Abstract
Transfer of nitrogen fixation (nif) genes from diazotrophs to amenable heterologous hosts is of increasing interest to genetically engineer nitrogen fixation. However, how the non-diazotrophic host maximizes opportunities to fine-tune the acquired capacity for nitrogen fixation has not been fully explored. In this study, a global investigation of an engineered nitrogen-fixing Escherichia coli strain EN-01 harboring a heterologous nif island from Pseudomonas stutzeri was performed via transcriptomics and proteomics analyses. A total of 1156 genes and 206 discriminative proteins were found to be significantly altered when cells were incubated under nitrogen-fixation conditions. Pathways for regulation, metabolic flux and oxygen protection to nitrogenase were particularly discussed. An NtrC-dependent regulatory coupling between E. coli nitrogen regulation system and nif genes was established. Additionally, pentose phosphate pathway was proposed to serve as the primary route for glucose catabolism and energy supply to nitrogenase. Meanwhile, HPLC analysis indicated that organic acids produced by EN-01 might have negative effects on nitrogenase activity. This study provides a global view of the complex network underlying the acquired nif genes in the recombinant E. coli and also provides clues for the optimization and redesign of robust nitrogen-fixing organisms to improve nitrogenase efficiency by overcoming regulatory or metabolic obstacles.
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Introduction
In nature, a variety of genes and islands can be rapidly and frequently horizontally transferred among bacteria, resulting in the acquisition of certain properties such as nitrogen fixation, antimicrobial resistance and pathogenesis, which help bacteria to succeed in altered habitats or new niches1,2,3. However, newly acquired genes or islands become a burden for bacteria if they are not properly integrated with host regulatory systems. A greater understanding of the physiological alterations that occur in an engineered cell following the insertion of large fragments of foreign DNA, especially from distant species, is needed to pave the way towards the goal of biological engineering.
Biological nitrogen fixation is catalyzed, in most cases, by the molybdenum nitrogenase encoded by a highly conserved nifHDK gene cluster. Previous studies have shown that the nif genes encoding active nitrogenase can be transferred to non-nitrogen-fixing prokaryotes to impart the ability to reduce atmospheric nitrogen gas into ammonia as a nitrogen source4,5,6,7,8,9,10,11,12. From the perspective of synthetic biology, one key goal of studying biological nitrogen fixation is to facilitate the introduction of this ability into organisms of great importance for human beings, for instance, engineering autonomous nitrogen-fixing cereal crops13,14,15. Eventually, a successfully engineered N2-fixing non-legume crop may significantly cut down the use of chemical fertilizers for a cleaner environment and higher yields16,17.
In recent years, the synthesis of nitrogen-fixing systems has become increasingly common due to advances in synthetic biology. Normally, a functional entity or pathway is first detected in prokaryotes before being transferred to eukaryotes and plants. Because of its well-studied genetic background, Escherichia coli is frequently used as the preferred first-step research model. Following the pioneering work on nitrogen-fixation engineering in 1970s4,5, several groups have reported successful gene transfer of nif genes to E. coli in the past five years11,12,18,19. However, these recombinant E. coli stains showed much lower nitrogenase activity compared with the original host11,12,, and the horizontally acquired ability was insufficient to enable diazotrophic growth on nitrogen-free medium, implying the presence of (i) regulatory coupling between the host and heterologous nitrogen-fixation systems, as well as (ii) a regulatory/or metabolic barrier that results in reduced nitrogenase activity in the engineered cells. To date, how the non-diazotrophic host maximizes opportunities to fine-tune the acquired capacity for nitrogen fixation has not yet been fully explored.
Pseudomonas stutzeri A1501 is a root-associated bacterium that exhibits an unusual feature, for a Pseudomonas strain, the ability to fix nitrogen20,21,22,23,24. The P. stutzeri A1501 genome contains a 49-kb nitrogen fixation island (NFI) that comprises the largest group of nif genes identified to date25. Within this island, a total of 52 nif-related genes are organized into 11 putative NifA-δ54-dependent operons24. nif gene expression in A1501 was revealed to be tightly regulated at both the transcriptional and post-transcriptional levels22,23,26,27. Given its natural integrity and well-studied regulation, the A1501 NFI is a promising model for studying the synthetic biology of nitrogen fixation systems.
We previously transferred the entire P. stutzeri A1501 NFI into E. coli and found that the nitrogenase activity of the engineered E. coli strain was dependent on the external ammonium concentration, oxygen tension and temperature12. Similar to previous reports, the nitrogenase activity of recombinant E. coli strain EN-01 was much lower than that of A1501. In the present study, to better understand the global regulatory effect of the host on NFI expression, we monitored the global transcriptional and proteomic profiles of recombinant E. coli grown anaerobically under nitrogen-fixation and nitrogen-repression conditions. Furthermore, the metabolic flux shift of E. coli EN-01 under nitrogen-fixation conditions was also determined by HPLC. To the best of our knowledge, this is the first report of a global investigation into the regulatory cascade of nif genes in an engineered nitrogen-fixing organism. These data are particularly useful for providing a more comprehensive understanding of how the E. coli host intervenes in the transcriptional regulation of the “foreign” NFI to support a functional nitrogenase complex. Our results will also help guide efforts to more successfully remodel and optimize similar systems in other species.
Results
Overview of the E. coli EN-01 transcriptome and proteome under nitrogen-fixation and nitrogen-repression conditions
The expression of 1156 genes was significantly altered (≥2-fold, P ≤ 0.05) under nitrogen-fixation conditions compared with nitrogen-repression conditions, including 789 up-regulated and 367 down-regulated genes. The up-regulated genes mainly belonged to three major functional categories: nitrogen metabolism, transport or membrane protein and unknown function, while the down-regulated genes were mainly involved in energy synthesis, transport, protein synthesis and regulation. These altered genes were further classified according to the COG functional classification system. As shown in Fig. 1, 259 genes, of which 102 were down-regulated and 157 were up-regulated, were involved in bacterial preservation and processing of genetic information (DNA replication, duplication, repair and gene transcription, expression, etc.). A total of 361 genes (31% of the total altered genes) were involved in transport and metabolic pathways. An additional 90 genes were involved in energy synthesis and transformation processes, with 67 genes induced under nitrogen-fixation conditions (Fig. 1A). Moreover, the expression of 151 genes encoding proteins of unknown function was also significant altered.
Transcriptome analysis has revealed that 255 genes were significantly up-regulated and 294 genes were severely down-regulated in P. stutzeri A1501 under nitrogen-fixation conditions25. Subsequent comparison of the E. coli EN-01 and P. stutzeri A1501 transcriptomes revealed that at least 24 genes in the E. coli EN-01 transcriptome showed a similar expression pattern to that in P. stutzeri A1501 (Supplementary Table S1), including the glnK-amtB operon, the two-component regulatory system ntrBC andthe serine protein kinase-coding gene prkA, implying that the overlapping genes or systems might be essential for NFI expression in E. coli.
Proteomic analysis of E. coli EN-01 was also performed under identical conditions. A total of 110 protein spots increased by more than 2-fold under nitrogen-fixation conditions; notably, 55 proteins were only expressed under nitrogen-fixation conditions. In addition, 96 proteins were significantly down-regulated under nitrogen-fixation conditions, and the expression of 24 protein spots was abolished on the 2D-PAGE gel (Supplementary Fig. S1). A total of 138 proteins were further identified by MALDI-TOF mass spectrometry, of which 63 were up-regulated and 75 were down-regulated (Supplementary Table S2). In addition, 132 identified proteins showed similar expression patterns to the transcriptional analysis. These proteins included the glutamine synthetase (GS) GlnA, nitrogen regulatory PII protein GlnK, hydroperoxidase II KatE, oligopeptide ABC transporter OppA, glucose-6-phosphate 1-dehydrogenase Zwf and the dihydrolipoamide dehydrogenase LpdA.
Taken together, the above data revealed dramatic changes to the global transcriptional regulatory networks and cellular metabolic pathways in the recombinant nitrogen-fixing E. coli EN-01 under nitrogen-fixation conditions. Subsequent analyses focused on the nitrogen regulatory system, energy production and conversion systems, and the oxygen protection-related pathway, which have been reported to be crucial for nitrogen fixation28.
Expression patterns of E. coli nitrogen metabolic pathways and regulatory system under nitrogen-fixation conditions
Expression of P. stutzeri NFI genes in recombinant E. coli strain EN-01
EN-01 exhibits nitrogenase activity under glucose-fermentation and nitrogen-fixation conditions, indicating the heterologous production of a functional nitrogenase complex12. To further test the heterologous expression of nif genes, we determined the induction ratio of 43 genes within the island using quantitative real-time RT-PCR (Table 1). Previously, we found that the A1501 NFI genes are organized into 11 NifA-σ54-dependent operons and are up-regulated under nitrogen-fixation conditions24. Similarly, except for a 7-gene operon (PST1302-1306), all NifA-σ54-dependent operons were strongly induced in E. coli, suggesting that the nif-specific transcriptional activator NifA is controlled by the E. coli nitrogen regulatory system. Consistent with the RT-PCR results, proteomic analysis also revealed that the NifS, NifD and PST1336 proteins encoded by the NFI genes were up-regulated under nitrogen-fixation conditions (Fig. 2). Why the PST1302-1306 operon (including the nifQ and nifB genes) was not regulated in EN-01 is still unclear. The nifQ mutant of Klebsiella pneumoniae is defective in nitrogen fixation due to an elevated requirement for molybdenum29. In addition, nifB has been long recognized as crucial for nitrogen fixation because NifB participates in the biosynthesis of the FeMo-co factor30. We therefore postulate that the relatively low nitrogenase activity of EN-01 might be partly attributed to low expression of these factors within the operon.
Regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island
Regulation of the nitrogen-fixation process in free-living diazotrophs facilitates the stringent control that is necessary to maximize the physiological benefits from diazotrophy28. In P. stutzeri A1501, the nitrogen regulatory cascade comprises the AmtB–GlnK–NtrBC global nitrogen regulation proteins, which sense the nitrogen signal and subsequently control the expression of the nif-specific regulatory proteins NifLA31. However, the nitrogen regulatory network in E. coli appears to be an expanded version compared to that in Pseudomonas, with one additional PII protein and cascade regulation achieved by a Nac regulatory protein32,33. Acquisition of a nif-specific regulatory system in the recombinant E. coli raises the question of nitrogen-fixation efficiency by these two regulatory systems of different evolutionary origins.
Changes in the expression of E. coli nitrogen regulatory system genes are summarized in Supplementary Table S3. Of these, the glnLG genes, which are homologs of Pseudomonas ntrBC, were up-regulated by more than 10-fold, the sigma factor rpoN was up-regulated by 3.64-fold, the ammonium transporter amtB was up-regulated by 55.28-fold, and the two PII protein genes glnK and glnB were both up-regulated under nitrogen-fixation conditions. Consistent with the transcriptional change, proteomic data indicated that GlnK and GlnA expression was enhanced (Supplementary Table S2). Based on the observations in the present study and those from previous experiments from a wide range of model systems, we propose a regulatory cascade that controls heterologous nif transcription in E. coli EN-01 (Fig. 3): The UTase/URase enzyme GlnD (glnD gene was up-regulated 5.27-fold) is activated by low internal glutamine concentrations and further modifies the PII protein GlnB (glnB was up-regulated 2.12-fold) and GlnK (glnK was up-regulated 86.88-fold) to PII-UMP. Subsequently, PII-UMP phosphorylates NtrC. As a global transcriptional regulator, NtrC activates the expression of a series of nitrogen metabolic genes, including the GS-encoding gene glnA (up-regulated 7.61-fold), the nitrogen transcriptional regulation (Ntr) genes, the ammonium transporter gene amtB (up-regulated 55.28-fold), the transcriptional regulator nac (up-regulated 124.98-fold) and the nif-specific regulator genes nifLA. In fact, we previously demonstrated cross-complementation of the P. stutzeri /or E. coli ntrC (glnG) mutant by the E. coli /or the P. stutzeri ntrC gene34. We have also shown that the purified E. coli NtrC protein can bind specifically to the nifLA operon promoter region12. In a sense, this working model supports a similar expression pattern for the NFI genes in E. coli with that observed in P. stutzeri A1501, in that the nif genes were subject to regulation by the NtrC protein via the nif-specific regulator NifA, suggesting a regulatory coupling of these two different evolutionary systems through a direct activating interaction.
