Cloning and Characterization of the Gene Encoding Clostridium butyricum rubredoxin

Anaerobe (2000) 6, 29±37 doi:10.1006/anae.1999.0315

MOLECUL AR BIOLOGY/GENETICS

Cloning and Characterization of the Gene Encoding Clostridium butyricum rubredoxin P. Ge¨rard1, B. Charpentier2, M. Young3, C. Branlant2 and H. Petitdemange1* 1

Laboratorie de biochimie des bacteÂries Gram‡, Domaine scientifique Victor Grignard, Universite Henri PoincareÂ, Faculte des sciences B.P.239 54506 Vandoeuvre-les-Nancy Cedex, France 2 Laboratorie de Maturation des ARN et Enzymologie MoleÂculaire UMR CNRS 7567 Universite Henri PoincareÂ, Faculte des sciences 54506 Vandoeuvre-les-Nancy Cedex, France 3 Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, U.K. (Received 21 June 1999; accepted in revised form 2 November 1999)

Using inverse PCR technique, a 831-bp DNA fragment containing the rubredoxin gene from Clostridium butyricum DSM 5431 was obtained. Sequencing of this fragment allowed us to deduce a 53-amino acid protein (molecular mass 5853 Da) with strong homology with other sequenced rubredoxins. Comparison of the sequences showed that the C. butyricum rubredoxin resembles most closely the rubredoxin of C. perfringens (73.6% of identity) whereas a higher degree of identity (73.1%) is found with the rubredoxin of the Gram-negative Desulfovibrio vulgaris Miyazaki strain than with other clostridial rubredoxins. No other truncated open reading frame were detected in this DNA segment. Expression of C. butyricum rubredoxin in Escherichia coli strain HB101 has been carried out by subcloning a 261-bp SspI±SspI fragment encompassing the rubredoxin gene. The latter gene was under the control of the lacZ promoter of pBluescript (SK)‡ leading to the production of 1.5 mg of pure protein/L of culture. Recombinant rubredoxin was purified and displayed the characteristic absorption spectrum of native rubredoxins. Transcript mapping of the rubredoxin gene revealed a unique potential transcriptional start site 92 nt upstream of the initiation codon but no sequence clearly similar to the consensus sequence of transcriptional promoter from Gram positive bacteria was detected. Following two potential stable stem-loop structures, 3'-end of the transcript was located about 190 nt downstream of the termination codon. These data strongly suggest that the rubredoxin gene is monocistronic in C. butyricum.

Key Words: Clostridium butyricum, rubredoxin, inverse PCR

Introduction Rubredoxins (Rd) are small non-haem iron proteins (6 kDa) which are used as cellular electron carriers. *Corresponding author. Tel: ‡33 3 83 91 2053. Fax: ‡33 3 83 91 2550. E-mail: [email protected]

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# 2000 Academic Press

The active site of Rd consists of a single iron atom that is coordinated to four cysteine sulfur atoms in a tetrahedral arrangement [1]. This protein has been discovered in many anaerobic bacteria including clostridia [2±4] and sulfate-reducing bacteria [5±7], and also in the aerobic bacteria Acinetobacter calcoaceticus [8] and Pseudomonas oleovorans [9]. Rubredoxin # 2000 Academic Press

