Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloningand Heterologous Expression in Thiocapsa roseopersicina�
Gergely Maroti,1† Yingkai Tong,1 Shibu Yooseph,1 Holly Baden-Tillson,1 Hamilton O. Smith,1Kornel L. Kovacs,2 Marvin Frazier,1 J. Craig Venter,1 and Qing Xu1*
J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850,1 and Department of Biotechnology,University of Szeged, Szeged, Hungary2
Received 10 March 2009/Accepted 18 July 2009
Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea thatencodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of aThiocapsa roseopersicina O2-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified bythe metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of anAlteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to expressour new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only twoaccessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF).These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonascounterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. Tofurther compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed comple-mentation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant.Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endopro-teolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate theenzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe]hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes fromenvironmental microbes in a culture-independent manner.
Hydrogen is a promising energy carrier for the future (10).Photosynthetic microbes such as cyanobacteria have attractedconsiderable attention, because they can split water photolyt-ically to produce H2. However, one major drawback of theprocesses is that their H2-evolving hydrogenases are extremelysensitive to O2, which is an inherent by-product of oxygenicphotosynthesis. Thus, transfer of O2-tolerant [NiFe] hydroge-nases into cyanobacteria might be one approach to overcomethis O2 sensitivity issue. A small number of O2-tolerant hydro-genases has been identified (9, 21, 47). However, they tend tofavor H2 uptake over evolution. Searching for novel O2-toler-ant [NiFe] hydrogenases from environmental microbes there-fore becomes an important part of the effort to construct suchbiophotolytic systems.
The oceans harbor an abundance of microorganisms with H2
production capability. Traditionally, new hydrogenases havebeen screened only from culturable organisms. However, sinceonly a few microbes can be cultured (14), many of them havenot been identified, and their functions remain unknown. Meta-genomics is a rapidly growing field, which allows us to obtaininformation about uncultured microbes and to understand thetrue diversity of microbes in their natural environments. Meta-genomics analysis provides a completely new approach foridentifying novel [NiFe] hydrogenases from the oceans in a
culture-independent manner. The Global Ocean Sampling(GOS) expedition has produced the largest metagenomic dataset to date, providing a rich catalog of proteins and proteinfamilies, including those enzymes involved in hydrogen metab-olism (45, 52, 56–58). Putative novel [NiFe] hydrogenase en-zymes that were identified from marine microbial met-agenomic data in these expeditions can be examined to findpotentially important new hydrogenases. Because source or-ganisms for metagenomic sequences are not typically known,these hydrogenases have to be heterologously expressed inculturable foreign hosts for protein and functional analyses.
Unlike most proteins, hydrogenases have a complex archi-tecture and must be assembled and matured through a multi-ple-step process (7, 11). Hydrogenases are divided into threedistinct groups based on their metal contents (54): Fe-S clus-ter-free hydrogenases (22, 23, 48), [FeFe] hydrogenases (1, 12,25), and [NiFe] hydrogenases (2, 3, 55). [NiFe] hydrogenasesare heterodimers composed of a large subunit and a smallsubunit, and their NiFe catalytic centers are located in thelarge subunits (2, 15, 19, 40). A whole set of accessory proteinsare required to properly assemble the catalytic centers (7). Theaccessory protein HypE first interacts with HypF to form aHypF-HypE complex, and the carbamyl group linked to HypFis then dehydrated by HypE in the presence of ATP to releasethe CN group that is transferred to iron through a HypC-HypD-HypE complex (6). The origin of the CO ligand that isalso bound to the iron is not clear, and possibly it comes fromformate, formyl-tetrahydrofolate, or acetate. The liganded Featom is inserted into the immature large subunit, in whichHypC proteins function as chaperones to facilitate the metal
* Corresponding author. Mailing address: J. Craig Venter Institute,9704 Medical Center Drive, Rockville, MD 20850. Phone: (301) 795-7248. Fax: (240) 268-2648. E-mail: [email protected].
† Present address: BayGen Institute, Bay Zoltan Foundation forApplied Research, H-6701 Szeged, Hungary.
insertion (5, 34, 36). Ni is delivered to the catalytic center bythe zinc-metalloenzyme HypA that interacts with HypB, anickel-binding and GTP-hydrolyzing protein. The final step inthe maturation process is endoproteolytic cleavage. Oncethe nickel is transferred to the active site, the endopepti-dase, such as HyaD or HynD, cleaves the C-terminal end ofthe large subunit (33, 43), which triggers a conformationalchange of the protein so that the Ni-Fe catalytic center canbe internalized.
Heterologous expression of functional [NiFe] hydrogenaseshas been demonstrated in several studies (4, 18, 31, 39, 44, 50),suggesting that it could be a feasible approach to express novelhydrogenases from the environment for functional analysis. Inthis study, we sought to prove the concept that metagenomi-cally derived environmental DNA can give rise to a functional[NiFe] hydrogenase through expression in a foreign host andthat novel [NiFe] hydrogenases from environmental microbescan be studied in a culture-independent manner. We clonedenvironmental DNA that harbors the genes of a putative novelhydrogenase that shows strong homology to a known O2-tol-erant hydrogenase, HynSL, from the phototrophic purple sul-fur bacterium Thiocapsa roseopersicina (21, 28, 41, 59). Weheterologously expressed the two structural genes (hyaA andhyaB) and two accessory genes (hupH and hyaD) of this novelenvironmental hydrogenase in T. roseopersicina, a foreign hostthat may already have the necessary machinery required toprocess the environmental hydrogenase since it carries thehomologous hydrogenase HynSL. We analyzed the new hydro-genase protein and its functions. In addition, we compared thematuration mechanisms between the two homolog hydroge-nases by performing complementation experiments.
MATERIALS AND METHODS
Bacterial strains, plasmids, growth conditions, and conjugation. Bacterialstrains and plasmids used in this study are listed in Table 1. Escherichia colistrains were grown in Luria-Bertani medium. T. roseopersicina strains weregrown under illumination in Pfennig’s mineral medium as described previously(11). For conjugation experiments, Pfennig’s mineral medium plates solidifiedwith Phytagel (7 g liter�1) (Sigma-Aldrich) and supplemented with sodiumacetate (2 g liter�1) was used. The GasPak gas generator (Becton Dickinson) wasused to anaerobically grow T. roseopersicina on plates. Conjugation experimentsto transfer expression vectors from E. coli to T. roseopersicina were performed aspreviously described (16). Antibiotics were used at the following concentrationsfor the bacteria: for E. coli, ampicillin (100 �g ml�1), kanamycin (25 �g ml�1),and streptomycin (25 �g ml�1); for T. roseopersicina, kanamycin (25 �g ml�1),streptomycin (5 �g ml�1), gentamicin (5 �g ml�1), and erythromycin (50 �gml�1).
Isolation of environmental DNAs. Impact filters (0.1 �m) containing environ-mental organisms were treated with lysozyme (2.5 mg/ml) in Tris-EDTA (TE)buffer (pH 8.0) for 1 h and then treated with proteinase K (200 �g/ml) for anadditional hour. After treatment with two freeze-thaw cycles, the samples werebrought to a final concentration of 1% sodium dodecyl sulfate, additional pro-teinase K (200 �g/ml) was added, and the samples were incubated at 55°C for 2 h.The reaction products were then extracted with aqueous phenol and phenol-chloroform. After centrifugation, the supernatant was precipitated with ethanoland dissolved in TE buffer. Finally, the DNA was further purified with cetyltri-methylammonium bromide (CTAB) to remove impurities that might inhibitfurther processing (37).
DNA library construction. DNA was randomly sheared by nebulization andwas then size selected by electrophoresis on a 1% low-melting-point agarose gel.The DNA was then cloned using BstXI adapters as described previously (51).The resulting library was transformed into E. coli by electroporation.
