ABSTRACT
Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. N2 fixation is energetically highly expensive, and an energy generating process such as photosynthesis can meet the energy demand of N2 fixation. However, synthesis and expression of nitrogenase is exquisitely sensitive to oxygen. Thus, engineering nitrogen fixation activity in photosynthetic organisms that produce oxygen is challenging. Cyanobacteria are oxygenic photosynthetic prokaryotes, and some of them also fix N2. Here, we demonstrate a feasible way to engineer nitrogenase activity in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. In addition, we have identified the minimal nif cluster required for such activity in Synechocystis 6803. Moreover, nitrogenase activity was significantly improved by increasing the expression levels of nif genes. Importantly, the O2 tolerance of nitrogenase was enhanced by introduction of uptake hydrogenase genes, showing this to be a functional way to improve nitrogenase enzyme activity under micro-oxic conditions. To date, our efforts have resulted in engineered Synechocystis 6803 strains that remarkably have more than 30% N2-fixation activity compared to that in Cyanothece 51142, the highest such activity established in any non-diazotrophic oxygenic photosynthetic organism. This study establishes a baseline towards the ultimate goal of engineering nitrogen fixation ability in crop plants.
IMPORTANCE Application of chemically synthesized nitrogen fertilizers has revolutionized agriculture. However, the energetic costs of such production processes as well as the wide spread application of fertilizers have raised serious environmental issues. A sustainable alternative is to endow crop plants the ability to fix atmospheric N2 in situ. One long-term approach is to transfer all nif genes from a prokaryote to plant cells, and express nitrogenase in an energy-producing organelle, chloroplast or mitochondrion. In this context, Synechocystis 6803, the non-diazotrophic cyanobacterium utilized in this study, provides a model chassis for rapid investigation of the necessary requirements to establish diazotrophy in an oxygenic phototroph.
Enabling crop plants the machinery to fix their own nitrogen via direct transfer of nitrogen fixation (nif) genes is envisioned to be key for the next agricultural revolution (1-3). However, engineering diazotrophic plants, attractive a proposition, will be an extreme challenge, due to the complexities in the biosynthesis of active nitrogenase, the enzyme that catalyzes nitrogen fixation, as well as the difficulty of coupling plant metabolism to supply energy and reducing power for the nitrogen fixation process (4). An additional impediment in the scenario is that photosynthesis produces O2, which is highly toxic to synthesis and activity of nitrogenase (5).
Diazotrophy occurs only in limited species of bacteria and archaea (6). Nitrogen fixation is mainly catalyzed by an iron and molybdenum-dependent nitrogenase enzyme complex, with two enzymatic components, an iron protein dinitrogenase reductase (NifH) and an iron-molybdenum protein dinitrogenase (NifDK) (7, 8). Three metal dependent cofactors, the F-cluster, P-cluster, and M-cluster, are necessary to form the holoenzyme for electron transfer to reduce atmospheric N2 to form ammonia, the biologically available form of N2 (9, 10). A significant number of additional nif genes are required for the biosynthesis of these metallocluster cofactors and for the maturation of nitrogenase to form a fully functional enzyme (11, 12).
Transferring nitrogen fixation to non-diazotrophs has been attempted for decades. To date, the heterotrophic bacterium Escherichia coli has been successfully engineered for nitrogen fixation activity through transfer of nif genes from various diazotrophic species (13-17). Engineering eukaryotic species for heterologous nitrogen fixation activity, including the yeast Saccharomyces cerevisiae and the green alga Chlamydomonas reinhardtii, have been unsuccessful. Limited success was reached only in expressing the NifH component as an active moiety in Chlamydomonas reinhardtii (18). While all the Nif components have been successfully expressed in yeast cells, the formation of a fully functional nitrogenase complex has not been achieved yet (19-22).
Expression of nitrogenase components into plants has also been attempted in a few studies. Individually expression of 16 Nif proteins targeted to the plant mitochondria has been reported recently, but none of the structural components showed enzymatic activity (23). Another recent study showed that an active NifH component can be formed in tobacco chloroplasts (24), indicating that expression of active nitrogenase in chloroplasts might be a viable way in the future to engineer crop plants to fix nitrogen(25). Since it is widely accepted that a cyanobacterial ancestor was the progenitor of chloroplasts (26), engineering a cyanobacterium to fix nitrogen may pave the way to achieve the final goal of engineering nitrogen fixing ability into crop plants. We have utilized the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) as a chassis to engineer nitrogen fixation activity into an oxygenic photosynthetic organism.
