upstream region (Fig. 3b). This corresponds to three phased A-tracts located at 67 nt upstream of the five A-tracts (Fig. 1a).



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Fig. 3. Predicted DNA paths of the per-fdx and pas-fdx genes. (a) The region from nucleotide positions -103 to +37 of the per-fdx gene. (b) The upstream region of the per-fdx gene extending from nucleotide positions -223 to -104. (c) The region from nucleotide positions -127 to +3 of the pas-fdx gene. DNA curvatures corresponding to the five promoter-proximal phased A-tracts of the per-fdx gene, the three promoter-distal phased A-tracts and the two phased A-tracts within the pas-fdx promoter are indicated by thick arrows, arrowheads and thin arrows, respectively. Adenines of phased A-tracts are circled. The predicted DNA path was calculated using the CURVATURE program (Shpigelman et al., 1993).

 
A DNA fragment in which the static DNA curvature is located in the centre shows maximum retardation as compared to fragments in which it is at the end, as shown by permutation gel assaying and theoretical investigation (Nair et al., 1994). Thus, to confirm the existence of promoter-proximal and promoter-distal curved DNAs, and also to map their centres, 11 different 160 or 161 bp DNA fragments, which were obtained by PCR amplification using 11 different sets of primers (Table 1), were examined for electrophoretic mobility. The length of each fragment was verified by the electrophoretic mobility at 55 °C (data not shown). When they were electrophoresed at 4 °C, some of the fragments showed anomalously slow mobility (Fig. 4). The RL value, the ratio of the molecular mass estimated from the mobility at 4 °C to that calculated from the nucleotide sequence, was plotted (data not shown). The finding that fragment 6 containing the five phased A-tracts at the centre gave the highest RL value clearly indicates that the bend centre is located at the centre of the five A-tracts (nucleotide position approx. -40). Fragment 2 containing the three phased A-tracts (nucleotide positions -155 to -130) at the centre showed only a shoulder peak on the RL value profile, suggesting these three phase A-tracts form a less prominent DNA curvature.



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Fig. 4. Analysis of the DNA curvature of the per-fdx gene and its upstream region by electrophoretic mobility. A 160 or 161 bp DNA fragment amplified by PCR using sets of primers (fragments 1–11, Table 1) was electrophoresed on a 10 % polyacrylamide gel at 4 °C as described in Methods. Lane M, 100 bp DNA ladder used as a molecular mass marker; lanes 1–11 correspond to the PCR-amplified DNA fragment numbers shown in Table 1.

 
Effects of deletions of the promoter upstream phased A-tracts on per-fdx promoter activity
The locations of the three promoter upstream A-tracts of the per-fdx gene are similar to those of the plc gene, suggesting that they could function in an analogous fashion to those in the plc gene. Furthermore, the two downstream A-tracts are located at positions similar to those within the pas-fdx promoter. Therefore, the five A-tracts of the per-fdx gene may be divided into the three upstream and two downstream A-tracts. To assess the possible effects of these elements on promoter activity, we attempted to compare the activities of various promoters with deleted or substituted phased A-tracts. For this purpose, the region between nucleotide positions -69 and +1 of the per-fdx gene and its derivatives was fused to the promoterless catP gene of the CAT reporter plasmid and then transformed into C. perfringens, of which the CAT activity has been shown to reflect the activity of a fused promoter well (Matsushita, C. et al., 1994; Bullifent et al., 1995). The CAT activity of C. perfringens carrying per-fdx : : catP, which has all the phased A-tracts, was compared with C. perfringens carrying per-fdx{Delta}1A : : catP, per-fdx{Delta}2A : : catP and per-fdx{Delta}3A : : catP, which lack one, two and three upstream A-tracts, respectively (Table 1). The deletion of the three A-tracts caused a drastic decrease in CAT activity (Table 2), suggesting that the three phased A-tracts of the per-fdx gene can stimulate the downstream promoter, as in the case of the three phased A-tracts of the plc gene (Katayama et al., 2001). Even deletion of only the most upstream A-tract had a drastic effect on CAT activity, the promoter activity being independent of the number of residual A-tracts. Therefore, the position of the bend centre seems to be critical for the stimulatory effect of the DNA curvature on promoter activity.


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Table 2. CAT activity of C. perfringens carrying pPSV with the catP gene fused to various promoters

C. perfringens cultures were harvested at an OD600 value of 0·8. CAT activity was determined for each cell lysate as described in Methods.

 
Effect of disruption of the most downstream A-tract on the promoter activity of the per-fdx gene
Using deletion or substitution derivatives of the two downstream phased A-tracts to assess their role is not reliable, since the fourth A-tract overlaps with the -35 region and the fifth one resides in the spacer, and such modification would affect their functions. Thus, we attempted to assess their function by replacing the most downstream A-tract with the sequence of the same region in the plc gene (AATTA). This replacement does not change the spacer length or the number of A+T, which may be involved in the function of the spacer, for example, strand separation upon transition from the closed to the open transcriptional initiation complex. The DNA fragment of the per-fdx gene extending from nucleotide positions -69 to +1 with such a replacement (Table 1, per-fdxR) was fused to the catP gene on pPSV. The CAT activity of the C. perfringens transformant with per-fdxR : : catP was much lower than that of the C. perfringens transformant with per-fdx : : catP (Table 2). This suggests a stimulatory effect of the most downstream A-tract on the promoter activity.

