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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank accession numbers for the C. perfringens and C. pasteurianum ferredoxin gene sequences discussed in this article are AP003194 and M11214, respectively.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-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., 19992000
; 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 BamHIKpnI 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.
|
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 [
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-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 polymerasepromoter 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-fdx3A 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barne, K. A., Bown, J. A., Busby, S. J. & Minchin, S. D. (1997). Region 2.5 of the Escherichia coli RNA polymerase 70 subunit is responsible for the recognition of the extended -10 motif at promoters. EMBO J 16, 40344040.
Bolshoy, A. & Nevo, E. (2000). Ecologic genomics of DNA: upstream bending in prokaryotic promoters. Genome Res 10, 11851193.
Bullifent, H. L., Moir, A. & Titball, R. W. (1995). The construction of a reporter system and use for the investigation of Clostridium perfringens gene expression. FEMS Microbiol Lett 131, 99105.[CrossRef][Medline]
Fujinaga, K., Taniguchi, Y., Sun, Y., Katayama, S., Minami, J., Matsushita, O. & Okabe, A. (1999). Analysis of genes involved in nitrate reduction in Clostridium perfringens. Microbiology 145, 33773387.
Gabrielian, A. E., Landsman, D. & Bolshoy, A. (19992000). Curved DNA in promoter sequences. In Silico Biol 1, 183196.
Graves, M. C. & Rabinowitz, J. C. (1986). In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms. J Biol Chem 261, 1140911415.
Graves, M. C., Mullenbach, G. T. & Rabinowitz, J. C. (1985). Cloning and nucleotide sequence determination of the Clostridium pasteurianum ferredoxin gene. Proc Natl Acad Sci U S A 82, 16531657.[Abstract]
Hatheway, C. L. (1990). Toxigenic clostridia. Clin Microbiol Rev 3, 6698.[Medline]
Kaji, M., Taniguchi, Y., Matsushita, O., Katayama, S., Miyata, S., Morita, S. & Okabe, A. (1999). The hydA gene encoding the H2-evolving hydrogenase of Clostridium perfringens: molecular characterization and expression of the gene. FEMS Microbiol Lett 181, 329336.[CrossRef][Medline]
Katayama, S., Matsushita, O., Jung, C.-M., Minami, J. & Okabe, A. (1999). Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature-dependent manner. EMBO J 18, 34423450.
Katayama, S., Matsushita, O., Tamai, E., Miyata, S. & Okabe, A. (2001). Phased A-tracts bind to the alpha subunit of RNA polymerase with increased affinity at low temperature. FEBS Lett 509, 235238.[CrossRef][Medline]
Kiyama, R. & Trifonov, E. N. (2002). What positions nucleosomes? A model. FEBS Lett 523, 711.[CrossRef][Medline]
Knight, E. J. & Hardy, R. W. (1966). Isolation and characteristics of flavodoxin from nitrogen-fixing Clostridium pasteurianum. J Biol Chem 241, 27522756.
Lovenberg, W., Buchanan, B. B. & Rabinowitz, J. C. (1963). Studies on the chemical nature of clostridial ferredoxin. J Biol Chem 238, 38993913.
Mahony, D. E. & Moore, T. J. (1976). Stable L-forms of Clostridium perfringens and their growth on glass surfaces. Can J Microbiol 22, 953959.[Medline]
Marczak, R., Ballongue, J., Petitdemange, H. & Gay, R. (1985). Differential levels of ferredoxin and rubredoxin in Clostridium acetobutylicum. Biochimie 67, 241248.[Medline]
Matsushita, C., Matsushita, O., Koyama, M. & Okabe, A. (1994). A Clostridium perfringens vector for the selection of promoters. Plasmid 31, 317319.[CrossRef][Medline]
Matsushita, C., Matsushita, O., Katayama, S., Minami, J., Takai, K. & Okabe, A. (1996). An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter. Microbiology 142, 25612566.[Abstract]
Matsushita, O., Jung, C.-M., Minami, J., Katayama, S., Nishi, N. & Okabe, A. (1998). A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. J Biol Chem 273, 36433648.