The unicellular diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 (hereafter Cyanothece 51142) uses temporal separation as its strategy to protect nitrogenase from O2 produced by photosynthesis (27, 28). Within Cyanothece, the two conflicting processes, photosynthesis and N2-fixation, occur sequentially during the diurnal periods, so that photosynthesis and O2 evolution is performed during the day whereas N2 is fixed at night (29). The energy requirements for nitrogenase are met in Cyanothece by the catabolism of glycogen. Glycogen is accumulated in the light as the storage form of fixed CO2, and later degraded in the dark to provide energy for nitrogenase. The provision of energy coupled with high rates of respiration ensures a low-oxygen intracellular environment and sufficient supplies of energy for N2 fixation (30). The Cyanothece 51142 genome contains the most complete contiguous set of nitrogen fixation and related genes to form a nif cluster, which contains 35 genes (cce_0545 to cce_0579), encoding structural proteins, metal cofactor synthesis proteins, ferredoxins, and genes for proteins with unknown but necessary functions (Fig. 1). All 35 genes exhibit similar oscillating diurnal pattern of transcription during light/dark cycles, showing a high level of transcription in the dark and notably reduced levels in light (27). Such a synchronized transcriptional pattern also confirms that all of these genes are related to nitrogen fixation.
In the current study, we have successfully transferred and expressed this large nif gene cluster in non-diazotrophic Synechocystis 6803 with a resultant N2-fixation activity. Subsequent engineering of the cluster as well its expression levels have led to nitrogenase activities as high as 30% of that in Cyanothece 51142.
RESULTS AND DISCUSSION
Introduction of nif genes into Synechocystis 6803
Synechocystis 6803 has a close phylogenetic relationship with Cyanothece 51142 (Fig. 1A) (31). The large nif cluster from Cyanothece 51142 (28.34 kilobase region of DNA) was transferred into wild type Synechocystis 6803 on a single extra-chromosomal plasmid. This large plasmid pSyNif-1 containing the entire nif cluster with 35 genes (Fig. 1D) was constructed using the DNA assembler method (32). The chassis of this vector was based on pRSF1010 (33), and this self-replicating plasmid pSyNif-1 was transferred into Synechocystis 6803 by conjugation, generating the engineered strain TSyNif-1. Remarkably, over the past four years since its introduction into the heterologous host, pSyNif-1 has been stably maintained in its entirety in Synechocystis (Fig. S1). Furthermore, all introduced Cyanothece genes were transcribed (Fig. 1E) as detected by RT-PCR, indicating that native promoters in the nif cluster from Cyanothece 51142 can drive transcription of genes in Synechocystis 6803. An acetylene reduction assay method for nitrogen fixation detected nitrogenase activity, under 12h/12h light/dark conditions for strain TSyNif-1 (Fig. 2). Nitrogen fixation reached an activity of 2% relative to that in Cyanothece 51142 grown under similar conditions. This is the first time that a non-diazotrophic phototroph has been engineered for biosynthesis of a fully functional nitrogenase enzyme and exhibits detectible and stable nitrogen fixation activity.
The minimal required gene cluster for nitrogen fixation activity
Gene expression parameters for Synechocystis 6803 are not as well understood as for E. coli. Thus, the refactoring of nif genes as performed in E. coli and Klebsiella (16, 34) to determine the minimal requirement of genes for nitrogen fixation in Synechocystis 6803 is impractical at this stage. Therefore, we approached the identification of a minimal nif cluster for nitrogen fixation using a “top-down” method, which determines the influence of a gene on nitrogenase activity by selectively removing individual genes from the nif cluster (Fig. 2). Extrapolating the genetic requirements for nitrogen fixation activity observed in studies in which nif genes were introduced in E. coli (14, 15), we determined that genes for all homologous proteins introduced into E. coli are present in the Cyanothece 51142 nif cluster between gene nifT and hesB (Fig. 1). Hence, our second plasmid, pSyNif-2, contains 24 genes in the nif cluster between nifT and hesB (Fig. 2). Eleven genes were removed that presumably encode three metal transporter proteins, two ferredoxins, and six proteins of unknown function, none of which have been analyzed previously, although they are associated with nitrogen fixation. Intriguingly, this second engineered strain TSyNif-2 with a reduced cluster of 24 genes has a 3-fold increase in nitrogen fixation activity when compared to strain TSyNif-1 (Fig. 2). Although plasmids pSyNif-1 and pSyNif-2 have the same plasmid backbone (Fig. S2), the transcriptional levels of the structural genes nifH, nifD and nifK are higher in the TSyNif-2 strain (Fig. 3). This improvement in nitrogenase activity could be the result of removal of one or more regulatory gene(s), which may encode protein(s) that repress expression of genes in the nif cluster.
However, further removal of genes from both directions, resulted in a decrease of nitrogen fixation activity by more than ten-fold for strains TSyNif-3 to TSyNif-6, in which the genes hesB, hesAB, nifT, and nifTZ, were removed, respectively (Fig. 2). Thus, this “top-down” approach determined that an essential minimal cluster from nifT to hesB is required for nitrogen fixation activity in Synechocystis 6803. Additionally, we investigated the removal of two more genes in the cluster, nifX and nifW, generating the two strains TSyNif-7 and TSyNif-8. Deletion of these two genes did not affect expression of surrounding genes (Fig. S3). However, nitrogen fixation activity dropped 100-fold and 10-fold, respectively, in these engineered strains (Fig. 2). We conclude that both nifX and nifW are important genes for nitrogen fixation. Notably, nifX exhibited a positive influence on N2 fixation in cyanobacteria, while it functions as a negative regulator for N2 fixation in the heterotrophic diazotroph Klebsiella oxytoca (35).
Improvement of nitrogen fixation activity
To increase the RNA expression levels of the nitrogenase related genes, we took advantage of three small endogenous plasmids in Synechocystis 6803, pCA2.4, pCB2.4, and pCC5.2. The expression of heterologous genes expressed from these endogenous plasmids maintains higher transcriptional levels than from an pRSF1010 based plasmid, because of the higher copy numbers of these three plasmids within Synechocystis (36, 37). First, we replaced the RSF1010 backbone of plasmid pSyNif-2 by the entire DNA segment of each of these endogenous episomes and then transferred the plasmids to Synechocystis 6803, generating three strains TSyNif-9, TSyNif-10, and TSyNif-11, with the chassis of pCA2.4, pCB2.4, and pCC5.2, respectively (Fig. 3A and Fig. S4). As expected, the expression levels of genes nifH, nifD and nifK in strain TSyNif-9 showed several fold higher transcription levels compared to in TSyNif-2 (Fig. 3B). In addition, nitrogen fixation activity was increased by another 2 to 3-fold, reaching 13% of the acetylene reduction activity in TSyNif-9 relative to that observed in Cyanothece 51142 (Fig. 3C). Next, nitrogenase activity was directly assayed in the engineered strains using a 15N assimilation assay method. Remarkably, the highest activity obtained was from strain TSyNif-9, reaching 31% of 15N assimilation relative to Cyanothece 51142 (Fig. 3D). The activity data presented here is comparable to published data from studies on nitrogen fixation activity in engineered in E. coli (Table 1). Additionally, the dinitrogenase protein NifD in whole cell extracts was detected via Western blotting by using antisera from Rhodospirillum rubrum (Fig. 3E). Although the MoFe-protein level in Cyanothece 51142 is significantly higher than in the engineered Synechocystis 6803 strains, the protein level in strain TSyNif-9 reached 10% of total cellular proteins (Fig. S4). It was also evident that the nitrogenase activities in the engineered strains were proportional to the level of nitrogenase structural proteins, which implied that optimizing the expression of nitrogenase proteins is critical for the activity. Most importantly from an evolutionary standpoint, these results highlight the potential for engineering plant chloroplasts to fix nitrogen at a high level of activity, since oxygenic cyanobacteria are the progenitors of chloroplasts.
Nitrogen fixation activity in Synechocystis 6803
Despite an additional metabolic load of expressing large cohorts of 35 or 24 genes related to nitrogen fixation being introduced in Synechocystis 6803, remarkably, the expression and activities of these heterologous proteins did not affect the growth of the engineered strains under diurnal light/dark conditions (Fig. S1). We used strain TSyNif-2 to assess the influence of oxygen and exogenous nitrate on nitrogenase activity under four conditions, BG11, BG110 (BG11 without nitrate), BG11 with 10 μM DCMU (no O2 evolution) and BG110 with 10 μM DCMU (Fig. S5). Interestingly, transcript levels of nifH, nifD and nifK genes in the TSyNif-2 strain were downregulated by nitrate, which is similar to that in Cyanothece 51142. Specifically, the depletion of nitrate improved the nitrogenase activity over 30-fold in BG110 with DCMU (Fig. S5). Nitrogen fixation activity was obtained only in anaerobic environment when DCMU was added to the testing culture, although the headspace of all cultures was flushed with pure argon. These data indicate that oxygen generated by photosynthesis directly blocks nitrogenase activity in TSyNif-2, highlighting that one of the biggest challenges for engineering nitrogen fixation in oxygenic phototrophs is the sensitivity of nitrogenase to oxygen.