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has been reported to participate in CO oxidation in Clostridium thermoaceticum and Acetobacterium woodi [10], whereas the larger Rd from Pseudomonas oleovorans is involved in alkane hydroxylation [11,12]. A possible involvement of Rd in an NAD(P)H dependent nitrate reduction was also suggested but not demonstrated in Clostridium perfringens [4]. Several NADH-rubredoxin-oxidoreductases have been isolated from different bacteria including species of Desulfovibrio [13], Peptostreptococcus [14], Pseudomonas [15] and Clostridium [16]. Recently, Rd was found to transfer electron to oxygen to form water in Desulfovibrio gigas with a rubredoxin-oxygenoxido-reductase (ROO), an homodimeric flavohaemo-protein containing two FAD and two distinct haem per monomer [17,18]. In this case, Rd and ROO are located in the same operon [19] whereas in Desulfovibrio vulgaris [20,21] and in Desulfoarculus baarsii [22], Rd is thought to be co-transcribed with desulfoferrodoxin, an iron±sulfur protein containing two mononuclear centers, which was found to act as an oxygen defence protein [23]. We previously described the presence of rub gene in eleven strains of C. butyricum, using PCR methods [24]. This allowed us to determine partial deduced amino acid sequences of Rd in these species. Such ubiquity probably indicates a major function for Rd in C. butyricum. Frequently, genes for proteins involved in the same metabolic pathway are organized as operons. Genetic studies on the entire rub gene in C. butyricum should give information about the potential redox partner of Rd and hence the function of Rd in the clostridia where it is still unknown. Contrary to the desulfovibrio, the only previous study in the clostridia demonstrated that the rubredoxin gene was monocistronic in C. pasteurianum [25]. In this paper, we report the cloning, sequencing and transcript mapping of the rub gene from C. butyricum DSM 5431, as well as expression of the protein in E. coli.

Materials and Methods Bacteria, plasmids and growth conditions Clostridium butyricum DSM 5431 was provided by H. Biebl (Braunschweig, Germany). Escherichia coli DH5a [recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 DlacU169 (F80 lacZDM15)] and E. coli HB101 (F hsdR hsdM supE44 proA2 leuB6 rpsL20 recA13 lacY1 galK2 thi-1 ara-14) were used as the recipient strains for transformation with recombinant plasmids. pXcmKm12, a T-vector based on those constructed by Cha et al. [26] in which a kanamycin-resistance gene has been incorporated between the two XcmI

sites (N.P. Minton, personal communication), was used to clone DNA fragments amplified by PCR. pBluescript (SK)‡ was used as a vector to express the recombinant protein. Clostridium butyricum was grown as previously described [24]. Escherichia coli was grown at 378C in Luria Bertani medium [27] supplemented with ampicillin (100 mg/mL). DNA and RNA isolation Chromosomal DNA and total RNA were isolated with the Qiagen RNA/DNA Midi kit (Qiagen). Plasmid DNA isolation was performed by the alkaline lysis method [27]. The total amount of extracted nucleic acid was determined by measuring the absorbance at 260 nm. DNA and RNA purities were determined by using ratio of absorbances 280/260. Cloning of the gene Two synthetic oligonucleotides, based on the sequence of the rub gene of Clostridium pasteurianum [28] were designed as primers for PCR analysis with C. butyricum chromosomal DNA as the template. The PCR fragment obtained with C. butyricum genomic DNA, was labeled with a digoxygenin DNA labelling and detection kit (Boehringer-Mannheim). Clostridium butyricum genomic DNA was digested to completion with restriction enzymes and the resulting fragments separated by agarose gel electrophoresis. DNA transfer from agarose gels to a Hybond-N membrane (Amersham) was carried out as described by Southern [29]. Hybridization and detection techniques were performed according to the manufacturer's instructions. For inverse PCR, 10 mg genomic DNA digested with NdeI and size fractionated by electrophoresis through a 0.8% agarose gel. DNA fragments in the size range of 0.7 to 0.9 kbp were excised from the gel and purified (Qiaquick gel extraction kit, Qiagen). After dilution of the fragment to favour intramolecular ligation, 1 U of T4 DNA ligase (GIBCO BRL) was added. Ligation was carried out for 3 h at room temperature. DNA was then ethanol precipitated and the pellet was dissolved in 10 mL of sterile water. Two microlitres were used in 30 mL PCR reactions containing RD 1 (5' - ACC AGG AAC AAA ATT TGA AGA TA-3') and RD 2 (5' - ACT GCT GCC TCA TCA TAA ATA - 3') oligonucleotides (1 mM each), deoxynucleotide triphosphate (dNTPs) (125 mM each) and Goldstar DNA polymerase (Eurogentec, 1 U) in 75 mM Tris-HCl (pH 9.0)±20 mM (NH4)2SO4±0.01% Tween 20±2.5 mM MgCl2. Amplification was performed in a DNA thermal cycler (Perkin Elmer, Cetus) using the

