The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii

BBRC Biochemical and Biophysical Research Communications 329 (2005) 966–975 www.elsevier.com/locate/ybbrc

The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii Qi Cheng

a,c,* ,

Anil Day b, Mandy Dowson-Day a, Gui-Fang Shen d, Ray Dixon

a

a Nitrogen Fixation Laboratory, John Innes Centre, Norwich NR4 7UH, UK School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK c Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK d Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China b

Received 2 February 2005

Abstract The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynthesis in the dark in Chlamydomonas reinhardtii, was precisely replaced by either the Klebsiella pneumoniae nifH (encoding the structural component of nitrogenase Fe protein) or the Escherichia coli uidA reporter gene encoding b-glucuronidase. Homoplasmic nifH or uidA transformants were identified by Southern blots after selection on minimal medium plates for several generations. All the uidA transformants had the ‘‘yellow-in-the-dark’’ phenotype characteristic of chlL mutants, whereas homoplasmic nifH transformants exhibited a partial ‘‘green-in-the-dark’’ phenotype. NifH protein was detected in the nifH transformants but not in the wild-type strain by Western blotting. Fluorescence emission measurements also showed the existence of chlorophyll in the dark-grown nifH transformants, but not in the dark-grown uidA transformants. The nifH transplastomic form of C. reinhardtii that lacks the chlL gene can still produce chlorophyll in the dark, suggesting that the nifH product can at least partially substitute for the function of the putative ‘‘chlorophyll iron protein’’ encoded by chlL. Thus, introducing nitrogen fixation gene directly into a chloroplast genome is likely to be feasible and providing a possible way of engineering chloroplasts with functional nitrogenase. Notably, to introduce foreign genes without also introducing selective marker genes, a novel two-step chloroplast transformation strategy has been developed.
2005 Elsevier Inc. All rights reserved. Keywords: Chlamydomonas reinhardtii; Chlorophyll biosynthesis pathway; Chloroplast transformation; Homologous recombination; Transplastomic; Homoplasmic; Heteroplasmic; Klebsiella pneumoniae; Fe protein; Light-independent protochlorophyllide reductase; Light-dependent protochlorophyllide reductase

Over the past two decades, attempt to introduce nitrogen fixation (nif) genes into the chloroplast has been proposed [1] but introducing dinitrogenase reductase subunit into chloroplast via transit peptides appeared to be problematic [2], and the general concern remains that nitrogen fixation is not compatible with photosynthetic oxygen evolution [3]. Meanwhile during *

Corresponding author. Fax: +44 1223 333953. E-mail address: [email protected] (Q. Cheng).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.064

the last two decades, it has become well known that, in addition to the light-dependent chlorophyll biosynthesis pathway, gymnosperms, green algae, and cyanobacteria have a light-independent pathway for chlorophyll biosynthesis in which chlorophyll a and b can be synthesized in complete darkness [4,5]. Three chloroplast genes termed chlL(frxC/gidB), chlN(gidA), and chlB are required for this alternative chlorophyll biosynthesis pathway in photosynthetic eukaryotes [6–8]. Photosynthetic bacteria such as Rhodobacter

Q. Cheng et al. / Biochemical and Biophysical Research Communications 329 (2005) 966–975

capsulatus also contain homologues of these genes which are designated bchL, bchN, and bchB for the light-independent protochlorophyllide reductase and bchX, bchY, and bchZ for chlorin reductase, a second enzyme specific for the bacteriochlorophyll synthesis pathway [9,10]. The amino acid sequences of both the prokaryotic and eukaryotic homologues (plastid) show significant similarities to those of the three subunits of nitrogenase [11,12]. In particular, dinitrogenase reductase Fe protein, NifH, and putative ‘‘chlorophyll Fe proteins’’ ChlL, BchL, and BchX exhibit 32–35% identity. So far, the mechanism of light-independent protochlorophyllide reduction and complex assembly has still largely remained unknown, since the enzyme has not yet been purified as a whole in an active form. Nevertheless, the similarities with nitrogenase suggest that protochlorophyllide reductase may be composed of two protein components analogous to the Mo–Fe protein encoded by the structural genes nifD and K, and the Fe protein encoded by the nifH structural gene. The structure of both nitrogenase components has been determined [13,14]. Fe protein contains a [4Fe–4S] cluster and plays a role in ATP-coupled electron transfer to the other component (Mo–Fe protein plus its co-factor) which carries out dinitrogen reduction (N2 to NH3) [15–17]. The sequence similarities between ChlL and NifH suggest that ChlL contains an ATP-binding site and conserved cysteine residues for liganding the [4Fe–4S] cluster, in addition to two Asp residues postulated to have a role in ATP hydrolysis [8,12]. In vitro reconstitution of the bacterial protochlorophyllide reductase from purified BchL, BchN, and BchB subunits reveals that this enzyme requires ATP and a reductant for activity [18], thus demonstrating that the enzyme has nitrogenase-like features. As a first step to investigate possible functional relationships between these two proteins, we have replaced the chlL gene in the chloroplast genome of C. reinhardtii with the K. pneumoniae nifH gene, thus introducing the structural subunit for nitrogenase Fe protein directly into the plastome of this organism. We find that nifH can functionally replace chlL in the biosynthesis of chlorophyllide to some extent. Such an in vivo system provides a useful tool for understanding the mechanism of nitrogenase-like complex in chloroplast as well as possibly engineering the plastid into a functional nitrogenasecontaining organelle.