Comparison of the promoter activities of the per-fdx, pas-fdx and plc genes
Although the most downstream A-tract is required for the stimulation of the promoter activity by the five A-tracts, it is uncertain whether the two A-tracts within the promoter function as an element independent of the three promoter upstream A-tracts. To assess this, we compared the promoter activities of the pas-fdx gene and substitution derivatives of it, and also the promoter activities between the plc, pas-fdx and per-fdx genes, which have phased A-tracts at the promoter upstream site, within the promoter and at both sites, respectively. The CAT activity determined for C. perfringens carrying pas-fdxR : : catP, in which the downstream A-tracts of the pas-fdx gene had been substituted with AATTA, was much lower than that determined for C. perfringens carrying pas-fdx : : catP (Table 2), suggesting that the two A-tracts within the promoter per se can stimulate the promoter activity. Determination of the CAT activity of C. perfringens carrying pPSV derivatives, in which the same region (nucleotide positions -69 to +1) of the per-fdx, pas-fdx and plc genes was transcriptionally fused to the catP gene, showed that the promoter strength became lower: per-fdx>pas-fdx>plc (Table 2). We concluded that the high activity of the per-fdx promoter could be due to the combined effects of the two types of A-tracts (i.e. those upstream of and those within the promoter).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Curved DNA is implicated in many biologically important processes, such as transcription initiation (Ohyama, 2001), illegitimate recombination (Mazin et al., 1994), DNA replication (Ueguchi et al., 1994) and nucleosome positioning (Kiyama & Trifonov, 2002). To the authors' knowledge, all curved DNAs studied so far are located outside promoters, and this is the first study characterizing a DNA curvature extending from upstream to downstream of the -35 region. Besides this curved DNA, another curved DNA, of which the function remains unknown, lies far upstream (nucleotide positions -155 to -130) of the per-fdx gene. It should also be noted that the per-fdx promoter possesses TG at nucleotide positions -15 and -14, namely the TG motif of the extended -10 element, which is recognized by and in contact with {sigma}70 region 3.0 (formerly designated as 2.5) and thereby compensates for weak -35 region contacts (Barne et al., 1997).

The five phased A-tracts can be regarded as consisting of two groups, the three upstream and two downstream A-tracts, because the former A-tracts correspond to those of the plc gene, of which the function has been well defined (Katayama et al., 2001), and the latter ones correspond to those of the pas-fdx gene, of which the function is suggested in this study. The molecular mechanism underlying the activation by the upstream per-fdx A-tracts is likely to involve the binding affinity of {alpha}-subunit C-terminal domain to the DNA minor groove of the phased A-tracts, as has been proposed for promoter upstream A-tracts (Katayama et al., 2001; Yasuno et al., 2001). The latter A-tracts might stimulate the promoter activity by a different mechanism. According to the recent model based on the three-dimensional structure and DNase I-hypersensitive site of the RNA polymerase–promoter open complex, the contacts of the three specific regions of the sigma factor with the -35 and -10 core elements and the TG motif induce DNA bends at nucleotide positions -45, -35 and -25 (Murakami et al., 2002). The bend centre of the DNA curvature formed by the two phased A-tracts within the promoter should be located at around -25, and this pre-formed bend may facilitate the formation of the open complex. Taken together, we suggest that the high activity of the per-fdx promoter is due to the combined effects of the two types of A-tracts. Along with the elucidation of the molecular mechanism underlying the stimulatory effect of the five phased A-tracts, these A-tracts have been successfully applied to obtain large amounts of C. perfringens sialidase (A. Takamizawa, S. Miyata, O. Matsushita, M. Kaji, Y. Taniguchi, E. Tamai, S. Shimamoto & A. Okabe, unpublished data).

All the promoter fusion experiments described here were performed on the plasmid-encoded fdx gene, not on the chromosomal gene. To prove that the results obtained reflect the situation on the chromosome, the experiments should be performed using the chromosomal fdx gene. Primer extension analysis showed that the transcriptional start sites of three different promoters, per-fdx, per-fdx{Delta}3A and per-fdxR, are the same as that determined for the chromosomal fdx gene (data not shown). Therefore, at least the transcription initiation site is not affected by cloning into the plasmid or by deletions/substitutions of the phased A-tracts. The relative concentrations of Fdx and flavodoxin in C. pasteurianum (Knight & Hardy, 1966) and Clostridium formicaceticum (Ragsdale & Ljungdahl, 1984) change depending on the extracellular iron concentration, implying that fdx gene expression in these two organisms is linked to the iron regulation system. Curved DNA is preferentially bound by some DNA-binding proteins (Azam & Ishihama, 1999; Rimsky et al., 2001), which may contribute to the regulation of fdx gene expression. This study has focused solely on the stimulatory effect of the phased A-tracts. Further studies on the response of fdx gene expression to environmental signals, such as temperature, iron concentration and oxidative stress, and on the role of the phased A-tracts including those residing far upstream of the promoter in fdx gene regulation are necessary.


   ACKNOWLEDGEMENTS
 
We wish to thank Nicholas J. Halewood for his invaluable assistance in the preparation of this manuscript. This work was supported by a Grant-in-Aid from MEXT under Exploratory Research Plan 14657069.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 21 May 2003; revised 22 August 2003; accepted 28 August 2003.



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