The Rubredoxin Gene of Clostridium butyricum following conditions: 948C for 5 min, 948C for 30 s, 558C for 30 s, 728C for 1 min for 30 cycles. The two synthetic oligonucleotides used for inverse PCR, RD 1 and 2 were designed from the partial sequence of the C. butyricum rub gene [24]. The fragment amplified with RD 1 and 2 was then cloned in pXcmKm12 and sequenced. Two synthetic oligonucleotides RD 3 (5'- ATA ATA ATT ATT GAT TAA TAA TGA ATT ATT TGG-3') and RD 4 (5'-TTA ATA TTT CGT TTA GTC TCT CTT CTG TAA-3') were then designed from the inverse PCR product sequence and used as primers for a PCR reaction performed in the conditions described above, except that the template was undigested genomic DNA. The resulting fragment was cloned in plasmid pXcmKm12 to create plasmid pXRUB.

Expression in E. coli and purification of the recombinant rubredoxin pXRUB was digested with SspI which cuts 32 bp upstream of the initiation codon and 70 bp downstream from the stop codon of the rub gene. The restriction fragment containing the rub gene was cloned into the SmaI site of the vector pBluescript (SK)‡ and the resultant plasmid was designated pBSRUB. Escherichia coli HB101 cells were transformed with pBSRUB and cultured for 12 h at 378C in 70 ml LB medium containing ampicillin (100 mg/ mL). Six flasks containing 1 L of LB medium were inoculated with 10 ml of this culture and incubated for 30 h at 378C with shaking. Cells were harvested by centrifugation at 40006g for 30 min. The cell pellet was suspended in 10 mM Tris-HCl (pH 7.6)-1 mM phenylmethanesulphonyl fluoride and sonicated at 28C for 10 min using a 150 W ultrasonic disintegrator (MSE, UK). The supernatant was collected from the cell lysate by centrifugation at 12 0006g for 15 min at 48C. Recombinant rubredoxin was isolated from this pinkish supernatant following the procedure of Le Gall and Forget [30].

Protein analysis Purity of rubredoxin was established by sodium dodecyl sulfate polyacrylamide electrophoresis [31]. The acrylamide concentration was 10% (w/v) in the stacking gel and 20% (w/v) in the separating gel. Staining was performed with the Silver Stain kit from Bio-Rad. UV-visible absorption spectrum was recorded at room temperature on a Shimadzu UV160A spectrophotometer using 1-cm quartz cells.

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Sequence comparisons A multiple sequence alignment was carried out for the sequences of Rds from the SwissProt database and the Rd of C. butyricum. Rd sequences from the following microorganisms were used (accession numbers are given in parentheses): Peptococcus aerogenes (P00267), C. pasteurianum (P00268), Desulfovibrio vulgaris Hildenborough (P00269), D. gigas (P00270), Megasphera elsdenii (P00271), D. desulfuricans (P04170), Butyribacterium methylotrophicum (P14071), C. perfringens (P14072), Chlorobium thiosulfatophilum (P09947), D. vulgaris Miyazaki (P15412), C. thermosaccharolyticum (P19500), C. sticklandii (P23474), Pyrococcus furiosus (P24297), Heliobacillus mobilis (P56263). The identity values were determined from the number of matches between C. butyricum and other Rds, divided by the length of shorter sequence, excluding gaps. S1 mapping experiments To analyse the 5' end of the rub transcript, a 5'-labelled probe named RD A was synthetized by PCR using primers RD 3 and RD 5 (5'-ACC CAA TCG TCT GGG ATA TCT TC-3') (Figure 1) and labelled with [g-32P]ATP (3000 Ci/mmol) (NEN Life Science Products) using T4 polynucleotide kinase (Biolabs). This probe contained 394 nucleotides upstream of the rub coding region and the first 113 nucleotides of the rub coding region. RD B probe used to analyse 3' end of the transcript was generated with primers RD 4 and RD 6 (5'-GTG GAG TTC CAA AAT CAG ATC TTG AAA-3') (Figure 1) which contained a BglII site. This fragment contained the last 35 nucleotides of the coding region and the 233 nucleotides downstream, of the coding sequence. The fragment was digested with BglII and labelled at the 3' end of the BglII site by filling, using T7 DNA polymerase (Pharmacia), [a-32P] dATP (3000 Ci/mmol) and dNTP (5 mM of each dCTP, dGTP and dTPP, 1 mM of dATP) (BoehringerMannheim) in a 10 mM MgCl2 ± 50 mM NaCl ± 40 mM Tris-HCl (pH 7.5) buffer. Labelling was followed by a cold chase using a dNTP mix (0.125 mM each). The probes were purified in a 1.2% (w/v) low-melting point agarose gel (NuSieve GTG agarose, TEBU), phenol extracted, and radioactivity was quantified. 86104 cpm of the probes were precipitated with total RNA from C. butyricum and pellets were resuspended in 30 mL of 1 mM EDTA±0.4 M NaCl±80% (v/v) formamide±40 mM PIPES (N, N'-bis piperazine, 2 ethanesulfonic acid) (pH 6.4) buffer. For annealing, RNA and probe (RD A or RD B) were heated to 858C for 20 min and incubated overnight at 428C. A 300 ml of 0.28 M NaCl±4.5 mM ZnSO4±0.05 M CH3COO Na (pH 4.5) containing 0.2 U of Sl nuclease (Pharmacia) were added in the

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Figure 1. Locations of the oligonucleotides used for inverse PCR (RD 1 and 2), cloning of the gene (RD 3 and 4), primer extension (RD 5) and probe synthesis (RD 3, 4, 5 and 6). Labeled extremities of the probes used for S1 nuclease mapping (RD A and B) are indicated by an asterisk.

annealing mix and incubated for 30 min at 308C. Reaction was stopped by addition of 80 mL of 50 mM EDTA±4 M CH3COONH4± 50 mg/mL tRNA. The samples were ethanol precipitated and the pellets were dissolved in a formamide dye solution [27]. The samples were subjected to electrophoresis on a 7% polyacrylamide±8 M urea sequencing gel with a size marker, obtained by hydrolysis of pBR322 plasmid by HpaII enzyme (Biolabs), dephosphorylation by calf alkaline phosphatase (Boehringer-Mannheim) and labeling by T4 polynucleotide kinase (Biolabs) with [g-32P] ATP (3000 Ci/mmol) (NEN Life Science Products).

DNA sequencing analysis DNA sequencing of both strands was conducted by the dideoxy-chain termination method [32] using [a-35S]dATP (1250 Ci/mmol) (NEN Life Science Products) and the T7 sequencing kit (Pharmacia LKB) with M13 reverse and universal primers. Database searches were performed with BLAST [33]. Nucleotide sequence accession number The sequence data reported here (Figure 2) have been deposited in the EMBL nucleotide sequence database under accession no. Y11875.

Primer extension analysis A synthetic oligonucleotide RD 5, complementary to nucleotide positions 91 to 113, was 5' end-labelled with [g-32 P]ATP (3000 Ci/mmol) (Amersham) and T4 polynucleotide kinase (Biolabs) and used as the primer for primer extension reactions. For annealing, 20 mg of total RNA and 20 ng of 5'-end labeled primer were heated to 658C for 10 min in 10 mL of 50 mM TrisHCl (pH 8.3)±40 mM KCl±6 mM MgCl2 and then cooled slowly to room temperature (10 min), cDNA synthesis was performed in the same solution for 30 min at 458C in the presence of the non-radioactive deoxyribonucleotide triphosphates (dNTPs) at a concentration of 225 mM each and 1 U of avian myeloblastosis virus reverse transcriptase (Life Science). The primer-extended fragment was ethanol precipitated and then dissolved in a formamide dye solution [27]. The samples were electrophoresed on a 6% polyacrylmide±8 M urea sequencing gel with dideoxy sequencing reaction products obtained with the same primer [32].

Results and Discussion Although several clostridial rubredoxins have been characterized and sequenced at the protein level, C. pasteurianum rub gene was the only one to be characterized [28]. Using synthetic oligonucleotides designed from the nucleotide sequence of rub gene of C. pasteurianum [28], we previously detected rub gene of C. butyricum by PCR [24]. We employed a digoxygenin-labelled PCR product, obtained with the same oligonucleotides, as a probe for Southern blot hybridization with C. butyricum DSM 5431 genomic DNA (Materials and Methods). For all the endonucleases tested, a single genomic DNA fragment hybridized with the probe. They had the following sizes with respect to the nuclease used: 6.7 kbp for BglII; 10.5 kbp for EcoRI; 7.5 kbp for HindIII; 0.8 kbp for NdeI; 17.9 kbp for ScaI; 6.7 kbp for SpeI; 2.6 kbp for BglII/EcoRI; 3.5 kbp for EcoRI/HindIII and 6.7 kpb for BglII/HindIII (data not shown). These

The Rubredoxin Gene of Clostridium butyricum

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Figure 2. Nucleotide sequence of the 831 bp fragment of C. butyricum DSM 5431 containing the rubredoxin ORF. The gene has been translated with the one-letter amino acid code with the symbols below the first nucleotide of the corresponding codon. The putative ribosome binding site is underlined and inverted repeats are indicated by open arrows representing the length and orientation of the stem. The translation stop signal is marked by an asterisk below the codon. The transcription start site identified by primer extension analysis is indicated (‡1).

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data strongly suggested that a single copy of the rub gene is present in the genome of C. butyricum. To acquire information on the nucleotide sequences that border the rubredoxin coding region, inverse PCR [34] was undertaken (Materials and Methods). As this requires a step of internal circularization of the DNA segment to be amplified, the small (0.8 kbp) NdeI fragment was used for a better efficiency of the intramolecular ligation. A PCR fragment of the expected size was obtained and cloned into plasmid pXcmKm12 (Materials and Methods). The nucleotide sequence of the cloned PCR product was determined on both strands and aligned with the established partial sequence of the rub gene [24]. Based on this alignment a nucleotide sequence of a 831bp fragment encompassing the entire rub open reading frame (ORF) was reconstructed (Figure 2). No other truncated ORF was detected in this DNA segment. As for other clostridial genes, the GC content is much higher in the coding region (36%) than in the intergenic region (20%) [35]. A potential ribosome-binding site (GGAGG) is located 7 bp upstream of the ATG start codon (Figure 2). Its sequence and location match well those found for other clostridial genes [35]. Using a RNA folding program, the potential secondary structures were analysed. This revealed two potential stable stem-loop structures downstream of the rub ORF (Figure 2). Based on the deduced amino acid sequence, the C. butyricum rubredoxin is a 53-amino acid protein whose calculated molecular mass is 5853 Da. This protein contains all the conserved residues found in other rubredoxins including the four cysteine ligands (C6, C9, C39, C42) of the iron atom. It is notable that the C. butyricum Rd does not contain, as other clostridial Rds, a glutamine at position 48 but an aspartic acid which was only found at this position in Peptococcus aerogenes Rd. We next compared the established sequence with that of other rubredoxins using a binary alignment method. A high degree of sequence conservation is observed. It ranges from 73.6% of identity (for Clostridium perfringens) to 54.7% of identity (for Peptococcus aerogenes). Surprisingly, a higher degree of identity is found for the Gramnegative Desulfovibrio vulgaris Miyazaki strain (73.1%) than for C. pasteurianum (71.7%) whereas the Rd of the thermophilic Archaean Pyrococcus furiosus is more similar (69.8%) than the ones from C. thermosaccharolyticum (69.2%) and C. sticklandii (66.0%). Escherichia coli HB101 cells were transformed with plasmid pBSRUB (Materials and Methods), in which rub ORF is inserted in the same orientation as the lacZ promoter of the vector. The crude cell extract obtained with these cells showed the characteristic pink colour of rubredoxin indicating an efficient expression of the rub ORF in the recombinant cells. Analysis of this crude extract by SDS-PAGE allowed us to detect the

recombinant rubredoxin. As it was shown for the C. pasteurianum recombinant Rd, it co-migrated with the front. After purification following the procedure of LeGall and Forget [30], an homogenous recombinant Rd giving a single band on SDS-PAGE was obtained (data not shown). The yield of purified recombinant Rd was about 1.5 mg/l of LB medium. The UV/visible absorption spectrum of the purified recombinant Rd showed the characteristic features of native rubredoxins (data not shown). In particular, the three absorption maxima at 279, 375, and 491 nm were observed indicating that E. coli was able to form the iron±sulfur centre. The purified recombinant Rd was found to be reduced by NADH-Rd-oxidoreductase from Clostridium acetobutylicum ATCC 824 with similar kinetic data than native Rd from this other clostridial species (data not shown). The fact that no truncated ORF was found downstream or upstream of the rub ORF strongly suggested that the rub gene is monocistronic. To verify this assumption we used the S1 nuclease mapping technique to identify the 5' and 3' ends of the rub transcripts produced in C. butyricum. To analyse the 5'

Figure 3. S1 nuclease mapping of the rubredoxin transcripts. (A) Mapping of the 5' extremity. Lanes: 1, 507-nt RD A probe hybridized with C. butyricum total RNA (50 mg) and treated with S1 nuclease as described in Materials and Methods; 2, 507-nt RD A probe control. (B) Mapping of the 3' extremity. 252-nt RD B probe hybridized with 50 and 100 mg of C. butyricum total RNA (lanes 1 and 2), 30 mg tRNA (lane 3) and treated with nuclease S1; 252-nt RD B probe control (lane 4). The molecular weight marker (lane M) is plasmid pBR322 digested with the HpaII nuclease.

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Figure 4. Primer extension analysis of the rub gene. Primer extension was performed with oligonucleotide RD 5 using 20 mg total RNA from C. butyricum DSM 5431 as described in Materials and Methods. PE, product of primer extension; T, G, C and A, products of the four sequencing reactions obtained with the same oligonucleotide. The position of the 5' end as well as the putative 735 promoter element are indicated.

extremity, a 507-bp 5' end labelled probe (RD A) was synthesized by PCR using primers RD 3 and RD 5 (Figure 1). After S1 nuclease digestion, two protected fragments of about 200 nt were obtained (Figure 3A), showing that the 5' end of the transcripts is located about 90 nt upstream from the rub translational start codon. To identify the 3' end, a 252-bp PCR fragment was 3' end-labelled (Materials and Methods) and used as the probe (RD B) (Figure 1). A protected fragment

of about 210 nt was observed (Figure 3B), which localized the 3' end of the rub transcript about 190 nt downstream from the stop codon. This location corresponds to the region just downstream of the two potential stem-loop structures determined by the RNA folding program. These structures could represent an unusual transcription termination signal; alternatively they could represent a possible signal for RNA maturation.

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Next we defined more precisely the 5' terminal extremity of the rub transcripts by use of primer extension analysis. A single cDNA was obtained (Figure 4). The 3' end of this cDNA was located 92 bp upstream of the rub start codon. Results of S1 mapping and primer extension analysis were in good agreement and suggested that the second minor band obtained (Figure 3A) was due to uncomplete digestion of the S1 nuclease. Altogether these results indicated that the rub gene may be transcribed from a unique start site. However no sequence clearly similar to the consensus sequence of transcriptional promoter from the Gram positive bacteria (5'-TTGACA [17 bp] TATAAT-3') [35] was detected upstream of the identified potential start site. Only a potential 735 element was identified (5'-TTCTAA-3') (Figure 4). These results differ from those obtained in C. pasteurianum in which three possible transcription initiation sites were found for the rubredoxin gene [25]. Moreover these three potential start sites were all preceded by sequences showing the characteristic features of clostridial promoters. The low homology of the sequence upstream of the C. butyricum rub potential transcriptional start site with consensus promoter sequence could imply the existence of alternative transcriptional factors involved in the regulation of rub gene expression or could reflect the existence of a RNA maturation site at this position. We showed here, as it was found in C. pasteurianum [25], that the rub gene is transcribed alone in C. butyricum DSM 5431. The fact that these two clostridial rubredoxin genes are not part of an operon could imply that Rd has a different function from that deduced in the sulfate-reducing bacteria. In fact, in sulfate reducing bacteria the rub gene was found to be part of an operon with genes encoding rubredoxinoxygen oxidoredictase (ROO) (Desulfovibrio gigas) [19] or desulfoferrodoxin (Desulfovibrio vulgaris [20,21] and Desulfoarculus baarsii [22]). In D. gigas, a complete redox chain linking the oxidation of NADH to the reduction of dioxygen was characterized. In this chain, Rd transfers an electron between an NADH oxidase and ROO [17,19]. Concurrently, deletion of gene encoding desulfoferrodoxin, the potential redox partner of Rd in other sulfate reducing bacteria, increased oxygen sensitivity of D. vulgaris [23] indicating that Rd could be widespread involved in oxygen tolerance of these `strict anaerobes'. Characterization of rub gene and its transcript allow us to study its expression under a wide range of culture conditions. Using continuous cultures, in which bacteria grow at the set dilution rate with stable environmental growth conditions, quantification of the rub transcripts should permit determination of physiological conditions associated with high rub