Materials and methods Algal strains and culture conditions Chlamydomonas reinhardtii chlL+ strains 137c and cc620 (originally from Chlamydomonas stock centre, Duke University, Durham, USA) were used as the recipient for chloroplast transformation, and petB mutant strain bDO1 was kindly provided by Dr. Michel Goldschmidt-

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Clermont (University of Geneva, Switzerland). Cells were grown on either Tris–acetate phosphate (TAP) medium or ‘‘high salt’’ minimal medium [19] at 25 C on a gyratory shaker at 200 rpm. A 1-liter Bioflo fermenter (New Brunswick Scientific, USA) was used for anaerobic growth. C. reinhardtii cultures were grown under controlled conditions (temperature 25 C, pH 7.5, agitation with DO at 30 mM O2/liter/h). When OD600 of about 1.0 was reached, usually after 4 days, DO (i.e., dissolved oxygen) was changed to 0 to maintain anaerobic conditions and the culture was kept in the dark (by wrapping the fermenter with foil), while temperature and pH were maintained. This culture was grown for 1 day and harvested by centrifugation at 4000g for 10 min. The cell pellet was stored in liquid nitrogen for further experiments. Construction of plasmids for C. reinhardtii chloroplast transformation Construction of a petB::aadA insertion. Plasmid p64A which carries a 7.8 kb EcoRI fragment of C. reinhardtii chloroplast DNA containing the clpP, trnL, petB, chlL, and oriB genes was obtained from Duke University and its complete sequence was provided by Dr. Liu. Xiangqin (Dalhousie University, Canada). The aadA gene cassette was originally constructed in pUC-atp-AAD (provided by Dr. Michel Goldschmidt-Clermont, University of Geneva, Switzerland). By digesting plasmid pUC-atp-AAD with the enzymes EcoRV and SmaI, subsequent isolation of a 1.9 kb fragment carrying ‘‘the atp-aadA-rbcL expression cassette,’’ and then ligating with vector p64A digested with Bst11071 (blunt end, unique in petB coding sequence), we obtained aadA insertions for subsequent C. reinhardtii chloroplast transformation. Plasmid pCQ3 contains aadA in the opposite transcriptional orientation to that petB gene (Fig. 1A) whilst bDO1 carries an aadA insertion in the opposite orientation. Construction of an expression vector for insertions within chlL. The oligonucleotides 1, 2, 3, 4, and were designed to amplify by PCR the 5 0 and 3 0 untranslated regions of chlL, yielding a multiple-cloning-site in expression vector pCQ5 (Table 1). Using oligonucleotides 1 and 2 as primers and plasmid p64A as template, we obtained a 0.7 kb Bst11071–SacI fragment from the PCR. A 3.0 kb C. reinhardtii EcoRI– Bst11071 fragment (extending from petB) was isolated from p64A. The two fragments were ligated into the high-copy vector pTZ18R (Stratagene) digested with EcoRI and SacI yielding plasmid pCQ4A which contains the chlL 5 0 region flanked with EcoRV, BamHI, and SacI sites. Using oligonucleotides 3 and 4 as primers and p64A as template, we obtained a 0.97 kb fragment from a PCR, subsequently isolating a smaller fragment (0.43 kb) by digestion with SacI and NsiI. A 1.1 kb C. reinhardtii NsiI–XbaI fragment (containing partial oriB sequence) was isolated from p64A and the two fragments were then ligated into pCQ4A digested with XbaI and SacI. Following transformation of Escherichia coli strain 71-18, we obtained the expression vector pCQ5 containing both the chlL 5 0 and 3 0 untranslated regulatory regions flanking a multiple-cloning-site, which allows insertion of foreign genes (Fig. 1A). Construction of C. reinhardtii chloroplast transformation vectors for the replacement of chlL with uidA and nifH. Oligonucleotides 5 and 6 were designed for PCR amplification of the 3 0 end of the nifH coding region (Table 1). Using oligonucleotides 5 and 6 as primers and pSA31 [20] as template, we obtained a 0.2 kb fragment carrying the 3 0 end of nifH which was subsequently digested with KpnI and BsmI. A 0.6 kb AhdI–KpnI fragment (containing the rest of the nifH coding region) was isolated from pSA31, and a single base at the 3 0 end of the AhdI site was removed by T4 DNA polymerase. The vector pCQ5 was cut with EcoRV and BsmI, and the three fragments were ligated to give pCQ9 after transformation of E. coli 71-18 (Fig. 1A). The construct pCQ7 (Fig. 1A) carries the entire uidA coding sequence from pBI201.3 [21] in the expression vector pCQ5. To construct this plasmid, pCQ5 was digested with BamHI and AvrII (blunted by fill-in), ligated with a 1.0 kb BamHI–BstI fragment and a 0.7 kb BstI–HinfI fragment (HinfI was also blunted by fill-in) from pBI201.3, and transformed into the uid
E. coli strain KW1.

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Fig. 1. Schematic diagrams of constructs and two-step chloroplast transformation. (A) (a) Chloroplast transformation vector pCQ3. The aadA cassette is inserted in the opposite orientation in the petB coding region; (b) expression vector pCQ5, containing multiple-cloning-site between chlL 5 0 and 3 0 untranslated regulatory sequences for insertion of foreign target genes; (c) secondary chloroplast transformation vector pCQ7, containing uidA gene driven by chlL promoter; (d) secondary chloroplast transformation vector pCQ9, containing nifH gene driven by chlL promoter. (B) Twostep chloroplast transformation via homologous recombination by bombardment vector pCQ3 to obtain petB mutant which was used as a recipient for the secondary transformation by delivering vector pCQ9 bearing nifH gene to obtain C. reinhardtii nifH transplastomic line. C. reinhardtii uidA transplastomic line was also achieved by this strategy.

DNA sequencing Dideoxynucleotide sequencing was performed on double-stranded DNA templates using the Pharmacia T7 Sequencing Kit with [a-35S]dATP as labelled nucleotide. All the PCR regions, except those replaced by the wild-type nifH sequence, were completely sequenced and confirmed to be correct. Chloroplast transformation of C. reinhardtii The transformation protocol was performed by using a home-made particle inflow gun according to previously published procedures [22,23]. All DNA was prepared on CsCl–ethidium bromide density gradients. After transformation, cells were plated on Tris–acetate phosphate (TAP) medium [19] containing 125 lg/ml spectinomycin and incubated under dim light. Homoplasmic lines were isolated by propagating single colonies in liquid cultures and solid medium containing spectinomycin over a period of 3 months. For the secondary chlL::uidA or chlL::nifH transformants, cells were plated on minimal medium and single colonies propagated in liquid and solid minimal media over a period of 6 months. Following this serial propagation on

minimal medium, transformed lines 3B (uidA) and 10E did not produce any dark green colonies when plated on Tap medium in the dark. The strategy is summarized in Fig. 1B. The average efficiency of homologous recombination is 30%. Southern blot hybridization Total cellular DNA was isolated according to a published protocol [24]. About 2 lg of DNA was digested with various restriction enzymes, the digested DNA fragments were separated by electrophoresis on a 0.8% agarose gel, and then transferred to Hybond N+ membranes in alkali, as recommended by the manufacturer (Amersham). Prehybridization (2 h) and hybridization (16–18 h) at 65 C with random-primed 32P-labelled probes were performed in 5· SSC, 5· Denhardt
s solution, and 0.5%(w/v) SDS with denatured sonicated salmon sperm DNA (100 C, 5 min). Filters were washed in 0.5% SDS, 2· SSC at room temperature for 5 min; 0.1% SDS, 2· SSC at room temperature for 15 min; and 0.5% SDS, 0.1· SSC at 37 C for 30 min, and repeated with a change of this solution at 65 C for 30 min. Finally, filters were washed briefly in 0.1· SSC for 1 min.

5 0 -CTÆTCCÆTTAÆAGCÆTGCÆATTÆCTCÆTTCÆCGCÆGGCÆGGTÆTTTÆGCCÆAATÆGATÆG-3 0 AflII BsmI SacII 5 0 -TTATTGCCCTGGCGGAAAAGCT-3 0 5 0 -ATGAAATTAGCTGTTTACGGAAAAGGTGG-3 0 5 0 -AATTTTAAGATAGAAATCTGATAAAAGAGTAAAAAGTTC-3 0

Sequence and restriction sites

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MUG assay

Oligonucleotide 1: (3 0 primer for PCR on pchlL: 56 mer with 29 homologous bases) Oligonucleotide 2: (5 0 primer for PCR of pchlL: 25 mer) Oligonucleotide 3: (5 0 primer for PCR on chlL terminator, 56 mer with 34 homologous bases) Oligonucleotide 4: (3 0 primer for PCR on chlL terminator: 26 mer) Oligonucleotide 5: (3 0 primer for PCR on nifH 3 0 end: 45 mer with 21 homologous bases) Oligonucleotide 6: (5 0 primer for PCR on nifH 3 0 end: 22 mer) Oligonucleotide 7: (5 0 primer for PCR on chlL coding sequence) Oligonucleotide 8: (3 0 primer for PCR on chlL coding sequence)

Name and description

Table 1 List of oligonucleotides designed for PCR

5 0 -CÆATGÆGAGÆCTCÆATGÆGATÆCCTÆAGAÆTATÆCGTÆCATÆAAAATCAAACTCCAGGAATAAAAAC-3 0 SacI BamHI EcoRV 5 0 -TTCTAAACGTTCTTCAAACCAATCG-3 0 5 0 -GAÆTCGÆAGCÆTCCÆTAGÆGTGÆAATÆGCAÆGCTÆTAAÆTAAÆGAAÆTAAÆAGCÆAGCÆTTTÆAAAÆTACÆTTTÆCCT-3 0 SacI AvrII BsmI 5 0 -ATAGGTTACTTACCTCCTTTTGGCGT-3 0

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The fluorogenic assays were performed in microplates. Algal culture (1 ml) was spun down at 10,000g for 5 min. GUS extraction buffer (Sigma) 200 ll was then added, vortexed, and centrifuged at 10,000g for 3–5 min. The supernatant was transferred to a clean tube and stored at
70 C. Assay buffer 180 ll was added to the wells of a microtiter plate and transferred to a 37 C water bath. Sample (20 ll) was added to the appropriate wells and assayed in triplicate. Stop buffer 180 ll was added to the wells of four black fluoroplates after 0, 15, 30, and 60 min incubation. Once all the required samples had been collected, the fluoroplates were read on a Fluorimeter (Tetertek Fluoroscan II). Western blot Proteins were extracted according to [24]. Protein concentrations were determined using the Bio-Rad protein assay reagent kit. Proteins were loaded and electrophoresed on a 10% SDS–PAGE gel and transferred to ECL membrane using a semi-dry transfer method. Blocking was performed with 5% milk in 0.1% TBST for 1 h. Polyclonal K. pneumoniae Fe protein anti-rabbit antiserum was used at 1:2000 dilution, incubated for 4 h at room temperature, and washed with 0.1% TBST for 10 min, three times, and then incubated with 1:10,000 diluted HRP-conjugated anti-rabbit secondary antibody (Amersham) for 1 h. The detection reaction was performed according to the protocol recommended by the manufacturer (Pierce). Pigment analysis Chlamydomonas reinhardtii cells were inoculated from TAP agar plates into 50 ml TAP liquid medium and shaken in flasks at 200 rpm under light. After a week, the cells were subcultured at 1/500 dilution into 50 ml TAP medium and grown for another week in complete darkness. Pigments were extracted according to the method of [7]. Pigments were analyzed for room temperature fluorescence emission spectra using a Perkin Elmer LS-5 luminescence spectrometer with an excitation wavelength at 438 nm. Dark-specific accumulation of protochlorophyllide was monitored at 627 nm, while the characteristic chlorophyll a and b emission spectrum was monitored at 666 and 648 nm, respectively.

Results Introduction of aadA, uidA, and nifH into the plastome of C. reinhardtii petB insertion mutant To create a useful selective marker for recombination within the chlL region of the C. reinhardtii plastome, the petB gene which is adjacent to chlL and encodes a subunit of the cytochrome b6/f complex necessary for photosynthesis [25] was disrupted by a bacterial aadA cassette [26], which encodes aminoglycoside 3 0 -adenylyltransferase conferring spectinomycin and streptomycin resistance. The resultant plastid transformation vector pCQ3 was introduced into the C. reinhardtii plastid genome via homologous recombination by chloroplast transformation using a particle gun (Fig. 1). Transformants were selected on spectinomycin (100 lg/ml), and subcultures were taken to obtain a homoplasmic line.

