Biosci. Biotechnol. Biochem., 69 (6), 1172–1177, 2005

Two Tandemly Arranged Ferredoxin Genes in the Hydrogenobacter thermophilus : Comparative Characterization of the Recombinant [4Fe–4S] Ferredoxins

Takeshi IKEDA,1 Masahiro YAMAMOTO,1 Hiroyuki ARAI,1 y Daijiro OHMORI,2 Masaharu ISHII,1 and Yasuo IGARASHI1;

1Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Department of Chemistry, School of Medicine, Juntendo University, Inba, Chiba 270-1695, Japan

Received February 14, 2005; Accepted March 17, 2005

A thermophilic, obligately chemolithoautotrophic has been reported that Hydrogenobacter and hydrogen-oxidizing bacterium, Hydrogenobacter ther- assimilate carbon dioxide via the reductive tricarboxylic mophilus TK-6, assimilates carbon dioxide via the acid cycle.6,7) Two key enzymes of this cycle, pyruvate: reductive tricarboxylic acid cycle. A gene encoding a ferredoxin (POR) and 2-oxoglutarate: ferredoxin involved in this cycle as an electron donor ferredoxin oxidoreductase (OGOR), catalyze carbox- (HtFd1) was cloned and sequenced. Interestingly, an- ylation of acetyl-CoA and succinyl-CoA respectively, other ferredoxin gene (encoding HtFd2) was found in which should require strong reducing power.8) Hence tandem with the HtFd1 gene. These two ferredoxin characterization of low-potential electron carriers, such genes overlapped by four bp, and transcriptional analy- as ferredoxins, which can act as electron donors for sis revealed that they are co-transcribed as an operon. these carboxylation reactions, is of particular importance The deduced amino acid sequences of HtFd1 and HtFd2 for a deeper understanding of the cycle. were 42.9% identical and each contained four cysteine In our previous studies, a ferredoxin (designated residues that serve as probable ligands to an iron-sulfur HtFd1) was purified from H. thermophilus9) and shown cluster. Spectroscopic analyses of the purified recombi- to act as an electron donor for POR and OGOR from this nant ferredoxins heterologously expressed in Escherich- strain.10) In this study, we cloned a gene encoding HtFd1 ia coli indicated that each ferredoxin contains a single and found that it was followed by another ferredoxin [4Fe–4S]2þ=1þ cluster. gene. We also report transcriptional analysis and heterologous expression in of the two Key words: Hydrogenobacter thermophilus; Aquifex newly identified tandemly arranged ferredoxin genes aeolicus; ferredoxin; operon; reductive tri- from the H. thermophilus genome, and an initial com- carboxylic acid cycle parative characterization of the recombinant ferredoxins HtFd1 and HtFd2. Ferredoxins are small iron-sulfur proteins that func- tion as electron carriers in diverse metabolic pathways. Materials and Methods They contain one or two iron-sulfur clusters as a prosthetic group(s), which can be divided into the Bacterial strains and growth conditions. H. thermo- following types depending on the structure: [2Fe–2S], philus strain TK-6 (IAM 12695, DSM 6534) was [3Fe–4S], and [4Fe–4S]. Ferredoxins containing one or cultivated as previously described.11) E. coli JM109 two [4Fe–4S]/[3Fe–4S] clusters have commonly been was used as a host for derivatives of pUC19. E. coli found in .1) EPI300 was used as a host for derivatives of the Hydrogenobacter thermophilus TK-6 is a facultative- CopyControl pCC1FOS vector (Epicentre, Madison, ly aerobic, thermophilic, obligately chemolithoauto- WI). E. coli BL21 (DE3) was used as a host for trophic hydrogen-oxidizing bacterium.2,3) Phylogenetic derivatives of pET21c (Novagen, Madison, WI). E. coli analyses of 16S ribosomal DNA have shown that the strains were grown in Luria-Bertani medium at 37 C. genus Hydrogenobacter is closely related to the genus When necessary, 100 mgml1 of ampicillin or 12.5 Aquifex and that the Hydrogenobacter-Aquifex branch is mgml1 of chloramphenicol was added to the medium. the deepest branching order in the Bacteria .4,5) It

y To whom correspondence should be addressed. Fax: +81-3-5841-5272; E-mail: [email protected] Abbreviations: EPR, electron paramagnetic resonance; ICP/AES, inductively coupled plasma/atomic emission spectroscopy; OGOR, 2- oxoglutarate:ferredoxin oxidoreductase; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; POR, pyruvate:ferredoxin oxidoreductase; RT-PCR, reverse transcription-polymerase chain reaction Two [4Fe–4S] Ferredoxins from H. thermophilus 1173 N-Terminal amino acid sequencing. HtFd1 was Construction of expression plasmids for HtFd1 and purified from H. thermophilus by a combination of HtFd2. The fdx1 gene was amplified by PCR using the anion-exchange and gel filtration chromatography as chromosomal DNA of H. thermophilus as a template previously described.9) The proteins were blotted onto a and a pair of primers, 1F (50-GAGGTATGCCATATG- Sequi-Blot PVDF membrane (Bio-Rad, Hercules, CA) GCACTCAGGACCATG-30), which introduced an NdeI with a semidry electroblotting system (HorizBlot site (underlined) at the initiation codon, and 1R (50- AE-6677; Atto, Tokyo, Japan). N-Terminal amino acid GAGAATTCTCATACTTCCTCTGTTA-30), which in- sequences were determined on a Procise 491 protein troduced an EcoRI site (in italics) after the stop codon. sequencer (Applied Biosystems, Foster City, CA). The nucleotide sequence of the PCR product was confirmed on both strands. The fragment obtained was Cloning and DNA sequencing. Colony and Southern digested with NdeI and EcoRI, and ligated to NdeI– hybridizations were performed using Hybond-N nylon EcoRI digested pET21c. The resultant plasmid was membranes (Amersham Biosciences, Piscataway, NJ) designated pET-Fd1. Similarly, pET-Fd2 was construct- and a DIG DNA labeling and detection kit (Roche ed with another pair of primers, 2F (50-AACAGAGGA- Diagnostics, Basel, Switzerland). Nucleotide sequences CATATGAGGATACTCATTGAC-30), which intro- were determined on an ABI 377 DNA sequencer duced an NdeI site (underlined) at the initiation codon, (Applied Biosystems) using a BigDye terminator cycle and 2R (50-AAGAATTCCTTATCCAACTTCTTCC-30), sequence kit (Applied Biosystems) for dideoxy chain- which introduced an EcoRI site (in italics) after the stop termination reactions. codon. On the basis of the nucleotide sequence of a putative ferredoxin gene reported by Aoshima et al.,12) a pair of Heterologous expression and purification of HtFd1 primers, 1F and 1R (sequences given below), was and HtFd2. E. coli BL21 (DE3) harboring pET-Fd1 or synthesized to amplify the fdx1 gene (encoding HtFd1) pET-Fd2 was cultivated aerobically in Luria-Bertani by polymerase chain reaction (PCR) using the chromo- medium containing 100 mgml1 of ampicillin at 37 C. somal DNA of H. thermophilus as a template. The When the optical density at 600 nm reached 0.6, 0.5 mM resultant PCR fragment was used as a probe to screen of isopropyl--D-thiogalactopyranoside and 1 mM of a fosmid library of the H. thermophilus genome. The FeSO4 were added to the medium. After cultivation for library was constructed by insertion of randomly another 4 h, cells were harvested at 10;000 g for 8 min, digested chromosomal DNA fragments of about 40 kb and washed with 50 mM HEPES buffer (pH 8.0) con- in length into the Eco72I site of pCC1FOS. Fourteen taining 1 mM MgCl2. The cells were resuspended in the positive colonies were obtained out of 576 clones. A 1.5- same buffer, and disrupted by sonication using a Sonifier kb Sau3AI fragment of the plasmid extracted from one 250 (Branson, Danbury, CT) at 40 W at a 50% duty cycle of the positive colonies hybridized with the probe by for 10 min. Cell debris was removed by centrifugation at Southern hybridization, and was ligated to BamHI 27;000 g for 15 min. The supernatant was transferred digested pUC19. For further investigation of the down- into a vial. After the vial was sealed with a rubber septum stream region, the fdx2 gene (encoding HtFd2) was and an aluminum cap, the headspace gas was replaced amplified by PCR with another pair of primers, 2F and with argon. Subsequently the vial was heat-treated at 2R (sequences given below), and used as a probe. A 2.3- 80 C for 10 min. The heat-denatured proteins were kb SacI fragment of the plasmid hybridized with removed by centrifugation at 27;000 g for 15 min. amplified fdx2 by Southern hybridization, and was Chromatography was performed aerobically with an ligated to SacI digested pUC19. The nucleotide se- A¨ KTApurifier system (Amersham Biosciences) at room quence reported in this paper was determined by temperature. The purity of HtFd1 or HtFd2 was moni- sequencing both strands and was deposited in the tored by the ratio of absorbance at 390 nm to that at DDBJ nucleotide sequence database under accession 280 nm (A390=A280) and by SDS–polyacrylamide gel no. AB185162. electrophoresis (PAGE). The heat-treated extract was applied onto a Q Sepharose fast flow column (Amersham Reverse transcription-polymerase chain reaction (RT- Biosciences; 1 11 cm) equilibrated with 10 mM Tris– PCR). Total RNA was extracted from H. thermophilus HCl buffer (pH 8.0) containing 1 mM MgCl2. Proteins using Isogen (Nippon Gene, Tokyo, Japan). The RNA were eluted with a linear gradient from 0 to 0.5 M NaCl at sample was treated with RNase-free DNase (Nippon a flow rate of 1.5 ml min1. HtFd1 and HtFd2 were eluted Gene) and further purified using Isogen. Approximately at about 0.33 and 0.27 M NaCl respectively. The brown- 1 mg of RNA was used for reverse-transcription reac- colored fractions were concentrated using an Ultrafree- tions with AMV reverse transcriptase XL (Life Sciences, 15 centrifugal filter device (Biomax-5 membrane; Milli- St. Petersburg, FL) and an fdx2-specific primer (50-AA- pore, Billerica, MA) and loaded onto a Superdex 75 HR GATTACCTTATCCAACTTCT-30). PCR was subse- 10/30 column (Amersham Biosciences) equilibrated quently performed using transcribed cDNA as a tem- with 10 mM Tris–HCl buffer (pH 8.0) containing 1 mM plate. MgCl2 and 0.1 M NaCl. Proteins were eluted with the same buffer at a flow rate of 0.5 ml min1. 1174 T. IKEDA et al.

Fig. 1. Physical Maps of the Ferredoxin Genes in the H. thermophilus Genome (A) and the Corresponding Region in the A. aeolicus Genome (base numbers are indicated) (B). The arrow boxes indicate the size and orientation of the transcription of the genes. The ferredoxin genes are shown in black. The relative positions of the cloned Sau3AI (1,480 bp) and SacI (2,268 bp) fragments are indicated. The deduced amino acid sequence of orf1 was 56.6% identical (in 198 aa) to the sequence of A. aeolicus AAC07519 (aq 1625, hypothetical protein). The deduced amino acid sequence of orf2 was 40.6% identical (in 106 aa) to the sequence of A. aeolicus AAC07821 (aq 2066, hypothetical protein). The deduced amino acid sequence of orf3 was 58.7% identical (in 126 aa) to the sequence of A. aeolicus AAC07630 (aq 1797, hypothetical protein). E, EcoRV; H, HindIII; K, KpnI; P, PstI; Sc, SacI; Su, Sau3AI.

Protein assay. Protein concentrations were deter- mined using an RC DC protein assay kit (Bio-Rad) with bovine serum albumin as the standard.

Spectroscopic measurements. UV–visible spectra were measured on a DU 7400 spectrophotometer (Beckman, Fullerton, CA). Electron paramagnetic reso- nance (EPR) spectra were measured on a JES-FE3XG spectrometer equipped with an ES-CT470 cryostat system (JEOL, Tokyo, Japan) and a digital temperature indicator/controller model 9650 (Scientific Instruments, West Palm Beach, FL).13) Iron contents were determined by inductively coupled plasma/atomic emission spec- troscopy (ICP/AES) at a wavelength of 238.204 nm on a P-4000 spectrometer (Hitachi, Tokyo, Japan). Fig. 2. Transcriptional Analysis of the Ferredoxin Genes by RT- Results and Discussion PCR. A, Arrows indicate primers used for RT-PCR. Sequences of the primers are given in ‘‘Materials and Methods’’. B, PCR was Cloning and transcriptional analysis of two ferredox- performed using transcribed cDNA as a template. The pairs of in genes primers used are depicted above each lane. Plus and minus signs HtFd1 was partially purified from H. thermophilus indicate that the reverse transcription reaction was performed with as previously described,9) and its N-terminal amino and without reverse transcriptase respectively. Lane M, molecular mass markers (100 bp ladder). The gel contained 2% agarose. DNA acid sequence was redetermined and found to samples were stained with ethidium bromide. be ALRTMVDPDTCTSCELCYDRVPEVYKNRGDGI. (The previously reported partial sequence9) appears to be due to a contaminating non-ferredoxin protein; data not bp Sau3AI and 2,268-bp SacI fragments were subcloned shown.) This redetermined partial sequence contained from the H. thermophilus genomic library and se- three putative cysteine residues (underlined) in a typical quenced. Unexpectedly, another putative ferredoxin arrangement for ferredoxins containing one or two [4Fe– gene (204 bp, corresponding to 67 aa), namely fdx2 4S]/[3Fe–4S] clusters.14) The sequence was identical to (encoding HtFd2), was found in tandem with the fdx1 the deduced amino acid sequence of a putative ferre- gene with a four-bp overlap (Fig. 1A). In the region doxin gene (orf9; 219 bp, corresponding to 72 aa) downstream of fdx2, three other open reading frames located in the region downstream of the cognate (ORFs) of unknown function (orf1, orf2, and orf3) were succinyl-CoA synthetase genes reported recently by identified. Aoshima et al.12) Hence we refer to this ferredoxin gene Because fdx1 and fdx2 overlapped by four bp, we as fdx1 hereafter. In HtFd1, the first methionine in the suspected that they might be co-transcribed as an operon. deduced amino acid sequence of fdx1 was removed by RT-PCR analysis (Fig. 2) clearly indicated that this was post-translational modification. indeed the case. The fdx1 and fdx2 genes apparently To investigate the region downstream of fdx1, 1,480- comprise a dicistronic operon, because the fdx1 and Two [4Fe–4S] Ferredoxins from H. thermophilus 1175

Fig. 3. Multiple Sequence Alignment of the Deduced Amino Acid Sequences of the Ferredoxins of H. thermophilus and A. aeolicus. Asterisks indicate residues conserved in all four sequences, and dots indicate residues conserved in three of the four sequences. @ indicates residues involved in the formation of an iron-sulfur cluster. Aa, A. aeolicus; Ht, H. thermophilus. upstream sucD genes were separated by a 73-bp inter- genic region (Fig. 1A), in which a putative promoter sequence was found (data not shown). Similarly, the fdx2 and downstream orf1 genes were separated by 139 bp (Fig. 1A), and appeared not to be co-transcribed.

Comparison with the corresponding region in the A. aeolicus genome Considering the close phylogenetic relationship be- tween H. thermophilus and A. aeolicus, we subjected the deduced amino acid sequences of fdx1 and fdx2 to a homology search against the whole genome sequence of A. aeolicus. We found two unannotated ORFs (base nos. 1,139,479–1,139,709 and 1,139,768–1,140,010 re- Fig. 4. SDS–PAGE (18%) of Recombinant HtFd1 and HtFd2. spectively) between the sucD2 and aq 1625 genes in the Lane 1, purified HtFd1; Lane 2, purified HtFd2; Lane M, mo- lecular mass markers. Proteins were stained with Coomassie brilliant A. aeolicus genome (Fig. 1B). Each ORF contained one blue R-250. [4Fe–4S]/[3Fe–4S] cluster binding motif,15) and was located in the region downstream of the sucC2 and sucD2 genes encoding succinyl-CoA synthetase,16) as cells showed a brownish color, indicating that the reported here for the fdx1 and fdx2 genes (Fig. 1). These ferredoxins were expressed as holoproteins. The re- putative ferredoxin genes were probably overlooked and combinant ferredoxins were purified to homogeneity left unassigned in the genomic database because of their under aerobic conditions by a combination of heat- short lengths (231 and 243 bp respectively). Because treatment, anion-exchange chromatography, and gel four genes ( fdx1–4) were initially annotated as ferre- filtration chromatography (Fig. 4). The thermostability doxins in the A. aeolicus genome16) and one more gene of the ferredoxins simplified the purification procedure encoding an Isc-type [2Fe–2S] ferredoxin ( fdx5) has because the heat-treatment process precipitated most of recently been identified,17) we refer to the upstream the host proteins. Although the calculated molecular putative ferredoxin gene as fdx6 and to the downstream masses of the HtFd1 and HtFd2 apoproteins based on the one as fdx7, and to their gene products as AaFd6 and corresponding amino acid sequences were approximate- AaFd7 respectively. ly 7.8 and 7.5 kDa respectively, the purified proteins Figure 3 shows the multiple sequence alignment of migrated as 12–13- and 5–6-kDa bands respectively on the deduced amino acid sequences of HtFd1, HtFd2, SDS–PAGE (Fig. 4). These size discrepancies were AaFd6, and AaFd7. The four cysteine residues (marked probably due to their highly acidic nature (the calculated with @) that form a typical [4Fe–4S]/[3Fe–4S] cluster pI values of HtFd1 and HtFd2 were 3.80 and 4.17 binding motif15) were strictly conserved among them. respectively) and low molecular masses, as seen with The very high sequence identities between AaFd6 and other acidic, small ferredoxins.1,18) AaFd7 (81.0% identity) and between HtFd1 and HtFd2 Purified HtFd1 and HtFd2 were dark brown in the air- (42.9% identity) suggest that all of them are sister oxidized state. The UV–visible spectra of HtFd1 and proteins derived from a common ancestral gene, HtFd2 showed a broad absorption maximum around following a gene duplication event. Their similar gene 390 nm (Fig. 5), which is characteristic of ferredoxins arrangements (Fig. 1) suggest that these ferredoxins play containing one or two [4Fe–4S] clusters.15) The similar roles in the bacterial cells. A390=A280 ratios of HtFd1 and HtFd2 were in the range of 0.60–0.62 and 0.80–0.82 respectively. Upon dithio- Heterologous expression, purification, and character- nite reduction at pH 9.4, both HtFd1 and HtFd2 showed ization of HtFd1 and HtFd2 simple, rhombic EPR spectra (gz;y;x ¼ 2:08, 1.94, 1.92 Recombinant HtFd1 or HtFd2 was expressed in and 2.06, 1.92, 1.88 respectively), which are attributed E. coli under the control of the T7 promoter of the to the S ¼ 1=2 [4Fe–4S]1þ cluster15) (Fig. 6). From pET vector. The cell extract of the recombinant E. coli these spectroscopic results, the identification of cluster 1176 T. IKEDA et al. HtFd1 has been reported to act as an electron donor for carboxylation reactions catalyzed by POR and OGOR from this strain,10) but the physiological role of newly identified HtFd2 has not yet been clarified. HtFd2 is probably functionally related to HtFd1 and involved in the same redox pathway(s), because the two ferre- doxin genes are co-transcribed as an operon. Since POR and OGOR energetically favor electron transfer from pyruvate and 2-oxoglutarate to ferredoxins respectively, the reverse reactions, i.e., the reductive carboxylation reactions, would require a cytosolic pool of reduced ferredoxins for catalytic efficiency. Therefore one plausible role of HtFd2 is to sustain the reductive pool for the assimilation of carbon dioxide. Further studies are being planned to address these highly important issues and to clarify possible functional differences of the two ferredoxins reported in this study. Fig. 5. UV–Visible Spectra of Recombinant HtFd1 (A) and HtFd2 (B) in the Air-Oxidized State. Acknowledgment The protein concentrations were 20 mM each. We thank Dr. Toshio Iwasaki of Nippon Medical School for his help in the EPR measurements and for helpful discussions.

References

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