The PII protein is recognized as a critical signal transduction protein in bacterial nitrogen metabolism35. E. coli encodes two PII counterparts, GlnB and GlnK, which show 68% and 78% identity, respectively, with the sole A1501 PII protein GlnK and share 67% identity with each other (Fig. 4). Under nitrogen-fixation conditions, E. coli EN-01 glnK mRNA expression was increased by 86.88-fold, while the glnB gene was up-regulated by 2.12-fold. GlnK protein was only detected under nitrogen-fixation conditions (Fig. 2). In A1501, an interaction between GlnK and the C-terminal domain of NifL was observed using the yeast two-hybrid system23, suggesting GlnK-dependent control of NifA activity by NifL. Based on the overall transcription of the nitrogen regulation system in E. coli EN-01, we conjectured that, as reported before35,36, GlnB and GlnK may constitute a two-tiered regulatory system and that their functions may depend on the timing of expression and the levels of accumulation, while glnK may play key roles in nitrogen regulation in E. coli to adjust to perturbations that occur under nitrogen-fixation conditions.
Rearrangement of the pathways for nitrogen assimilation and alternative nitrogen sources
Glutamate and glutamine provide approximately 75% and 25% of cellular nitrogen, respectively. Nitrogen assimilation must therefore result in the synthesis of these two nitrogen donors37. The GDH pathway, encoded by the glutamate dehydrogenase gene gdhA, is generally associated with a nitrogen-rich, aerobic environment, whereas the GS-GOGAT pathway, encoded by the GS gene glnA and the glutamate synthase (GOGAT) genes gltBD, is often associated with a nitrogen-limited, microaerobic environment37,38. Transcriptomic analyses showed that both operons of the GS-GOGAT system were up-regulated more than 7-fold under nitrogen-fixation conditions, however, the gdhA gene was down-regulated by 2.83-fold (Supplementary Table S3). In accordance with the mRNA results, we also observed that GlnA was up-regulated 7.61-fold and GdhA, encoding GDH, was down-regulated 9.1-fold in the proteomic analysis (Supplementary Table S2). Clearly, the recombinant E. coli EN-01 chooses the high-affinity GS-GOGAT pathway rather than the GDH pathway when environmental nitrogen is depleted.
Zimmer et al. previously concluded that E. coli use a range of pathways to scavenge for nitrogen-containing compounds as a first line of defense against nitrogen starvation39,40. In this work, we observed a rearrangement of transport capacity by EN-01 under nitrogen-fixation conditions, with alterations of 299 transport or membrane protein genes. Notably, the ammonium transporter gene amtB, which forms a transcriptional operon with the PII protein-coding gene glnK, showed a 55-fold enhancement (Fig. 3). Glutamate uptake systems, including the gltIJKL operon, which encodes a glutamate/aspartate uptake system; the glnHPQ operon, which is responsible for a high-affinity glutamine transport system; the gadC gene, which encodes a glutamic acid/γ-aminobutyrate antiporter; and the gltP gene, which encodes a proton/glutamate-aspartate symporter, were up-regulated under nitrogen-fixation conditions. Alternative nitrogen-transport systems, for instance, lysine, arginine, ornithine, histidine, serine, polyamine, peptide, dipeptide and oligopeptide transporter genes, were all significantly induced. Additionally, the astABCDE operon, which is responsible for the degradation of arginine into N2-succinyl-L-ornithine, CO2 and ammonia, was also up-regulated (Supplementary Table S3).