Improvement of O2 tolerance by introduction of uptake hydrogenase
In order to test the oxygen sensitivity of nitrogen fixation activity in TSyNif-2, a measured amount of oxygen was added to the headspace to cultures grown in BG110 media, to generate micro-aerobic conditions of 0.5% and 1.0% of O2 in the sealed testing bottles. The activity dropped more than 10-fold and 60-fold (Fig. 4A), respectively, demonstrating that as expected, nitrogen fixation activity in engineered Synechocystis 6803 is highly sensitive to O2. To enhance O2 tolerance under these same conditions, genes coding for the uptake hydrogenase enzyme from Cyanothece 51142 were introduced into the chromosome of the TSyNif-2 strain. The uptake hydrogenase is conserved in diazotrophic cyanobacteria (38), and has been shown to be necessary for nitrogen fixation under aerobic conditions in Cyanothece (39). The structural genes hupS and hupL, present together in a single operon in Cyanothece 51142, were transformed into TSyNif-2, generating strain TSyNif-12 (Fig. 4B). In addition to the structural genes hupSL, a protease encoded by hupW is also present in Cyanothece 51142. HupW is required for the maturation of HupL protein through the processing of its C-terminus (40). So, the hupSLW genes organized in two operons were transformed into TSynif-2 to generate the TSyNif-13 strain (Fig. 4B). The expression of hup genes in TSyNif-12 and TSyNif-13 was assessed by RT-PCR (Fig. S6). The introduction of the uptake hydrogenase did not affect nitrogen fixation activity under anaerobic conditions (Fig. 4C). Interestingly, under micro-aerobic conditions, nitrogen fixation activity markedly improved with the expression of uptake hydrogenase, especially for strain TSyNif-13, a 2-fold and a 6-fold increase in TSyNif-2 for O2 levels of 0.5% and 1.0%, respectively. The above results suggest that expression of uptake hydrogenase proves to be highly effective in enhancing O2 tolerance of nitrogen fixation activity in the engineered Synechocystis strain.
In this study, introduction of the nif gene cluster from Cyanothece 51142 has enabled nitrogen fixation activity in Synechocystis 6803. The minimal cluster for 24 genes (Fig. 2) for nitrogenase activity will provide a useful framework for future studies to further enhance such activity by refactoring genes as done in Klebsiella oxytoca (34). Although uptake hydrogenase is a complex enzyme, introduction of its structural genes and a protease works as a protecting mechanism from the toxicity of O2. The O2 toxicity to nitrogenase is likely the most difficult aspect to overcome to achieve nitrogen fixation activity under aerobic conditions.
A fully functional nitrogenase holoenzyme, requires 8 electrons and 16 ATPs to reduce one molecule of N2 to ammonia. Thus, metabolism within cells needs to be adjusted to supply enough reducing power and energy for nitrogen fixation. Biosynthesis of fully functional nitrogenase is a complex process. This complexity increases the difficulty to find the minimal genes and best ratios between each protein expressed from the nif cluster in Synechocystis 6803. The same gene designations in different species occasionally have alternative functions (Table S1). An example is the gene nifX, which functions as a negative regulator in Klebsiella oxytoca (35). Gene nifX is of importance in Cyanothece, since deletion of nifX affects nitrogenase activity.
Although multiple challenges and many barriers exist to enable plants to efficiently fix atmospheric nitrogen, we have engineered an oxygenic photosynthetic cell to fix N2 by reconfiguring the genetic processes for nitrogen fixation from Cyanothece 51142 to function in Synechocystis 6803. Our studies to date have established the highest rate of engineered nitrogen fixation activity in any non-diazotrophic oxygenic organism.
MATERIALS AND METHODS
Microorganisms, culture conditions and media
All cyanobacterial strains, including Cyanothece 51142, Synechocystis 6803, and the engineered strains (listed in Table S2), were cultured in 100-mL flasks of fresh BG11 medium (41) with appropriate antibiotics (20 μg/ml kanamycin, 15 μg/ml chloramphenicol, or 20 μg/ml spectinomycin). As a pre-culture, cells were grown at 30°C, with 150 rpm shaking, and under 50 μmol photons · m−2 · s−1 constant light. For the nitrogen fixation activity assay, unless otherwise stated, pre-cultured cells were collected and washed with fresh BG11 without nitrate (BG110) medium and resuspended in 500 ml fresh BG110 medium. Cells were then grown at 30°C with air bubbling under 12h light/dark conditions with 50 μmol photons · m−2· s−1 of light. Yeast and E. coli strains (listed in Table S2) used for construction of recombinant plasmids were grown with 200 rpm shaking in YPAD (42) and LB medium at 30°C and 37°C, respectively.
Construction of recombinant plasmids and engineered strains
Plasmids and strains used in this study are listed in Table S2, and all primers are listed in Table S3. Two methods were used to construct the plasmids, DNA assembler (32) and Gibson assembly (43). To build the large pSyNif-1 and pSyNif-2 plasmids containing the nif genes, genomic DNA from Cyanothece 51142 was used as the template for PCR, and all DNA fragments were combined using the DNA assembler method to construct the plasmids in the yeast S. cerevisiae. For the other recombinant plasmids listed in Table S2, Gibson assembly method was used to construct them with DNA fragments amplified by PCR. Genomic DNA from Cyanothece 51142 and the large plasmid pSyNif-2 were used as templates for PCR to construct the plasmids for backbone replacements, the plasmids containing the uptake hydrogenase genes, and the plasmids used to remove specific nif genes, respectively.
Plasmids pSyNif-1 and pSyNif-2 were introduced into Synechocystis 6803 wild-type strain through the method of tri-parental conjugation (44) to form strains TSyNif-1 and TSyNif-2, respectively. The other recombinant plasmids were transformed into Synechocystis 6803 by natural transformation (45), and double homologous recombination integrated the fragments into the chromosome (for the uptake hydrogenase genes) or the plasmid (for the plasmid backbone replacement, and specific removal of nif genes).
Reverse transcription-PCR (RT-PCR) and Quantitative-PCR (q-PCR)
RT-PCR analysis was performed using RNA samples isolated from culture grown in BG110 medium at time point D1 (1 hour into the dark period) under light/dark conditions. After extraction and quantification of RNA (46), 100 ng of DNase-treated RNA samples were used for reverse transcription, using the Superscript II Reverse Transcriptase and random primers (Invitrogen) according to the manufacturer’s instructions. cDNA generated after reverse transcription was used as the template for PCR to evaluate the transcription levels of various individual genes.
The q-PCR was performed on RNA samples extracted from culture grown in BG110 medium under light/dark conditions as previously described (27). Briefly, QRT-PCR SYBR green dUTP mix (Abgene) was used for the assay on an ABI 7500 system (Applied Biosystems). Each reaction was performed in three replicates, and the average CT was used to calculate the relative transcriptional levels for the amount of RNA. All primers used for RT-PCR and q-PCR are listed in Table S3.
Measurement of nitrogen fixation activity
Nitrogen fixation activity was measured by an acetylene reduction assay (47), modified from a previously published method (48). Unless otherwise stated, the activity assay was performed as follows: 25 ml of cyanobacterial culture were grown in BG110 medium with air bubbling under light/dark conditions as mentioned above, and transferred to a 125 ml air-tight glass vial. 10 μM DCMU was added to the culture, vials were flushed with pure argon, and cultures were incubated in 12h light/dark conditions. Cells in the sealed vials were cultured overnight and at the time point D1, 5 ml acetylene was added into the sealed vials, followed by 3 hours of incubation in light at 30°C. 200 μl of gas was sampled from the headspace and injected into an Agilent 6890N Gas Chromatograph equipped with a Poropak N column and a flame ionization detector, using argon as the carrier gas. The temperature of the detector, injector, and oven were 200°C, 150°C and 100°C, respectively.
Total protein levels were determined on a plate reader (Bio-Tek Instruments, Winooski, VT) using a BCA-assay kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. Total chlorophyll a was methanol extracted and quantified on an Olis DW2000 spectrophotometer (On-Line Instrument Systems, Inc., GA).
In vivo15N2 incorporation assay
All strains were grown under light/dark conditions as mentioned above, and 50 mL cultures were transferred into 125 mL airtight glass vials. 10 μM DCMU was added to the cultures, vials were flushed with pure N2, and cultures were incubated under light/dark conditions. Cells in the sealed vials were cultured overnight and at the time point D1, 8 mL of headspace gas was removed, followed by injection of 8 mL of 15N2 gas (98%+; Cambridge Isotope Laboratories, Inc.). After 8 h of incubation at 30°C in light (50 μmol photons · m−2 · s−1), the cultures were collected and dried in a laboratory oven at 50°C-60°C for 24 h. The dried pellets were ground, weighed, and sealed into tin capsules. Isotope ratios were measured by Elemental Analyzer-Isotope Ratio Mass Spectrometry (EA-IRMS, Thermo Fisher Scientific), and are showed as δ15N (‰), of which the number is a linear transform of the isotope ratios 15N/14N, representing the per mille difference between the isotope ratios in a sample and in atmospheric N2 (49). Data presented are mean values based on at least two biological replicate cultures.
Western blot analysis
The coding region of the nifD gene (cce_0560) from Cyanothece 51142 was PCR-amplified from the genomic DNA of Cyanothece 51142, using the primers shown in Table S3. The PCR fragment was ligated into the expression vector pET28a cleaved by NdeI and BamHI. The resulting plasmid pET28a-nifD was used to produce the NifD protein with an N-terminal His6 tag. For overproduction of the NifD protein, Escherichia coli BL21 (DE3) was transformed with plasmid pET28a-nifD and cultivated in LB medium at 37 °C to an optical density at 600 nm (A600) of 0.3. Protein expression was induced by the addition of 0.2 mM isopropyl-β-D-thiogalactopyranoside, and the culture was incubated for another 18 h at 20 °C. After the cells were harvested, purification of NifD by nickel-nitrilotriacetic acid affinity chromatography was performed. Briefly, harvested E. coli cells were resuspended in 20 mM HEPES buffer (pH 7.0) containing 100 mM NaCl, and 2 mM β-mercaptoethanol supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Lysozyme was added to a concentration of 1 mg/ml, and the cells were lysed by freezing-thawing, followed by sonication. After cells were centrifuged at 13,000 rpm, Tris-HCl buffer (pH 8.0) was added to the supernatant (final concentration, 50 mM), and it was loaded onto a Ni-NTA agarose column (0.2 ml). After bound proteins were washed with the starting buffer containing 1 M NaCl, they were eluted with 0.3 ml of the starting buffer containing 250 mM imidazole. The purified protein was stored at -20 °C and used as the positive control for western blot assay.
Cyanobacterial cells cultured in N-free medium under L/D cycles were collected at time point D4 (4 hours after dark phase) and resuspended in 0.5 mL TG buffer (10 mM Tris-HCl [pH 8.0], 10% glycerol) containing a protease inhibitor cocktail (Sigma-Aldrich). A 0.5 mL volume of sterilized, acid-washed glass beads was added to the cells, and the mixture was disrupted using a bead beater (BioSpec Products). The resultant mixture was centrifuged for 10 min at 7,500 × g, and the supernatant was transferred into a new tube. The amount of protein was determined using a bicinchoninic acid (BCA) protein assay reagent (Thermo Scientific).
Fifteen μg of total protein extract from each sample was solubilized with 8 × sample buffer (10 ml of 0.5 M Tris [pH 6.8], 15 ml of 70% glycerol, 8 ml of 20% sodium dodecyl sulfate, 4 ml of β-mercaptoethanol, 4 ml of 0.1% bromophenol blue) at 70 °C for 10 min and fractionated on a sodium dodecyl sulfate (0.1%, wt/vol)-polyacrylamide (12.5%, wt/vol) gel by electrophoresis. After electrophoresis, proteins were eletrotransferred to a polyvinylidene fluoride (PVDF) membrane (Millipore), blocked with 5% bovine serum albumin (BSA) for 2 hours at room temperature, and then incubated with the primary antibodies against the NifD protein from Rhodospirillum rubrum (50, 51) diluted in 1.5% BSA (1:2,000) overnight at 4 °C. The HRP-conjugated secondary antibody Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (Bio-Rad) was diluted at 1:5,000 in 1.5% BSA. Immunodetection was performed using Western blotting Luminol Reagent (Millipore).
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
This study was supported by the National Science Foundation (MCB-1331194). We thank Prof. Huimin Zhao and his research group (University of Illinois) for introducing us to the use of the DNA Assembler method; Lingxia Zhao, Xiujun Duan, and Alicia Lohman for expert technical assistance and members of the research groups of Himadri Pakrasi, Costas Maranas, Tae Seok Moon, and Fuzhong Zhang for critical scientific discussions. D.L., M.L., J.Y., H.B.P. and M.B.P. designed the experiments; D.L., M.L., J.Y. and M.B.P. performed the experiments; D.L., M.L., H.B.P. and M.B.P. wrote the paper.