Mazin, A., Milot, E., Devoret, R. & Chartrand, P. (1994). KIN17, a mouse nuclear protein, binds to bent DNA fragments that are found at illegitimate recombination junctions in mammalian cells. Mol Gen Genet 244, 435438.[Medline]
Meyer, J. (2000). Clostridial ironsulphur proteins. J Mol Microbiol Biotechnol 2, 914.[Medline]
Morris, J. G. (1991). Characteristics of anaerobic metabolism. In Anaerobes in Human Disease, pp. 1637. Edited by B. I. Duerden & B. S. Drasar. London: Edward Arnold.
Mortenson, L. E., Valentine, R. C. & Camahan, J. E. (1962). An electron transport factor from Clostridium pasteurianum. Biochem Biophys Res Commun 7, 448452.[Medline]
Moulis, J.-M. & Davasse, V. (1995). Probing the role of electrostatic forces in the interaction of Clostridium pasteurianum ferredoxin with its redox partners. Biochemistry 34, 1678116788.[Medline]
Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O. & Darst, S. A. (2002). Structural basis of transcription initiation: an RNA polymerase holoenzymeDNA complex. Science 296, 12851290.
Nair, T. M., Madhusudan, K., Nagaraja, V., Kulkarni, B. D., Majumdar, H. K. & Singh, R. (1994). On the mobility behavior of a curved DNA fragment located in circular permutation. FEBS Lett 351, 321324.[CrossRef][Medline]
Ohyama, T. (2001). Intrinsic DNA bends: an organizer of local chromatin structure for transcription. Bioessays 23, 708715.[CrossRef][Medline]
Pedersen, A. G., Jensen, L. J., Brunak, S., Staerfeldt, H. H. & Ussery, D. W. (2000). A DNA structural atlas for Escherichia coli. J Mol Biol 299, 907930.[CrossRef][Medline]
Pérez-Martín, J., Rojo, F. & de Lorenzo, V. (1994). Promoters responsive to DNA bending: a common theme in prokaryotic gene expression. Microbiol Rev 58, 268290.[Medline]
Rabinowitz, J. (1972). Preparation and properties of clostridial ferredoxins. Methods Enzymol 24, 431446.[Medline]
Ragsdale, S. W. & Ljungdahl, L. G. (1984). Characterization of ferredoxin, flavodoxin, and rubredoxin from Clostridium formicoaceticum grown in media with high and low iron contents. J Bacteriol 157, 16.[Medline]
Rimsky, S., Zuber, F., Buckle, M. & Buc, H. (2001). A molecular mechanism for the repression of transcription by the H-NS protein. Mol Microbiol 42, 13111323.[CrossRef][Medline]
Saint-Amans, S., Girbal, L., Andrade, J., Ahrens, K. & Soucaille, P. (2001). Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures. J Bacteriol 183, 17481754.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shaw, W. V. (1975). Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol 43, 737755.[Medline]
Shimizu, T., Ohtani, K., Hirakawa, H. & 7 other authors (2002). Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci U S A 99, 9961001.
Shpigelman, E. S., Trifonov, E. N. & Bolshoy, A. (1993). CURVATURE: software for the analysis of curved DNA. Comput Appl Biosci 9, 435440.[Abstract]
Songer, J. G. (1996). Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 9, 216234.
Toyonaga, T., Matsushita, O., Katayama, S., Minami, J. & Okabe, A. (1992). Role of the upstream region containing an intrinsic DNA curvature in the negative regulation of the phospholipase C gene of Clostridium perfringens. Microbiol Immunol 36, 603613.[Medline]
Ueguchi, C., Kakeda, M., Yamada, H. & Mizuno, T. (1994). An analogue of the DnaJ molecular chaperone in Escherichia coli. Proc Natl Acad Sci U S A 91, 10541058.[Abstract]
Yasuno, K., Yamazaki, T., Tanaka, Y., Kodama, T. S., Matsugami, A., Katahira, M., Ishihama, A. & Kyogoku, Y. (2001). Interaction of the C-terminal domain of the E. coli RNA polymerase alpha subunit with the UP element: recognizing the backbone structure in the minor groove surface. J Mol Biol 306, 213225.[CrossRef][Medline]
Received 21 May 2003;
revised 22 August 2003;
accepted 28 August 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |