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

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Two Tandemly Arranged Ferredoxin Genes in the Hydrogenobacter Thermophilus Genome: Comparative Characterization of the Recombinant [4Fe–4S] Ferredoxins Biosci. Biotechnol. Biochem., 69 (6), 1172–1177, 2005 Two Tandemly Arranged Ferredoxin Genes in the Hydrogenobacter thermophilus Genome: 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 Aquifex 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 oxidoreductase (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 Escherichia coli 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 bacteria.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 mgmlÀ1 of ampicillin or 12.5 Aquifex and that the Hydrogenobacter-Aquifex branch is mgmlÀ1 of chloramphenicol was added to the medium. the deepest branching order in the Bacteria domain.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 mgmlÀ1 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 Â 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 Â 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 Â 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 Â 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 minÀ1. 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.
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