Library screening. The library was screened using a digoxigenin (DIG)-basednonradioactive method. DIG-11-dUTP (50 �M) (Roche) was used in the PCRmixtures to partially replace dTTP. A 1.2-kb DIG-labeled DNA probe was madein PCR using primers 5�-AAATGCATCACGTGATCATGC and 5�-TAGGCGTGCGGCGATGAACG with plasmid pSZAT5 as the template. After the probewas purified by Qiagen quick-spin columns, the labeled probe was used forcolony lift hybridization according to the manufacturer’s directions. The hybrid-ization blots were then subjected to immunological detection using anti-DIG
TABLE 1. Bacterial strains and plasmids used in this study
Bacterial strain or plasmid Relevant genotype or phenotype or description Referenceor source
StrainsThiocapsa roseopersicina
BBS Wild type 8GB112131 hynSL�::Sm hupSL�::Gm hoxH�::Em; Smr Gmr Emr 42GB2131 hupSL�::Gm hoxH�::Em; Gmr Emr 38GB21 BBS �(hupS-hupL)::�Gm; Gmr 38DYDG2 hupSL�::Gm �hynD; Gmr This workM539 hypF::Tn5Km in BBS; Kmr 16MGAmDHSL pAmDHSL in GB112131 This workDYDAmD pAmD in DYDG2 This work
Escherichia coliS17-1 (�pir) 294 (recA pro res mod) Tpr Smr (pRP4-2-Tc::Mu-Km::Tn7) �pir 24HB101 F� mcrB mrr hsdS20(rB
PlasmidspUC19 Cloning vector; Ampr InvitrogenpSSYK1 2,698-bp fragment of the Alteromonas hydrogenase gene locus in pBR322; Ampr This workpSZAT5 7,226-bp fragment of the Alteromonas hydrogenase gene locus in pBR322; Ampr This workpAmMG1 Alteromonas hyaD, hupH, hyaA, and hyaB genes (4,325 bp) in pUC19; Ampr This workpMHE6crtKm Broad-host-range vector; Kmr 17pAmDHSL-a Alteromonas hyaD and hupH genes and partial hyaA gene in pMHE6crtKm; Kmr This workpAmDHSL Alteromonas hyaD, hupH, hyaA, and hyaB genes in pMHE6crtKm; Kmr This workpAmD Alteromonas hyaD gene in pMHE6crtKm; Kmr This workpDYD In-frame-deleted hynD in pK18mobsacB; Kmr This work
antibody conjugated with alkaline phosphatase (Roche). Alkaline phosphataseactivity was detected by a chemiluminescence method using the CDP-Star de-tection kit (Roche).
Plasmid construction. The two structural genes and two accessory genes of themetagenomically identified [NiFe] hydrogenase were located in two separateclones, pSZAT5 (carrying hyaD, hupH, hyaA, and the partial hyaB gene) andpSSYK1 (carrying the complete hyaB gene). The genes were joined together intoa single cluster through the MluI site in hyaB and cloned in pUC19 (Fig. 1) Theresulting plasmid, pAmMG1, carries a 4,325-bp insert that possesses the hyaD,hupH, hyaA, and hyaB genes of Alteromonas in their original genomic arrange-ment (Fig. 1). This plasmid served as a template for creating expression con-structs pAmDHSL and pAmD in this study.
The expression construct pAmDHSL was created in two steps. In the first step,pAmMG1 was digested with AseI and BamHI to release an 1,810-bp DNAfragment that contains the hyaD and hupH genes and a partial hyaA gene (Fig.1). This fragment was further treated with Klenow DNA polymerase to produceblunt ends. The fragment was then ligated into the XbaI-digested/Klenow-filledpMHE6crtKm expression vector (Fig. 1) (17) to create a 7,678-bp construct,pAmDHSL-a. In the next step, pAmMG1 was digested with KpnI to release a2,540-bp DNA fragment containing the 3�-end region of the hyaA gene and theentire hyaB genes, and this 2,540-bp fragment was then ligated with KpnI-digested pAmDHSL-a to create the 10,218-bp construct pAmDHSL (Fig. 1). Forconstruction of pAmD, two primers 5�-TAATGTGAGTTAGCTCACTC and5�-CTCCCGGGGAGTTCTTCGGTCATCGTGC were used to amplify thehyaD gene from pAmMG1. The PCR product was ligated into the XbaI-digestedKlenow-filled pMHE6crtKm vector to create a 6,771-bp construct, pAmD.
To make in-frame deletions in the hynD gene of T. roseopersicina, the con-struct pDYD was created as follows. The upstream and downstream regions ofhynD were amplified by PCR using two pairs of primers, primers 5�-TCCGAGAACGATTTCGATCG and 5�-ACGGCGCTGGACCTTATGCC and primers 5�-CCGCAGTCGAGGCGGCGATT and 5�-ATATCGAGCACGATCACCTG. Theresulting 974-bp and 931-bp PCR fragments were ligated and inserted into thepK18mobsacB vector through PstI and SphI sites, creating pDYD. The aboveconstructs were examined by both PCR and DNA sequencing to confirm theiridentities.
In vitro hydrogenase activity assays. In vitro hydrogen uptake assays wereperformed spectrophotometrically using oxidized benzyl viologen (BV) (Sigma)as an artificial electron receptor. Reaction mixtures (2.0 ml) were composed of20-�l whole-cell extracts (10 mg of total protein ml�1) or 100-�l membranefractions (20 mg of total protein ml�1) of T. roseopersicina, 1.76 ml of potassiumphosphate buffer (pH 7.0) (20 mM), and 40 �l of oxidized BV (20 mM). Thereaction mixtures were placed into glass cuvettes (Allen Scientific Glass, Inc.),which were then sealed with rubber stoppers. After the rubber stopper waspierced with a needle, the cuvettes were flushed with argon for 10 min and thenwith 10% H2 for another 10 min. During the reaction, the decrease of H2 coupledto the BV reduction was detected at 600 nm by a spectrophotometer. Sampleswere incubated at 55°C during the whole measurement. An extinction coefficientof 7,400 M�1 cm�1 (32) was used to calculate hydrogen uptake activity.
In vitro hydrogen evolution assays were performed using reduced methylviologen (MV) (Sigma) as an artificial electron donor. Reaction mixtures (2.0ml) contained 20-�l whole-cell extracts (10 mg of total protein ml�1) or 100-�l
membrane fractions (20 mg of total protein ml�1) of T. roseopersicina, 1.76 ml ofpotassium phosphate buffer (pH 7.0) (20 mM), and 40 �l of oxidized MV (20mM). Sodium dithionite (Sigma) (final concentration, 2.5 mg/ml) was added toreduce MV and to initiate reactions. After the reaction mixtures were incubatedfor 1 h at 22°C, the reactions were terminated by trichloroacetic acid (TCA). Gassamples from the headspace were injected into a gas chromatograph (Varian) todetermine the amount of evolved H2.
In vivo hydrogen evolution assay. T. roseopersicina cells were anaerobicallygrown under argon in sealed 100-ml Hypo-Vial bottles (Pierce). At various timepoints during growth, samples were taken from the headspaces, and their hy-drogen content was measured by gas chromatography. The volume of the cellcultures (50 ml) and the ratio of the gas/liquid phases (1:1) were kept constantthrough all the experiments.
Protein techniques. Approximately 50 �g of total proteins was loaded ontoeach lane of a sodium dodecyl sulfate-polyacrylamide gel, and electrophoresiswas performed as described previously (46). Coomassie blue staining of proteinsin the gels were carried out by using SimplyBlue SafeStain reagent (Invitrogen).Western blotting was performed as previously described (46). Polyclonal rabbitantibodies were raised against T. roseopersicina HynL protein and used fordetection of both native T. roseopersicina and heterologously expressed Altero-monas [NiFe] hydrogenases with 1:10,000 dilution. Goat anti-rabbit immuno-globulin G serum conjugated to horseradish peroxidase (Amersham) was used assecondary antibody with 1:5,000 dilution.
RNA isolation and reverse transcription-PCR (RT-PCR). Total RNA wasisolated from T. roseopersicina cells as described previously (38). Isolated totalRNA was treated with RNase-free DNase I (Invitrogen) for 15 min prior toreverse transcription. cDNA was synthesized in a reaction mixture containingSuperScript III reverse transcriptase (Invitrogen) and a primer 5�-GACAAAGCTCTTCATTTGCG located in the middle of the Alteromonas hyaB gene. Theno-RT control reaction mixture contained all the components except SuperScriptIII reverse transcriptase. PCR was then carried out on the cDNA by using twohyaB-specific primers, 5�-GGGCATCTTCGAATAGAAGC and 5�-AAATGCATCACGTGATCATGC. PCR products were separated on agarose gels.
Partial purification of hydrogenases from T. roseopersicina. T. roseopersicinaculture (50 ml) was harvested by centrifugation at 5,000 g. The cells weresuspended in 1 ml of 20 mM potassium phosphate buffer (pH 7.0) and sonicatedfor 1 min on ice at a power level of 15 W with a mechanical amplitude of 50 �m.The sonicated cells were centrifuged at 5,000 g for 10 min to remove cell debrisand sulfur crystals. The supernatant was centrifuged at 100,000 g for 3 h toseparate the membrane fraction. The membrane pellet containing a mixture ofcytoplasmic and periplasmic membranes was washed twice with 1 ml of 20 mMpotassium phosphate buffer (pH 7.0) and then resuspended in 1 ml of the samebuffer. The 100,000 g supernatant was saved as the soluble fraction. Bothfractions were tested for hydrogenase activity. The protein concentrations weredetermined using the DC method (Bio-Rad) based on the Lowry method.
RESULTS
Cloning the genes of an environmental [NiFe] hydrogenasefrom the Sargasso Sea. In the metagenomic data collection
FIG. 1. Schematic representation of expression construct pAmDHSL. The hyaD-hupH-hyaA-hyaB locus in pAMDHSL was not drawn to scale.Restriction endonuclease sites used for construction of pAmMG1 and pAmDHSL are indicated (the restriction endonuclease site labeled with anasterisk is no longer active in pAmDHSL). Modular expression units are also labeled. Pcrt and PT7/lac represent the promoter of crtD gene fromT. roseopersicina and the T7 promoter lac operator fusion, respectively. RBS, ribosomal binding site; Term, transcription terminator; TAG,optional C-terminal Strep-tag II and FLAG tags.
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generated from the Sargasso Sea sampling project of the J.Craig Venter Institute (JCVI), three sequences with GenBankidentifier (GI) numbers 137178577, 142721499, and 142721500(45, 52, 56) showed 59 to 66% identity to T. roseopersicina[NiFe] hydrogenase HynSL, a hydrogenase that is notably re-sistant to oxygen and is remarkably active at high temperatures(21, 28, 41, 59). These three sequences appeared to encode thesame novel environmental [NiFe] hydrogenase, but they wereonly partial sequences (195 amino acids [aa] for GI 137178577,216 aa for GI 142721499, and 179 aa for GI 142721500) thatspanned different regions of the large and small subunits (Fig.2) (45, 52).
To further study this novel environmental [NiFe] hydroge-nase, we first sought to obtain the complete sequence. Onlypaired-end reads were obtained from plasmid clones in theSargasso Sea genomic DNA libraries (3). Consequently, mostof the cloned inserts were not completely sequenced. Wetraced back to one of the original clones (pSZAT5) that gen-erated the sequence of GI 142721499 and sequenced its entireinsert. The pSZAT5 clone contained a 7,226-bp genomic insertthat harbors a complete structural gene for the small subunit,hyaA, but an incomplete structural gene (1,104 bp) for thelarge subunit, hyaB (Fig. 2). To obtain the complete hyaB gene,we constructed a genomic DNA library in pBR322 from theoriginal DNA samples. After the library was screened with aDIG-labeled and hynL-specific probe, a clone carrying thecomplete hyaB gene, named pSSYK1, was identified. ThepSSYK1 clone carried a 2,670-bp insert that partially overlapswith the insert of the pSZAT5 clone (Fig. 2). The genomicsequence from these two clones were combined to provide a8,744-bp DNA sequence in the gene locus of the novel hydro-genase (Fig. 2). Analysis revealed two structural genes of thehydrogenase, hyaA (999 bp) and hyaB (1,878 bp) (Fig. 2).These may encode a 332-aa small subunit (HyaA) and a 625-aalarge subunit (HyaB), respectively. This hydrogenase shows60% identity to the large subunit (576 aa) and 64% identity tothe small subunit (369 aa) of T. roseopersicina hydrogenaseHynSL. Upstream of hyaA and hyaB, two hydrogenase acces-sory genes in the same orientation were identified, hupH (435-bp) and hyaD (554-bp) genes (Fig. 2), which could encode an[NiFe] hydrogenase expression protein, HupH (35), and anessential endopeptidase, HyaD, for maturing the hydrogenaselarge subunit (49), respectively. Four additional genes wereidentified further upstream of hyaA and hyaB (Fig. 2); these
genes include an incomplete gene for NADH oxidoreductase,hmp; a gene for a cytochrome protein, cyt; and two hypothet-ical genes, orf1 and orf2.
After we finished cloning the [NiFe] hydrogenase from theDNA samples from the Sargasso Sea, the JCVI SequencingCenter sequenced the entire genome of a heterotrophic ma-rine bacterium Alteromonas macleodii Deep ecotype (AltDE),a deep-sea ecotype strain isolated from the Mediterranean Sea(26). Analysis of its genome revealed the gene operon of an[NiFe] hydrogenase, which contains two structural genes (hyaAB)and eight accessory genes (hyaD, hupH, and hypCABDFE), all inthe same orientation with each other. The 8,744-bp DNA se-quence from our hydrogenase gene locus (Fig. 2) showed 99%identity to that of the A. macleodii hydrogenase operon, whilethe predicted protein sequences of all the genes in the locusshowed 95% to 100% identity to those of their counterparts inA. macleodii. Overall, these data indicate that the Sargasso Sea[NiFe] hydrogenase originated from the Alteromonas clade,i.e., A. macleodii or its close relatives.
Expression of the Alteromonas hydrogenase in T. roseopersi-cina. To investigate whether the structural genes (hyaA andhyaB) and accessory genes (hyaD and hupH) (Fig. 2) couldencode a functional hydrogenase, we sought to express them inT. roseopersicina. We first subcloned these genes from plasmidspSZAT5 and pSSYK1 into pUC19 to create pAmMG1 (seeMaterials and Methods). We then excised from pAmMG1 a4,325-bp segment that contained the hyaD-hupH-hyaA-hyaBgene cassette. In addition, the segment contained a 337-bpnoncoding region upstream of hyaD and a 110-bp region down-stream of hyaB. This was cloned into the broad-host-rangeexpression vector pMHE6crtKm (Fig. 1) (17) that can replicatein T. roseopersicina to create the new plasmid, pAmDHSL.Hydrogen metabolism and hydrogenase processing have beenstudied in detail in T. roseopersicina, and various mutants areavailable. We used a T. roseopersicina hydrogenase mutantstrain GB112131 (HynSL� HupSL� HoxH�) as a host strain;in this strain, all three native hydrogenases, including one sol-uble bidirectional hydrogenase (Hox), one O2-tolerant hydro-genase (Hyn), and one periplasmic uptake hydrogenase (Hup),were knocked out (13, 41, 42). The expression vector pAmDHSLwas introduced from E. coli into T. roseopersicina by conjuga-tion, thus generating a kanamycin-resistant T. roseopersicinastrain, MGAmDHSL. The expression of the transferred hydro-genase genes in strain MGAmDHSL can be driven by a con-
FIG. 2. Schematic representation of the gene locus of a novel environmental hydrogenase. The figure was not drawn to scale. The solid arrowsrepresent the open reading frames of the genes, and the arrows show the direction of deduced gene transcription. pSZAT5 and pSSYK1 wereplasmids used to obtain the nucleotide sequence in the region. hyaA and hyaB are structural genes of the novel hydrogenase. hyaD and hupH areaccessory genes of the novel hydrogenase. hmp is a NADH oxidoreductase gene, cyt is a cytochrome gene, and orf1 and orf2 are hypothetical genes.GI #1, GI #2, and GI #3 represent the locations of three peptide sequences (GI 137178577, GI 142721499, and GI 142721500, respectively) fromthe Sargasso Sea metagenomic data.
stitutive Thiocapsa native promoter for the crtD gene carriedby pAmDHSL (Fig. 1) (27). It is possible that the 337-bpnoncoding region upstream of hyaD may also contain a pro-moter.
Heterologous expression of the Alteromonas hydrogenase inT. roseopersicina was examined at the transcription level. RT-PCR amplification of the RNA transcripts from T. roseopersi-cina strain MGAmDHSL generated a 422-bp hyaB-specificDNA fragment as expected. No products were generated fromthe non-RT control (Fig. 3A), indicating that the hyaB-specificproduct was transcribed from RNAs, not from contaminatingDNAs. In contrast, the RT-PCR amplification of the RNAsfrom strain GB112131 (a negative control) failed to generateany PCR products (Fig. 3A), indicating that hyaB transcriptswere truly transcribed from the transferred Alteromonas hyaBgene. RT-PCR amplification was also performed for hyaA,hyaD, and hupH. Specific PCR products were generated fromthe cDNA samples from strain MGAmDHSL, but not thosefrom the negative control (data not shown), indicating thathyaA, hyaD, and hupH were transcribed as well.
Expression of the hydrogenase in T. roseopersicina was fur-ther examined at the translational level by Western blotting.Immunological cross-reactions of T. roseopersicina hydroge-nase antibodies with different [NiFe] hydrogenases have beendemonstrated before (29). We therefore sought to use poly-clonal antibodies of Thiocapsa HynL protein to detect its ho-molog protein Alteromonas HyaB. T. roseopersicina strainGB112131 was used as a negative control, and T. roseopersicinastrain GB2131 (38) (expressing only O2-tolerant hydrogenaseHynSL) was used as a positive control. As expected, no proteinsignals were detected in the negative-control strain, whereasexpression of T. roseopersicina hydrogenase large-subunitHynL was detected in the positive-control strain (Fig. 3B). Twodifferent forms of HynL proteins in the positive-control strain,the 61-kDa mature form (bottom band) and the 64-kDa im-
mature form (top band), were distinguished on the blot (Fig.3B), which were generated from the C-terminal cleavage of thelarge subunit in the maturation process. The Alteromonas hy-drogenase large-subunit HyaB was detected in T. roseopersi-cina strain MGAmDHSL (Fig. 3B), which is consistent withthe RT-PCR result (Fig. 3A) and indicates that the Alteromo-nas hydrogenase was expressed at the protein level in Thio-capsa. Both mature and immature forms of the HyaB proteinswere also detected in the mid-log-phase MGAmDHSL cells(Fig. 3B), and their sizes are consistent with those predictedon the basis of the HyaB’s protein sequence (66.5 kDa versus69 kDa).
It was previously demonstrated (16) that maturation of thelarge-subunit HynL in the T. roseopersicina hypF mutant (strainM539) was blocked at an early stage of the process and only theimmature form of HynL accumulates in this mutant. We usedmutant strain M539 as a control for parallel Western blottingand detected only one protein band (immature HynL, 64 kDa)(data not shown). This result confirms that observed bottomand top bands for HynL and HyaB were not caused by proteindegradation during sample processing and were indeed theimmature and mature forms of the large subunits.
Functional analysis of expressed Alteromonas hydrogenase.To determine whether the Alteromonas hydrogenase was activewhen expressed in T. roseopersicina, we performed in vitro(using whole-cell extracts) and in vivo analyses. Figure 4Ashows the result of in vitro hydrogen evolution assays in whichreduced MV was used as an electron donor. As expected, noactivities were detected in the negative control (strainGB112131), whereas H2 evolution activity (0.28 �l H2 h�1
mg�1 total protein, or 0.012 �mol H2 � h�1 � mg�1 total pro-tein) was detected in strain MGAmDHSL which contained theAlteromonas hydrogenase. Figure 4B shows the result of H2
uptake assays in which oxidized BV was used as an electronacceptor. The hydrogen uptake activity (5.04 �l H2 h�1 mg�1
FIG. 3. Detection of heterologous expression of environmental Alteromonas hydrogenase in T. roseopersicina. (A) RT-PCR to detect thetranscription of heterologous Alteromonas hyaB gene. Total RNAs isolated from strains GB112131 and MGAmDHSL were used for reversetranscription reactions. RT �, the RT reactions with SuperScript III reverse transcriptase added; RT �, the RT reactions without reversetranscriptase added (no-RT control). (B) Western blotting to detect heterologous expression of Alteromonas HyaB protein. Anti-T. roseopersicinaHynL primary antibody was used for Western blotting. Two forms of HynL and HyaB proteins were detected, the mature form (bottom band) andthe nonmature form (top band). GB112131 (negative control) is a T. roseopersicina �hynSL �hupSL �hoxH strain, GB2131 (positive control) isa T. roseopersicina �hupSL �hoxH strain, and MGAmDHSL is T. roseopersicina GB112131 strain carrying plasmid pAmDHSL.
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total protein, or 0.225 �mol H2 � h�1 � mg�1 total protein) wasagain detected in strain MGAmDHSL, while no activities weredetected in the negative control. Overall, these results demon-strate that the heterologously expressed Alteromonas hydroge-nase is functional in both H2 evolution and uptake activities.This indicates the proper assembly of the catalytic center in theheterologously expressed protein and suggests that the matu-ration mechanism of HyaAB is similar to that of HynSL.
In vitro hydrogenase activities detected in strain MGAmDHSLare in the normal range observed for membrane-associated[NiFe] hydrogenases, but they were only 20% of the hydrogenevolution activity (Fig. 4A) and 15% of the uptake activity (Fig.4B) detected for the native T. roseopersicina hydrogenaseHynSL. The cellular localization of the heterologously ex-pressed hydrogenase was examined. Approximately 60%( 10%) of the heterologous hydrogenase activity was found inthe membrane fraction, and �40% was in the soluble fraction.This distribution of activity appears to be similar to that of thenative Thiocapsa O2-tolerant hydrogenase (30, 38), suggestingthat the Alteromonas hydrogenase (HyaAB) is also a looselymembrane-bound hydrogenase just like the Thiocapsa hydro-genase HynSL (30, 38). This is consistent with the fact thatthese two hydrogenases carry membrane-targeting signal pep-tides at the N-terminal ends of their small subunits.
To investigate whether heterologously expressed Alteromo-nas hydrogenase can function in T. roseopersicina, in vivo hy-drogen uptake activity assays were further performed. Underthe N2-fixing condition that allows T. roseopersicina nitroge-nase to produce H2, we measured in vivo H2 uptake activity instrain MGAmDHSL by indirectly monitoring the changes ofthe H2 level in the headspace of the cell cultures. Again, T.roseopersicina mutant strain GB112131 was used as a negativecontrol, and strain GB2131 served as a positive control. Aconsiderable amount of H2 (16.7 1.67 �l 50 ml�1 day�1) wasconsumed in the positive-control strain GB2131, which isequivalent to about 75% of the total amount of H2 producedby the native nitrogenase of T. roseopersicina (data notshown). In contrast, no H2 consumption was detected instrain MGAmDHSL that carried the heterologously expressed
hydrogenase, similar to what we observed in the negative-control strain GB112131, indicating that this heterologouslyexpressed hydrogenase is not functional in the H2 oxidationdirection under in vivo conditions.
Complementation of Thiocapsa endopeptidase HynD withAlteromonas HyaD. Introduction of only two accessory genes(hyaD-hupH) of the Alteromonas hydrogenase into T. roseop-ersicina was sufficient to create an active Alteromonas [NiFe]hydrogenase in this phototrophic bacterium, indicating that thenative accessory proteins from T. roseopersicina were able tocomplement functions of the Alteromonas HypABCDEF pro-teins. The sequence of the endopeptidase HyaD of Alteromo-nas hydrogenase shows only 37% identity and 57% similarity tothat of its T. roseopersicina counterpart HynD, a homologymuch weaker than those observed from comparison of thehydrogenase structural subunits. To further compare the mat-uration machineries of the Alteromonas and T. roseopersicinahydrogenases, we created a T. roseopersicina hynD knockoutmutant strain (DYDG2) to perform complementation experi-ments by introducing pDYD (a construct carrying hynD dele-tion) into T. roseopersicina mutant strain GB21. As confirmedby PCR results, in-frame deletion was introduced into thehynD gene in strain DYDG2 through homologous DNA re-combination. We then cloned the environmental AlteromonashyaD gene into expression vector pMHE6crtKm to createpAmD, which contains exactly the same upstream promoterregion as that of pAmDHSL. The pAmD plasmid was thenintroduced into the T. roseopersicina hynD mutant to createstrain DYDAmD. To examine the cleavage activity of intro-duced HyaD, Western blotting was performed. T. roseopersi-cina strain GB2131 containing the native endopeptidase HynDwas used as a positive control. As expected, both mature andimmature forms (m-HynL and Pre-HynL, respectively) of thelarge-subunit HynL of the Thiocapsa hydrogenase were de-tected in this strain (Fig. 5). In the T. roseopersicina hynDmutant strain DYDG2, however, only the immature form (Pre-HynL) was detected (Fig. 5). After introducing hyaD into thismutant, the mature form (m-HynL) was observed in the com-plemented DYDAmD strain (Fig. 5), demonstrating restored
FIG. 4. Detection of activity of heterologously expressed Alteromonas hydrogenase under in vitro conditions. (A) Measurement of H2 evolutionactivity of heterologously expressed Alteromonas hydrogenase. (B) Measurement of H2 uptake activity of heterologously expressed Alteromonashydrogenase. T. roseopersicina whole-cell extracts were used for the measurements. GB112131 (negative control) is a T. roseopersicina �hynSL�hupSL �hoxH strain, MGAmDHSL is a T. roseopersicina GB112131 strain carrying plasmid pAmDHSL, and GB2131 is a T. roseopersicina�hupSL �hoxH strain.
cleavage of the T. roseopersicina large subunit. This result in-dicates that the Alteromonas endopeptidase HyaD comple-mented T. roseopersicina HynD in the mutant strain DYDG2and that the two hydrogenases share similar maturation mech-anisms (Fig. 5).
To determine whether the enzyme activities of T. roseoper-sicina hydrogenase were also restored in the complementedDYDAmD strain, in vitro H2 evolution assays were performedusing membrane fractions of strains DYDG2, GB2131, DY-DAmD, and GB112131. H2 evolution activities were detectedin the positive-control strain GB2131, whereas no activitieswere detected in the negative-control strain GB112131, as weobserved previously (Fig. 4A). The enzyme activities in thehynD mutant strain DYDG2 decreased significantly, but theywere slightly higher than the basal level in the negative control.These activities may be explained by the presence of a traceamount of Hox, a soluble hydrogenase carried in strainDYDG2, but not in strain GB112131. In strain DYDAmD thatwas complemented with the environmental HyaD, however,the activities were fully restored to the same level as in strainGB2131 (data not shown), indicating that the environmentalAlteromonas HyaD can fully complement the T. roseopersicinaHynD in the hynD mutant strain.
DISCUSSION
This study represents the first report of converting an envi-ronmental DNA into a functional [NiFe] hydrogenase. Tradi-tionally, O2-tolerant hydrogenases are screened from microbesthat can be cultured in the laboratory. However, less than 1%of environmental microbes can be cultured under laboratoryconditions (14). Metagenomics allows new genes to be discov-ered even though the underlying organisms are not known orcannot be cultured. Thus, it provides a completely new ap-
proach for identifying potential O2-tolerant [NiFe] hydroge-nases from environmental microbes, which may be used forconstructing biophotolytic systems in cyanobacteria for energyproduction. Our research on the environmental Alteromonashydrogenase demonstrates a model for how to discover newenzymes and to functionally study these enzymes in a culture-independent manner.
As described before, [NiFe] hydrogenases require a wholeset of accessory proteins to properly assemble their catalyticcenters (7). In previous studies (4, 18, 31, 50) that reportsuccessful expression of functional [NiFe] hydrogenases innonnatural hosts, all of the accessory genes were coexpressedwith the structural genes of the hydrogenases. In two cases (39,44), however, active [NiFe] hydrogenases have been formedwhen an incomplete set of the maturation genes were coex-pressed. One case is that an active [NiFe] hydrogenase fromDesulfovibrio gigas was formed in Desulfovibrio fructosovoransby coexpressing only two accessory genes (hynCD) (44), andanother case is that a Rhodococcus opacus [NiFe] hydrogenasewas expressed in Ralstonia eutropha using only one accessorygene (an endopeptidase gene, hoxW) (39, 44). In our study,an active Alteromonas hydrogenase was also expressed in T.roseopersicina using only two of the Alteromonas accessorygenes. A common feature observed in these three studies isthat the structural subunits of heterologously expressed hydro-genase share strong homology (�60% identity) with those ofnative hydrogenases in the foreign host. Homology of theiraccessory proteins, however, varies dramatically for differenthydrogenases. D. gigas and R. opacus accessory proteins, HynCand HoxW, demonstrate high levels of homology (67 and 74%identities, respectively) to those in D. fructosovorans or R.eutropha. In our case, Alteromonas accessory proteins HynD,HupH, HypF, HypD, HypE, and HypC show only 37 to 45%identity to their counterparts in T. roseopersicina. These studiesimply that the level of the homology between the foreign andnative hydrogenase structural proteins seems more importantfor expressing an active hydrogenase in foreign hosts than thelevel of homology between accessory proteins.
Heterotrophic A. macleodii and phototrophic purple sulfurT. roseopersicina display different GC contents in their ge-nomes (44.9% versus 63.7% GC), although they both belong tothe group of gammaproteobacteria (26) (K. L. Kovacs et al.,unpublished data). Consequently, codon usage differs in thesetwo organisms. Since we were able to express a functionalAlteromonas hydrogenase in T. roseopersicina, codon usage wasnot a barrier. However, the activity of the Alteromonas hydro-genase is only 15 to 20% of that of the native T. roseopersicinahydrogenase. Besides possible difference in kinetic character-istics, several other factors could contribute to this lower ac-tivity. First, the T. roseopersicina hydrogenase was assembledby its own native accessory proteins, whereas the Alteromonashydrogenase was assembled by a set of heterologous accessoryproteins, except those two Alteromonas proteins HyaD andHupH that were cotransferred into the host. It is possible thatthe T. roseopersicina assembly machinery could not fully re-place the functions of some of the Alteromonas accessory pro-teins. Second, elements regulating the expression of the twohydrogenases could account for the difference. T. roseopersi-cina hydrogenase genes were regulated by their own promoteris located upstream of the hynS gene. On the other hand, the
FIG. 5. Complementation of T. roseopersicina hynD mutant withheterologous Alteromonas hyaD. The large subunit of T. roseopersicinahydrogenase HynSL was detected by Western blot hybridization in thewhole-cell extracts of strains GB2131, DYDG2, and DYDAmD. Pre-HynL, the nonmature form of the large subunit; m-HynL, the matureform of the large subunit; GB2131 is a T. roseopersicina �hupSL�hoxH strain, DYDG2 is a T. roseopersicina �hupSL �hynD strain, andDYDAmD is a T. roseopersicina DYDG2 strain carrying plasmidpAmD.
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Alteromonas hydrogenase genes were regulated by the crtDpromoter of the T. roseopersicina photosynthetic pigment bio-synthesis operon. Finally, the Alteromonas hydrogenase genescarried their original ribosomal binding site from Alteromonas,which may not be recognized as well as the ribosomal bindingsite from T. roseopersicina is.
Although Alteromonas hydrogenase in extracts showed bothH2 evolution and uptake activities, no uptake activity was de-tected in vivo in T. roseopersicina. To catalyze the redox reac-
tion in the living cells, the hydrogenase requires specific elec-tron transfer proteins. The lack of in vivo activity suggests thatheterologously expressed Alteromonas hydrogenase is not cou-pled to the native electron transfer system in T. roseopersicina.It has been demonstrated that in T. roseopersicina the electrontransfer proteins specific for the T. roseopersicina hydrogenaseHynSL are proteins Isp1 and Isp2 (their genes, isp1 and isp2,form an operon with structural genes hynS and hynL ofHynSL) (38, 41). However, the Alteromonas counterparts of
FIG. 6. Alignment of the amino acid sequences of various endopeptidases. Amino acids highlighted are analogues of Glu16, Asp62, and His93 of E.coli endopeptidase HybD (20), which are considered indispensable for the enzymatic activity of hydrogenase-specific endopeptidases. The VRVFE aminoacid stretch (boxed) is conserved only in two proteases (Thiocapsa HynD and Alteromonas HyaD) examined in this study. Residues that are identical (*),conserved (:), or semiconserved (.) in the sequences are indicated below the sequences. The species is indicated after the enzyme as follows: T. r.,Thiocapsa roseopersicina; T. d., Thiobacillus denitrificans; A. m., Alteromonas clade; A. s., Aeromonas salmonicida; E. c., Escherichia coli K-12; R. p.,Rhodopseudomonas palustris; R. l., Rhizobium leguminosarum; R. r., Rhodospirillum rubrum; R. e., Ralstonia eutropha H16; R. m., Ralstonia metallidurans;M. c., Methylococcus capsulatus; G. l., Geobacter lovleyi; and D. v., Desulfovibrio vulgaris.
Isp1 and Isp2 could not be identified in the complete genomeof Alteromonas strain AltDE, implying that different electrontransfer proteins may be used in this bacterium.
Based on sequence alignments and functionality, [NiFe] hy-drogenases are divided into four groups (53). Two highly con-served regions including two pairs of cysteines that ligate theNiFe site are present at the N- and C-terminal ends of largesubunits from each group (53). Thiocapsa and Alteromonashydrogenases fall into group 1, a group of membrane-associ-ated hydrogenases. In addition to the two highly conservedregions, homologous domains can be found throughout theentire large subunits of these two hydrogenases. This homol-ogy is consistent with the fact that the Alteromonas large sub-unit immunologically cross-reacted with antibodies of theThiocapsa large subunit. The existence of strong similarity be-tween these two subunits (75% similarity) suggests these twohydrogenases may have similar enzyme characteristics. It re-mains to be determined whether the levels of its thermostabil-ity and O2 stability are similar to those of the Thiocapsa hy-drogenase.
Endopeptidases that act in the last maturation step of [NiFe]hydrogenase large subunits are normally hydrogenase specific.Even in the same bacterial organism, different [NiFe] hydro-genases have their own endopeptidases. In this study, we dem-onstrate that Alteromonas endopeptidase HyaD complementsThiocapsa HynD. This is the only example we are aware of inwhich an endopeptidase for one [NiFe] hydrogenase is able tocomplement an endopeptidase for a different hydrogenase.Interestingly, the sequence homology between these two en-dopeptidases is not typically higher than those among otherendopeptidases. For example, the Alteromonas endopeptidaseHyaD shows only 37% identity and 57% similarity to the T.roseopersicina HynD. This is even lower than that between theT. roseopersicina HynD and several other endopeptidases, suchas the Thiobacillus denitrificans endopeptidase (45% identity)and the Geobacter lovleyi endopeptidase (38%). An alignmentof 13 different [NiFe] hydrogenase endopeptidases is shown inFig. 6. Amino acids important for the catalytic activity of en-dopeptidases, such as the amino acids corresponding to Glu16,Asp62, and His93 in E. coli endopeptidases HybD (20), arepresent in all 13 sequences. A stretch of amino acids VRVFEin the middle regions of the proteins is unique to and con-served only in the Alteromonas and T. roseopersicina endopep-tidases. Overall, the sequence alignment of the endopeptidasesdoes not provide a clear explanation why these two proteinsare commutable in the assembly process of the T. roseopersi-cina hydrogenase HynSL. It is possible that despite differencesin the primary sequences, the Alteromonas and T. roseopersi-cina endopeptidases share a similar three-dimensional struc-ture, which allows the Alteromonas endopeptidase to act on theT. roseopersicina Hyn, a homolog of the Alteromonas hydroge-nase. It remains to be determined whether the complementa-tion of these two endopeptidases is bilateral, i.e., whether theT. roseopersicina endopeptidase HynD can perform cleavageon the Alteromonas immature large subunit.
ACKNOWLEDGMENTS
This work was supported by Synthetic Genomics, Inc., and the Hy-drogen, Fuel Cells, and Infrastructure Technology Program (DE-FG36-05GO15027) of the U.S. Department of Energy.
We thank Pin-Ching Maness, Mike Seibert, Maria L. Ghirardi, andLin-ping Zhang for helping us with setting up the biohydrogen labo-ratory and for their helpful discussions. We thank the JCVI Sequenc-ing Center team and the JCVI Microbial and Environmental Genom-ics group for tracing original clones and DNA samples for the SargassoSea sampling project. Finally, we thank Laura Sheahan for kindlyediting the manuscript.
REFERENCES
1. Adams, M. W., L. E. Mortenson, and J. S. Chen. 1980. Hydrogenase. Bio-chim. Biophys. Acta 594:105–176.
2. Albracht, S. P. 1994. Nickel hydrogenases: in search of the active site.Biochim. Biophys. Acta 1188:167–204.
3. Albracht, S. P., E. G. Graf, and R. K. Thauer. 1982. The EPR properties ofnickel in hydrogenase from Methanobacterium. FEBS Lett. 140:311–313.
4. Bascones, E., J. Imperial, T. Ruiz-Argueso, and J. M. Palacios. 2000. Gen-eration of new hydrogen-recycling Rhizobiaceae strains by introduction of anovel hup minitransposon. Appl. Environ. Microbiol. 66:4292–4299.
5. Blokesch, M., A. Magalon, and A. Bock. 2001. Interplay between the specificchaperone-like proteins HybG and HypC in maturation of hydrogenases 1, 2,and 3 from Escherichia coli. J. Bacteriol. 183:2817–2822.
6. Blokesch, M., A. Paschos, A. Bauer, S. Reissmann, N. Drapal, and A. Bock.2004. Analysis of the transcarbamoylation-dehydration reaction catalyzed bythe hydrogenase maturation proteins HypF and HypE. Eur. J. Biochem.271:3428–3436.
7. Bock, A., P. W. King, M. Blokesch, and M. C. Posewitz. 2006. Maturation ofhydrogenases. Adv. Microb. Physiol. 51:1–71.
8. Bogorov, L. V. 1974. The properties of Thiocapsa roseopersicina, strain BBS,isolated from an estuary of the White Sea. Mikrobiologiia 43:326–332. (InRussian.)
9. Burgdorf, T., O. Lenz, T. Buhrke, E. van der Linden, A. K. Jones, S. P.Albracht, and B. Friedrich. 2005. [NiFe]-hydrogenases of Ralstonia eutrophaH16: modular enzymes for oxygen-tolerant biological hydrogen oxidation. J.Mol. Microbiol. Biotechnol. 10:181–196.
10. Cammack, R., M. Frey, and R. Robson. 2001. Hydrogen as a fuel: learningfrom nature. Taylor & Francis, London, United Kingdom.
11. Casalot, L., and M. Rousset. 2001. Maturation of the [NiFe] hydrogenases.Trends Microbiol. 9:228–237.
12. Chen, J. S., and L. E. Mortenson. 1974. Purification and properties ofhydrogenase from Clostridium pasteurianum W5. Biochim. Biophys. Acta371:283–298.
13. Colbeau, A., K. L. Kovacs, J. Chabert, and P. M. Vignais. 1994. Cloning andsequence of the structural (hupSLC) and accessory (hupDHI) genes forhydrogenase biosynthesis in Thiocapsa roseopersicina. Gene 140:25–31.
14. Colwell, R. R., and D. J. Grimes (ed.). 2000. Nonculturable microorganismsin the environment. ASM Press, Washington, DC.
15. Fauque, G., H. D. Peck, Jr., J. J. Moura, B. H. Huynh, Y. Berlier, D. V.DerVartanian, M. Teixeira, A. E. Przybyla, P. A. Lespinat, I. Moura, et al.1988. The three classes of hydrogenases from sulfate-reducing bacteria of thegenus Desulfovibrio. FEMS Microbiol. Rev. 4:299–344.
16. Fodor, B., G. Rakhely, A. T. Kovacs, and K. L. Kovacs. 2001. Transposonmutagenesis in purple sulfur photosynthetic bacteria: identification of hypF,encoding a protein capable of processing [NiFe] hydrogenases in �, , and �subdivisions of the proteobacteria. Appl. Environ. Microbiol. 67:2476–2483.
17. Fodor, B. D., A. T. Kovacs, R. Csaki, E. Hunyadi-Gulyas, E. Klement, G.Maroti, L. S. Meszaros, K. F. Medzihradszky, G. Rakhely, and K. L. Kovacs.2004. Modular broad-host-range expression vectors for single-protein andprotein complex purification. Appl. Environ. Microbiol. 70:712–721.
18. Friedrich, B., C. G. Friedrich, M. Meyer, and H. G. Schlegel. 1984. Expres-sion of hydrogenase in Alcaligenes spp. is altered by interspecific plasmidexchange. J. Bacteriol. 158:331–333.
19. Friedrich, B., and E. Schwartz. 1993. Molecular biology of hydrogen utili-zation in aerobic chemolithotrophs. Annu. Rev. Microbiol. 47:351–383.
20. Fritsche, E., A. Paschos, H. G. Beisel, A. Bock, and R. Huber. 1999. Crystalstructure of the hydrogenase maturating endopeptidase HYBD from Esch-erichia coli. J. Mol. Biol. 288:989–998.
21. Gogotov, I. N., N. A. Zorin, L. T. Serebriakova, and E. N. Kondratieva. 1978.The properties of hydrogenase from Thiocapsa roseopersicina. Biochim. Bio-phys. Acta 523:335–343.
22. Hagemeier, C. H., L. Chistoserdova, M. E. Lidstrom, R. K. Thauer, and J. A.Vorholt. 2000. Characterization of a second methylene tetrahydromethanop-terin dehydrogenase from Methylobacterium extorquens AM1. Eur. J. Bio-chem. 267:3762–3769.
23. Hartmann, G. C., A. R. Klein, M. Linder, and R. K. Thauer. 1996. Purifi-cation, properties and primary structure of H2-forming N5,N10-methyl-enetetrahydromethanopterin dehydrogenase from Methanococcus thermo-lithotrophicus. Arch. Microbiol. 165:187–193.
24. Herrero, M., V. de Lorenzo, and K. N. Timmis. 1990. Transposon vectorscontaining non-antibiotic resistance selection markers for cloning and stablechromosomal insertion of foreign genes in gram-negative bacteria. J. Bacte-riol. 172:6557–6567.
VOL. 75, 2009 A [NiFe] HYDROGENASE FROM ENVIRONMENTAL MICROBES 5829
25. Horner, D. S., B. Heil, T. Happe, and T. M. Embley. 2002. Iron hydrogenas-es–ancient enzymes in modern eukaryotes. Trends Biochem. Sci. 27:148–153.
26. Ivars-Martinez, E., A. B. Martin-Cuadrado, G. D’Auria, A. Mira, S. Ferri-era, J. Johnson, R. Friedman, and F. Rodriguez-Valera. 2008. Comparativegenomics of two ecotypes of the marine planktonic copiotroph Alteromonasmacleodii suggests alternative lifestyles associated with different kinds ofparticulate organic matter. ISME J. 2:1194–1212.
27. Kovacs, A. T., G. Rakhely, and K. L. Kovacs. 2003. Genes involved in thebiosynthesis of photosynthetic pigments in the purple sulfur photosyntheticbacterium Thiocapsa roseopersicina. Appl. Environ. Microbiol. 69:3093–3102.
28. Kovacs, K. L., G. Maroti, J. Balogh, S. Arvani, and G. Rakhely. 2002.Hydrogenases, accessory genes and the regulation of [NiFe]-hydrogenasebiosynthesis in Thiocapsa roseopersicina. Int. J. Hydrogen Energy 27:1463–1469.
29. Kovacs, K. L., L. C. Seefeldt, G. Tigyi, C. M. Doyle, L. E. Mortenson, andD. J. Arp. 1989. Immunological relationship among hydrogenases. J. Bacte-riol. 171:430–435.
30. Kovacs, K. L., G. Tigyi, and H. Alfonz. 1985. Purification of hydrogenase byfast protein liquid chromatography and by conventional separation tech-niques: a comparative study. Prep. Biochem. 15:321–334.
31. Lenz, O., A. Gleiche, A. Strack, and B. Friedrich. 2005. Requirements forheterologous production of a complex metalloenzyme: the membrane-bound[NiFe] hydrogenase. J. Bacteriol. 187:6590–6595.
32. Lester, R. L., and J. A. DeMoss. 1971. Effects of molybdate and selenite onformate and nitrate metabolism in Escherichia coli. J. Bacteriol. 105:1006–1014.
33. Magalon, A., M. Blokesch, E. Zehelein, and A. Bock. 2001. Fidelity of metalinsertion into hydrogenases. FEBS Lett. 499:73–76.
34. Magalon, A., and A. Bock. 2000. Analysis of the HypC-HycE complex, a keyintermediate in the assembly of the metal center of the Escherichia colihydrogenase 3. J. Biol. Chem. 275:21114–21120.
35. Manyani, H., L. Rey, J. M. Palacios, J. Imperial, and T. Ruiz-Argueso. 2005.Gene products of the hupGHIJ operon are involved in maturation of theiron-sulfur subunit of the [NiFe] hydrogenase from Rhizobium leguminosa-rum bv. viciae. J. Bacteriol. 187:7018–7026.
36. Maroti, G., B. D. Fodor, G. Rakhely, A. T. Kovacs, S. Arvani, and K. L.Kovacs. 2003. Accessory proteins functioning selectively and pleiotropicallyin the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina. Eur.J. Biochem. 270:2218–2227.
37. Murray, M. G., and W. F. Thompson. 1980. Rapid isolation of high molec-ular weight plant DNA. Nucleic Acids Res. 8:4321–4325.
38. Palagyi-Meszaros, L. S., J. Maroti, D. Latinovics, T. Balogh, E. Klement,K. F. Medzihradszky, G. Rakhely, and K. L. Kovacs. 2009. Electron-transfersubunits of the NiFe hydrogenases in Thiocapsa roseopersicina BBS. FEBS J.276:164–174.
39. Porthun, A., M. Bernhard, and B. Friedrich. 2002. Expression of a functionalNAD-reducing [NiFe] hydrogenase from the gram-positive Rhodococcusopacus in the gram-negative Ralstonia eutropha. Arch. Microbiol. 177:159–166.
40. Przybyla, A. E., J. Robbins, N. Menon, and H. D. Peck, Jr. 1992. Structure-function relationships among the nickel-containing hydrogenases. FEMSMicrobiol. Rev. 8:109–135.