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Colonies of petB
transformants were photosynthetic mutants and therefore dependent on acetate for growth. The homoplasmic petB
Chlamydomonas transformant was not able to grow in minimal medium and was termed cc620::pCQ3. We also used a second strain, bDO1 [27], for the isolation of secondary transformants in which the aadA cassette in petB was in the opposite orientation to that of pCQ3. Subsequent experiments revealed that the orientation of the aadA cassette did not affect the efficiency of selection for petB+ transformants on minimal medium. The overall efficiency of homologous recombination is about 30%. Replacement of chlL with uidA and nifH The petB::aadA photosynthetic mutants were transformed with plasmids containing the wild-type petB gene together with a modified flanking region in which the coding region of chlL was precisely replaced with either that of nifH or uidA, leaving the 5 0 and 3 0 regulatory regions of chlL intact. The introduction of uidA is to show that the chlL regulatory elements are able to direct expression of a foreign gene but would not expect to complement chlL. Homologous recombination between the plasmid and recipient chloroplast genome should replace the petB::aadA insertion with a functional copy of petB and this should allow transformants to photosynthesize and grow on a medium lacking acetate. Since these transformants are expected to lack aadA, they should be spectinomycin sensitive. The nifH or uidA coding region was introduced into the chloroplast genome of a proportion of petB transformants by a double recombination event in which one event flanks petB and the other occurs upstream of the 5 0 chlL regulatory region. Homologous recombination between the plasmid and recipient chloroplast genome will replace the petB::aadA insertion with a functional copy of petB. Two outcomes are possible depending on the sites of the cross-over event. Only a proportion of events will replace chlL coding regions with uidA or nifH sequences. Cross-over events that flank petB and nifH will introduce both petB and nifH into the genome. Cross-over events that only flank petB will restore the mutant petB gene without swapping the adjacent chlL gene with nifH. All of the transformants selected for further study grew on minimal medium but many exhibited a yellow or partial green phenotype when cultured on acetate medium and incubated in the dark, indicative of replacement of the wild-type chlL sequence by the uidA or nifH sequences. In order to enrich for homoplasmic lines, transformants were subcultured several times on minimal medium prior to further analysis. Inactivation of chlL results in yellow-in-the-dark phenotype [7]. Because plastid DNA is a multicopy sequence initial petB restored transformants containing uidA or nifH will also contain resident plastid genomes containing an inactive

petB [aadA inserted] and chlL. Single colonies of pCQ7 and pCQ9 transformants isolated on minimal medium were re-streaked once on minimal medium. The resulting single colonies were cultured on acetate medium and incubated in the dark. Two phenotypes were observed. Colonies were either dark green or a pale yellow-green. Pale yellow green colonies were subcultured repeatedly in liquid minimal medium and single colonies were isolated after plating. DNA blot analysis showed that 8 of 19 original pCQ7 transformants isolated contained uidA genes while 8 of 37 pCQ9 transformants contained nifH genes. The efficiencies of homologous recombination for uidA and nifH transformations are about 40% and 20%, respectively. No hybridizing bands were detected when DNA from wild-type C. reinhardtii or the petB::aadA insertion mutants were tested with the uidA probe (Fig. 3A, lanes 5 and 6). When total DNA from putative uidA chloroplast transformants was digested with PstI, the 15 kb fragment was detected with the uidA probe (Fig. 3A, lane 3) but not with an internal chlL probe (Fig. 3B, lane 3), in contrast to heteroplasmic uidA transformants in which the chlL signal was still present at 16 kb (Fig. 3B, lanes 2 and 4). We chose one uidA transformant termed 3B for more extensive analysis. When probed with a 1.1 kb BamHI–BstBI internal uidA probe, all the expected hybridizing fragments were detected in the plastome DNA, including the BamHI site introduced as a consequence of the introduction of the uidA coding sequence (Fig. 3C). Similarly, when digested with PstI and probed with the internal chlL probe, no signal could be detected in ‘‘nifH’’ transformant 10E (Fig. 4A, lane 2), in contrast to heteroplasmic nifH transformants and heteroplasmic uidA transformants (Fig. 4A, lanes 3, 4, and 6). We chose one nifH transformant termed 10E to perform digestions with several enzymes and probe with an internal nifH probe. Bands of the expected size were detected with this probe when DNA was digested with various restriction enzymes including the AhdI and BsmI, which cut within the nifH insertion (Fig. 4B). To distinguish homoplasmic and heteroplasmic transformants more clearly, we also used a 3.4 kb probe which flanks the chlL region, extending from the NcoI site in petB to an XbaI site downstream of chlL (Fig. 2). An EcoRI fragment of 7.8 kb is expected in DNA which contains chlL. This fragment should be absent in homoplasmic transformants when digested with a second enzyme which specifically cuts within nifH or uidA, since two shorter fragments are then expected (Figs. 3D and 4C). When total cellular DNA from the nifH transformant 10E was digested with EcoRI and AhdI, and probed with the 3.4 kb probe, the expected two smaller bands (3.7 and 4.1 kb) were detected and no wild-type band (7.8 kb EcoRI fragment) was evident (even after a very long exposure time), suggesting that

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Fig. 2. Restriction sites in PstI fragments of C. reinhardtii wild-type, petB mutant, and C. reinhardtii nifH and uidA transformant strains. Probes used in Southern blot hybridizations are all indicated by bars.

Fig. 3. Southern blot analysis of uidA chloroplast transformants. Total cellular DNA was isolated from C. reinhardtii uidA strains. (A) Hybridization with uidA probe; (B) hybridization with chlL probe. Lane 1, pCQ7/EcoRI; lane 2, uidA strain [pCQ7] 2A/PstI; lane 3, 3B/ PstI; lane 4, 3H/PstI; lane 5, BDO1/PstI; lane 6, cw15+/PstI. (C) Total cellular DNA was isolated from C. reinhardtii uidA strain 3B, digested with various restriction enzymes, and probed with uidA-specific probe. Lane 1, PstI; lane 2, EcoRI; lane 3, EcoRI and BamHI; lane 4, XbaI and BamHI; lane 5, BstBI and BamHI. (D) Total cellular DNA was isolated from homoplasmic C. reinhardtii uidA strain 3B and 3E, digested with EcoRI + BamHI, and probed with the 3.4 kb NcoI–XbaI probe. Lane 1, 7.8 kb EcoRI fragment from plasmid p64A; lane 2, C. reinhardtii uidA strain 3B; lane 3, C. reinhardtii uidA strain 3E.

this nifH transformant is indeed homoplasmic (Fig. 4C). A similar analysis of two uidA transformants 3E and 3B revealed that they are also homoplasmic lines, since digestion with EcoRI and BamHI gave two hybridizing

Fig. 5. Dark-grown C. reinhardtii wild-type strain, uidA and nifH chloroplast transformants show phenotypes both in solid and liquid media. (A) C. reinhardtii wild-type strain (outer circle), uidA (second circle), and nifH (inner circle plus flagella-like Fe letters) chloroplast transformants grown on the same TAP plates after 2 months in the complete darkness; (B) C. reinhardtii wild-type strain, uidA and nifH chloroplast transformants were grown in liquid TAP medium for a week; (C) C. reinhardtii wild-type strain, uidA, and nifH chloroplast transformants grown on separate TAP plates illustrating the best results.

bands of 5.1 and 3.7 kb, and the wild-type 7.8 kb band was absent (Fig. 3D). As precaution, we also performed PCR using chlL-specific oligonucleotides 7 and 8

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fraction of C. reinhardtii, as we never detected it in the pellet fraction following cell-breakage. This is consistent with results obtained previously with the chlL (frxC) gene product from liverwort [6]. The chlL::uidA transformant is deficient in light-independent chlorophyll biosynthesis and the chlL::nifH transformant restores the capacity to synthesize chlorophyll in the dark

Fig. 4. Southern and Western blot analyses of nifH chloroplast transformants. (A) Total cellular DNA was isolated, digested with PstI only and probed with the chlL-specific probe. Lane 1, 32P-labelled 1 kb ladder marker; lane 2, nifH strain 10E; lanes 3 and 4, heteroplasmic nifH strains; lane 5, uidA strain 3B; lane 6, heteroplasmic uidA strain. (B) Total cellular DNA was isolated from homoplasmic C. reinhardtii nifH strain 10E, digested with various restriction enzymes and probed with the nifH-specific probe. Lane 1, PstI; lane 2, EcoRI; lane 3, EcoRI and AhdI; lane 4, XbaI and AhdI; lane 5, BsmI and AhdI. (C) Total cellular DNA was isolated from homoplasmic C. reinhardtii nifH strain 10E and wild-type C. reinhardtii strain, digested with EcoRI + AhdI, and probed with the 3.4 kb NcoI–XbaI probe. Lane 1, Wild-type C. reinhardtii strain cc620; lane 2, C. reinhardtii nifH strain 10E. (D) Western blot of anaerobically grown C. reinhardtii samples. Lane 1, pure Fe protein (1 ng); lane 2, wild-type C. reinhardtii strain cc620; lane 3, C. reinhardtii nifH strain 10E.

(Table 1) on nifH homoplasmic line and no band was picked up even after 45 cycles additionally proving the deficiency of the chlL gene, while on heteroplasmic line a very bright band was detected indicating the existent of wild-type chlL (data not shown). Expression of nifH and uidA We have used the chlL::uidA transformants to investigate the level of expression from the chlL promoter under various growth conditions. The average GUS activities of C. reinhardtii uidA strains were about 10 U (nmol 4-MU/mg/h) under light-grown conditions; 20 U (nmol 4-MU/mg/h) under dark-grown conditions (no significant difference with or without adding 5 lg/ml ALA). GUS activities can reach as high as 50 U (nmol 4-MU/mg/h) under anaerobic conditions. This may explain why the nifH product was only detected in extracts from chlL::nifH 10E transformant grown anaerobically (Fig. 4D). No NifH protein was detected in aerobically grown chlL::nifH 10E cells (not shown). The nifH product is likely to be located in the soluble

Dark-grown C. reinhardtii wild-type strain, uidA and nifH chloroplast transformants show ‘‘green-in-thedark’’, partial ‘‘green-in-the-dark’’ or ‘‘yellow-in-thedark’’ phenotypes both in solid and liquid media (Figs. 5A–C). Disruption of chlL inactivates the dark-dependent pathway of chlorophyll biosynthesis leading to a ‘‘yellow-in-the-dark’’ phenotype [7,28]. Accordingly, the two homoplasmic uidA transformants, 3B and 3E, were yellow when cultured in the dark in either liquid or solid TAP medium, in contrast to strain cc620 and the petB::aadA insertion mutants which were green in the dark (Fig. 5C). However, the homoplasmic nifH transformant was partially green in the dark, suggesting that it may be able to partially substitute for the function of chlL. In order to investigate pigment biosynthesis by the transformants, strains were grown in liquid TAP medium in complete darkness and harvested after 1 week. Pigments were emulsified into diethyl ether and scanned for fluorescence emission. Two different chl+ strains, cc620 and 137c, exhibited the characteristic chlorophyll a and b emission spectrum with maxima at 666 and 648 nm, respectively (Figs. 6A and C). Although cc620, in contrast to 137c, accumulated some protochlorophyllide in the dark, as shown by the minor emission peak at 627 nm, it is competent to synthesize the two chlorophyll species in complete darkness. However, the chlL::uidA transformant 3B accumulated only protochlorophyllide, since the peak at 627 nm was the only major fluorescence emission peak (Fig. 6D). Similar results were obtained previously with a KIXX cassette disruption in chlL [7]. Although, the chlL::nifH transformant 10E accumulated protochlorophyllide to a greater extent than strain cc620, it exhibited an emission maximum at 660 nm and as well as a peak at 647 nm indicating conversion of protochlorophyllide to chlorophyll a and b (Fig. 6B). These results show that the chlL::uidA transformant is deficient in light-independent chlorophyll biosynthesis and chlL::nifH transformant was able to partially restore the capacity for chlorophyll biosynthesis in the dark.

Discussion We have used homologous recombination events to replace precisely the entire chlL coding region in the

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Fig. 6. Fluorescence emission spectral analysis of pigments extracted from dark-grown C. reinhardtii chlL+ strains, uidA, and nifH chloroplast transformants. Pigments were extracted into diethyl ether and scanned for fluorescence emission. (A) C. reinhardtii chlL+ strain cc620; (B) the chlL::nifH transformant 10E; (C) C. reinhardtii chlL+ strain137c; (D) the chlL::uidA transformants 3B.

C. reinhardtii plastome with the eubacterial genes uidA and nifH. As expected, replacement of chlL with uidA inactivates the light-independent pathway of chlorophyll biosynthesis, but remarkably nifH can partially restore the capacity for chlorophyll biosynthesis in the dark. Since K. pneumoniae nifH can complementarily replace the function of C. reinhardtii chlL, by analogy ChlL might also function in ATP-coupled electron transfer to the other components of the light-independent protochlorophyllide reductase (DPOR) encoded by ChlN and ChlB, which have sequence similarities with NifD and NifK [29]. Recent in vitro reconstitution of DPOR with purified BchL, BchN–BchB subunits [18] also indicates the structural and mechanistic similarity between DPOR and nitrogenase. However, the details of the subunit structures, biochemical properties, and requirements for biosynthesis of DPOR are as yet largely unknown. Structural similarities between NifH and the chlorophyll iron proteins
noted previously include: (1) strict conservation of the Walker-type A and B nucleotidebinding motifs and cysteines for liganding the [4Fe–4S] cluster, (2) an arginine or tyrosine residue at position 100 which is critical for electron transfer activity, and (3) mapping of conserved residues onto the Azotobacter vinelandii Fe protein structure suggests common matches within the subunit interior and at the subunit–subunit interface of the homodimer, in addition to the potential for similar ionic interactions with NifK