gene expression and then further our understanding of the biological function of this clostridial Rd. Acknowledgements We are grateful to the MinisteÁre des Affaires EtrangeÁres and to the British Council for travel funds under the Alliance programme number 96004. We thank G. Raval for his helpful technical assistance.

References 1. Sieker L.C., Stenkamp R.E. and LeGall J. (1994) Rubredoxin in crystalline state. Methods Enzymol 243: 203±216 2. Lovenberg W. and Sobel B.E. (1965) Rubredoxin: a new electron transfer protein from Clostridium pasteurianum. Proc Natl Acad Sci USA 54: 193±199 3. Ragsdale S.W., Ljungdahl L.G. (1984) Characterization of ferrodoxin, flavodoxin, and rubredoxin from Clostridum formicoaceticum grown in media with high and low iron contents. J Bacteriol 157: 1±6 4. Seki S., Ikeda A. and Ishimoto M. (1988) Rubredoxin as an intermediary electron carrier for nitrate reduction by NAD(P)H in Clostridium perfringens. J Biochem 103: 583±584 5. Bruschi, M. (1976a) The amino acid sequence of rubredoxin from Desulfovibrio vulgaris. Biochim Biophys Acta 434: 4±17 6. Bruschi, M. (1976b) The amino acid sequence of rubredoxin from the sulfate reducing bacterium, Desulfovibrio gigas. Biochem Biophys Res Commun 70: 615±621 7. Shimizu F., Ogata M., Yagi T., Wakabayashi S. and Matsubara H. (1989) Amino acid sequence and function of rubredoxin from Desulfovibrio vulgaris Miyazaki. Biochimie 71: 1171±1177 8. Aurich H, Sorger D. and Asperger O. (1976) Isolierung und charakterisierung eines rubredoxins aus Acinetobacter calcoaceticus. Acta Biol Med Germ 35: 443±451 9. Peterson J.A., Kusunose M., Kusunose E. and Coon M.J. (1967) Enzymatic o-oxidation. II. Function of rubredoxin as the electron carrier in o-hydroxylation. J Biol Chem 242: 4334±4340 10. Ragsdale S.W., Ljungdahl L.G. and DerVartanian D.V. (1983) Isolation of carbon monoxide dehydrogenase from Acetobacterium woodi and comparison of its properties with those of the Clostridium thermoaceticum enzyme. J Bacteriol 155: 1224±1237 11. Eggink G., Lageveen R.G., Altenburg B. and Witholt B. (1987) Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli. J Biol Chem 262: 17712±17718 12. Eggink G., Engel H., Vriend G., Terpstra P. and Witholt B. (1990) Rubredoxin reductase of Pseudomonas oleovorans. Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints. J Mol Biol 212: 135±142 13. LeGall J. (1968) Purification partielle et eÂtude de la NAD: rubreÂdoxine oxydo-reÂductase de D. gigas. Ann Inst Pasteur (Paris) 114: 109±115 14. Mayhew S.G. and Peel J.L. (1966) Rubredoxin from Peptostreptococcus elsdenii. Biochem J 100: 80P 15. Ueda T., Lode E.T. and Coon M.J. (1972) Enzymatic o-oxidation: isolation of homogeneous reduced diphosphophyridine nucleotide-rubredoxin reductase. J Biol Chem 247: 2109±2116 16. Petitdemange H., Marczak R., Blusson H. and Gay R. (1979) Isolation and properties of reduced nicotinamide adenine dinucleotide-rubredoxin oxidoreductase of Clostridium acetobutylicum. Biochem Biophys Res Commun 91: 1258±1265 17. Chen L., Kiu M.Y., LeGall J., Fareleira P., Santos H. and Xavier A.V. (1993) Rubredoxin oxidase, a new flavo-hemo-protein, is the site of oxygen reduction to water by the ``strict anaerobe'' Desulfovibrio gigas. Biochem Biophys Res Commun 193: 100±105 18. Timkovich R., Burkhalter R.S., Xavier A.V., Chen L. and LeGall J. (1994) Iron uroporphyrin I and a heme c-derivative are prosthetic groups in Desulfovibrio gigas rubredoxin oxidase. Bioorg Chem 22: 284±293