41. Rakhely, G., A. Colbeau, J. Garin, P. M. Vignais, and K. L. Kovacs. 1998.Unusual organization of the genes coding for HydSL, the stable [NiFe]hy-drogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS. J.Bacteriol. 180:1460–1465.
42. Rakhely, G., A. T. Kovacs, G. Maroti, B. D. Fodor, G. Csanadi, D. Latinovics,and K. L. Kovacs. 2004. Cyanobacterial-type, heteropentameric, NAD�-reducing NiFe hydrogenase in the purple sulfur photosynthetic bacteriumThiocapsa roseopersicina. Appl. Environ. Microbiol. 70:722–728.
43. Rossmann, R., M. Sauter, F. Lottspeich, and A. Bock. 1994. Maturation ofthe large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickelincorporation followed by C-terminal processing at Arg537. Eur. J. Biochem.220:377–384.
44. Rousset, M., V. Magro, N. Forget, B. Guigliarelli, J. P. Belaich, and E. C.Hatchikian. 1998. Heterologous expression of the Desulfovibrio gigas [NiFe]hydrogenase in Desulfovibrio fructosovorans MR400. J. Bacteriol. 180:4982–4986.
45. Rusch, D. B., A. L. Halpern, G. Sutton, K. B. Heidelberg, S. Williamson, S.Yooseph, D. Wu, J. A. Eisen, J. M. Hoffman, K. Remington, K. Beeson, B.Tran, H. Smith, H. Baden-Tillson, C. Stewart, J. Thorpe, J. Freeman, C.Andrews-Pfannkoch, J. E. Venter, K. Li, S. Kravitz, J. F. Heidelberg, T.Utterback, Y. H. Rogers, L. I. Falcon, V. Souza, G. Bonilla-Rosso, L. E.
Eguiarte, D. M. Karl, S. Sathyendranath, T. Platt, E. Bermingham, V.Gallardo, G. Tamayo-Castillo, M. R. Ferrari, R. L. Strausberg, K. Nealson,R. Friedman, M. Frazier, and J. C. Venter. 2007. The Sorcerer II GlobalOcean Sampling expedition: northwest Atlantic through eastern tropicalPacific. PLoS Biol. 5:e77.
46. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratorymanual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, NY.
47. Schneider, K., R. Cammack, H. G. Schlegel, and D. O. Hall. 1979. Theiron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus. Bio-chim. Biophys. Acta 578:445–461.
48. Thauer, R. K., A. R. Klein, and G. C. Hartmann. 1996. Reactions withmolecular hydrogen in microorganisms: evidence for a purely organic hy-drogenation catalyst. Chem. Rev. 96:3031–3042.
49. Theodoratou, E., A. Paschos, W. Mintz, and A. Bock. 2000. Analysis of thecleavage site specificity of the endopeptidase involved in the maturation ofthe large subunit of hydrogenase 3 from Escherichia coli. Arch. Microbiol.173:110–116.
50. Umeda, F., H. Min, M. Urushihara, M. Okazaki, and Y. Miura. 1986.Conjugal transfer of hydrogen-oxidizing ability of Alcaligenes hydrogenophi-lus to Pseudomonas oxalaticus. Biochem. Biophys. Res. Commun. 137:108–113.
51. Venter, J. C., M. D. Adams, E. W. Myers, P. W. Li, R. J. Mural, G. G. Sutton,H. O. Smith, M. Yandell, C. A. Evans, R. A. Holt, J. D. Gocayne, P. Amanati-des, R. M. Ballew, D. H. Huson, J. R. Wortman, Q. Zhang, C. D. Kodira,X. H. Zheng, L. Chen, M. Skupski, G. Subramanian, P. D. Thomas, J.Zhang, G. L. Gabor Miklos, C. Nelson, S. Broder, A. G. Clark, J. Nadeau,V. A. McKusick, N. Zinder, A. J. Levine, R. J. Roberts, M. Simon, C.Slayman, M. Hunkapiller, R. Bolanos, A. Delcher, I. Dew, D. Fasulo, M.Flanigan, L. Florea, A. Halpern, S. Hannenhalli, S. Kravitz, S. Levy, C.Mobarry, K. Reinert, K. Remington, J. Abu-Threideh, E. Beasley, K. Bid-dick, V. Bonazzi, R. Brandon, M. Cargill, I. Chandramouliswaran, R. Char-lab, K. Chaturvedi, Z. Deng, V. Di Francesco, P. Dunn, K. Eilbeck, C.Evangelista, A. E. Gabrielian, W. Gan, W. Ge, F. Gong, Z. Gu, P. Guan, T. J.Heiman, M. E. Higgins, R. R. Ji, Z. Ke, K. A. Ketchum, Z. Lai, Y. Lei, Z. Li,J. Li, Y. Liang, X. Lin, F. Lu, G. V. Merkulov, N. Milshina, H. M. Moore,A. K. Naik, V. A. Narayan, B. Neelam, D. Nusskern, D. B. Rusch, S. Salzberg,W. Shao, B. Shue, J. Sun, Z. Wang, A. Wang, X. Wang, J. Wang, M. Wei, R.Wides, C. Xiao, C. Yan, et al. 2001. The sequence of the human genome.Science 291:1304–1351.
52. Venter, J. C., K. Remington, J. F. Heidelberg, A. L. Halpern, D. Rusch, J. A.Eisen, D. Wu, I. Paulsen, K. E. Nelson, W. Nelson, D. E. Fouts, S. Levy, A. H.Knap, M. W. Lomas, K. Nealson, O. White, J. Peterson, J. Hoffman, R.Parsons, H. Baden-Tillson, C. Pfannkoch, Y. H. Rogers, and H. O. Smith.2004. Environmental genome shotgun sequencing of the Sargasso Sea. Sci-ence 304:66–74.
53. Vignais, P. M., B. Billoud, and J. Meyer. 2001. Classification and phylogenyof hydrogenases. FEMS Microbiol. Rev. 25:455–501.
54. Vignais, P. M., and A. Colbeau. 2004. Molecular biology of microbial hydro-genases. Curr. Issues Mol. Biol. 6:159–188.
55. Volbeda, A., M. H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C.Fontecilla-Camps. 1995. Crystal structure of the nickel-iron hydrogenasefrom Desulfovibrio gigas. Nature 373:580–587.
56. Yooseph, S., G. Sutton, D. B. Rusch, A. L. Halpern, S. J. Williamson, K.Remington, J. A. Eisen, K. B. Heidelberg, G. Manning, W. Li, L. Jarosze-wski, P. Cieplak, C. S. Miller, H. Li, S. T. Mashiyama, M. P. Joachimiak, C.van Belle, J. M. Chandonia, D. A. Soergel, Y. Zhai, K. Natarajan, S. Lee,B. J. Raphael, V. Bafna, R. Friedman, S. E. Brenner, A. Godzik, D. Eisen-berg, J. E. Dixon, S. S. Taylor, R. L. Strausberg, M. Frazier, and J. C. Venter.2007. The Sorcerer II Global Ocean Sampling expedition: expanding theuniverse of protein families. PLoS Biol. 5:e16.
57. Yutin, N., M. T. Suzuki, H. Teeling, M. Weber, J. C. Venter, D. B. Rusch, andO. Beja. 2007. Assessing diversity and biogeography of aerobic anoxygenicphototrophic bacteria in surface waters of the Atlantic and Pacific Oceansusing the Global Ocean Sampling expedition metagenomes. Environ. Mi-crobiol. 9:1464–1475.
58. Zhang, Y., and V. N. Gladyshev. 2008. Trends in selenium utilization inmarine microbial world revealed through the analysis of the global oceansampling (GOS) project. PLoS Genet. 4:e1000095.
59. Zorin, N. A., and I. N. Gogotov. 1982. Stability of hydrogenase from thepurple sulfur bacteria Thiocapsa roseopersicina. Biokhimiia 47:827–833. (InRussian.)
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