and by implication, the other components of the protochlorophyllide reductase [12]. It has been proposed that the ‘‘chlorophyll iron proteins’’ evolved from nitrogenase Fe protein as a consequence of a gene duplication event. Our results provide the first evidence that these two proteins are similar in function as well as structure. It is notable that K. pneumoniae NifH is apparently able to function with the other components of protochlorophyllide reductase since, although combinations of Fe protein and Mo–Fe protein from diverse bacteria can result in substantial substrate reduction [30,31], some combinations are not effective [32]. For example, the recent structure of the A. vinelandii Mo–Fe protein:Fe protein co-complex indicates that intersubunit contacts are critical for catalysis and the redox center facilitating electron transfer, signal, and energy transduction [33], but residues determining the specific of the interaction have not yet been defined. The ability of NifH to function partially in the darkdependent chlorophyll biosynthesis pathway raises a number of questions concerning the requirements for the biosynthesis and maintenance of activity of the Fe protein within the chloroplast environment. Fe protein is the most oxygen-sensitive component of nitrogenase and its ability to replace ChlL partially suggests that oxygen-sensitive enzymes may function in chloroplasts when C. reinhardtii is grown in the dark. This is perhaps not surprising since hydrogenase, another oxygen-sensi-

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tive enzyme located in the chloroplast, is active in darkgrown Chlamydomonas or in anaerobically adapted cells grown in the light [34,35]. A further requirement for Fe protein activity is a suitable low potential electron donor. A variety of ferredoxins and flavodoxins can function with nitrogenase [36,37] and since reducing equivalents are generally abundant in the chloroplast this is not likely to present a problem. Assuming that a [4Fe–4S] cluster is required for NifH and ChlL function in the protochlorophyllide reductase complex, the biosynthesis of this cluster is presumably achieved by ancillary proteins. In diazotrophs, the products of two genes, nifS and nifU, are necessary for Fe protein activity, being required for the mobilization of sulfur and iron for Fe–S cluster formation [38–40]. However, homologues of these genes have been found in non-diazotrophic eubacteria, as well as in yeast and humans, suggesting that they may provide a ubiquitous pathway for Fe–S cluster assembly [41,42]. The product of a third gene, nifM, is required for activation and stability of the Fe protein in nitrogen-fixing organisms. When nifH is expressed in either E. coli or yeast in the absence of nifM, a very low level of dimeric Fe protein is synthesized [43–45]. NifM is suggested to have a chaperone-like role in maintaining the apoFe protein in the correct conformation to accept the [4Fe–4S] cluster and the carboxyl-terminal region of the protein.shares homology with peptidyl-proline cis/trans isomerases [46]. The structural similarities between Fe protein and the chlorophyll iron proteins
suggest that an equivalent of nifM must be present in organisms which contain chlL. Since homologues of nifU, nifS of nifM are not present in the liverwort chloroplast genome, we have searched updated Chlamydomonas genome database and identified nifU, nifS of nifM equivalents, respectively, and are currently locating their mutants in order to investigate relevant phenotypes. It is interesting to note that Fe protein not only has a role in transferring electrons to the Mo–Fe protein of nitrogenase, it is also required as an electron donor for the biosynthesis of the Mo–Fe co-factor [47]. If, by analogy, protochlorophyllide reductase contains a metallocluster which participates in substrate reduction, then chlorophyll iron protein
may perform an equivalent role. Our next approach will be the replacement of the putative nifDK-like DPOR component ChlN and ChlB genes with that of Mo-nitrogenase structural nifDK genes. Based on the similar assumption that the ChlNB complex may resemble the NifDK complex harboring a similar metal scaffold, provided by yet unknown DPOR biogenesis proteins in chloroplast, the expectation is to alter the enzymatic DPOR structure towards a functional nitrogenase in vivo. It will no doubt stimulate further studies on the expression of nitrogenase components in plants which ultimately could be aiming

for an auto-fixing N2 chloroplast and also biochemical studies on the structure and function of protochlorophyllide reductase. On the other hand, it would be interesting to investigate what effect it may cause if the C. reinhardtii chlL gene replaces the nifH gene in K. pneumoniae. Nevertheless, this is the first report of the potential function of one of the major nif genes in a eukaryotic cell. Additional aspect of such research may also open avenues for studying evolutionary relationship among the light-independent protochlorophyllide reductase and the highly evolved light-dependent protochlorophyllide reductase (LPOR), and moreover, the so far non-existence of a light-utilizing nitrogenase [48].

Acknowledgments We thank Jacqueline Girard-Bascou and Francis-Andre´ Wollman (Paris) for strain bDO1. We thank John Gray for reading the manuscript and Alison Smith for continuous support. C.Q. is funded by EU (European Union) and BBSRC (Biotechnology and Biological Sciences Research Council).

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