The Rubredoxin Gene of Clostridium butyricum 19. Gomes C.M., Silva G., Oliveira S., LeGall J., Liu M.Y., Xavier A.V., Rodrigues-Pousadas C. and Teixeira M. (1997) Studies on the redox centers of the terminal oxidase from Desulfovibrio gigas and evidence for its interaction with rubredoxin. J Biol Chem 272: 22502±22508 20. Brumlik M.J., Leroy G., Bruschi M. and Voordouw G. (1989) Analysis of the transcriptional unit encoding the genes for rubredoxin (rub) and a putative rubredoxin oxidoreductase (rbo) in Desulfovibrio vulgaris Hildenborough. J Bacteriol 171: 4996±5004 21. Kitamura M., Koshino Y., Kamikawa Y., Kohno K., Kojima S., Miura K., Sagara T., Akutsu H., Kumagai I. and Nakaya T. (1997) Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F) - comparison of the primary structure of desulfoferrodoxin. Biochim Biophys Acta 1351: 239±247 22. Pianzzola M.J., Soubes M. and Touati D. (1996) Overproduction of the rbo gene product from Desulfovibrio species supresses all deleterious effects of lack of superoxide dismutase in Escherichia coli. J Bacteriol 178: 6736±6742 23. Voordouw J.K. and Voordouw G. (1998) Deletion of the rbo gene increases the oxygen sensitivity of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Appl Environ Microbiol 64: 2882±2887 24. GeÂrard P., Amine J., Raval G. and Petitdemange H. (1999) Distribution of the rubredoxin gene among the Clostridium butyricum species. Curr Microbiol 38: 264±267 25. Mathieu I. And Meyer J. (1993) Transcript mapping of the rubredoxin gene from Clostridium pasteurianum. FEMS Microbiol Lett 112: 223±228

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26. Cha J., Bishai W. and Chandrasegaran S. (1993) New vectors for direct cloning of PCR products. Gene 136: 369±370 27. Sambrook J., Fritsch E.F. and Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn, New York, Cold Spring Harbor Laboratory, Cold Spring Harbor 28. Mathieu I., Meyer J. and Moulis J.M. (1992) Cloning, sequencing and expression in Escherichia coli of the rubredoxin gene from Clostridium pasteurianum. Biochem J 285: 255±262 29. Southern E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503±517 30. LeGall J. and Forget N. (1978) Purification of electron-transfer components from sulfate reducing bacteria. Methods Enzymol 53: 613±634 31. Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680±685 32. Sanger F., Nicklen S. and Coulson A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463±5467 33. Altschul S.F., Gish W., Miller W., Myers E.W. and Lipman D.J. (1990) Basic local alignment search tool. J Mol Biol 215: 403±410 34. Ochman H., Gerber A.S. and Hartl D.L. (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120: 621±623 35. Young M., Minton N.P. and Staudenbauer W.L. (1989) Recent advances in the genetics of the clostridia. FEMS Microbiol Rev 63: 301±326