Nac is a glnK-controlled Ntr protein widely existed in enterobacteria41. Nac is well known to be involved in regulating the expression of multiple pathways to modulate the levels of important cellular metabolic intermediates and thus integrates nitrogen assimilation with other aspects of metabolism41,42,43,44. As expected, nac gene expression was drastically up-regulated (over 100-fold) under nitrogen-fixation conditions. gabDTP (γ-aminobutyrate transport and degradation), dppABD (dipeptide transport), and fklB (peptidyl-prolyl cis-trans isomerase) gene expression was also increased (Supplementary Table S3). However, codBA (cytosine metabolism), nupC (nucleoside transport) and ydcSTUVW (putative putrescine transport and degradation) gene expression did not appear to be significantly altered. These genes were recognized to be controlled by the nac gene in E. coli39. The unclear inconsistent gene expression obtained in this study may provide important clues into the regulatory disturbances of the NFI island by the nac-conferred nitrogen stress response in recombinant E. coli EN-01.
Metabolic flux shift of E. coli EN-01 under nitrogen-fixation conditions
Biological nitrogen fixation is an energy-dependent process that requires ATP, produced by the catalytic reaction of carbon sources, to break the N≡N bond45. Heterologous NFI expression may place a considerable metabolic burden on the host E. coli. In the present work, EN-01 was observed to shift its metabolic flux to overcome the energy barrier for nitrogen fixation. As the central carbon pathways constitute the backbone of cell metabolism by providing energy, metabolic building blocks, and reducing power for biomass synthesis, we investigated the responses of central carbon metabolism in EN-01. As shown in Fig. 5, a stronger flux toward the pentose phosphate pathway (PPP) was found under nitrogen-fixation conditions. Thus, the PPP was postulated to serve as the primary route for glucose catabolism, since the key genes encoding the rate-limiting enzymes glucose-6-phosphate 1-dehydrogenase (zwf), transketolase (tktAB), transaldolase (talA) and glucose-6-phosphate isomerase (pgi) were highly induced. The enhanced expression of glucose-6-phosphate 1-dehydrogenase was also detected in proteome analyses (Supplementary Table S2). Conversely, the glyoxylate shunt and Entner-Doudoroff pathway were found to be inhibited in EN-01 under nitrogen-fixation conditions. Furthermore, the electron transfer systems commonly involved in energy production46,47 were up-regulated, such as the appABC (encoding cytochrome bd-II oxidase), hyaABCDE (encoding the hydrogenase), fhlA (encoding the transcriptional activator of formate hydrogenlyase), fdhF and hycABCDEFGH (encoding formate hydrogenlyase complex), ndh (encoding the NADH dehydrogenase), napH (encoding the ferredoxin-type protein), and adhE (encoding the bifunctional acetaldehyde-CoA/ alcohol dehydrogenase) genes. Meanwhile, both the transcription and translation of the formate acetyltransferase 1 gene pflB were enhanced (Supplementary Table S2). Additionally, three alcohol dehydrogenase genes that are reportedly involved in ethanol fermentation were up-regulated.
E. coli is an anaerobic fermentative bacterium that produces a variety of organic acids by utilizing glucose under nitrogen-fixation conditions; however, these acids are harmful to nitrogenase activity. In contrast to E. coli, P. stutzeri A1501 is an alkali-producing strain that supplies an optimal environment for nitrogen fixation at pH 7.0–9.0 (Supplementary Fig. S2). The organic acid content in E. coli wild-type and EN-01, as well as P. stutzeri A1501, were determined by HPLC after growth under nitrogen-fixation conditions. E. coli wild-type and EN-01 produced a large number of organic acids under nitrogen-fixation conditions (Supplementary Table S4), including pyruvic acid, succinic acid, lactic acid, formic acid and acetic acid, whereas A1501 does not generate these organic acids. The presence of these organic acids could certainly have negative effects on nitrogenase activity in the recombinant EN-01 strain. Altering the intracellular acid environment may be a feasible method to increase the nitrogen-fixing ability of E. coli EN-01.
Oxygen protection strategy of E. coli EN-01 to cope with O2 toxicity to nitrogenase
Nitrogenase is extremely oxygen sensitive. In fact, purified nitrogenase, regardless of its source, undergoes extremely rapid and irreversible inactivation by O248. E. coli EN-01 can grow under both aerobic and anaerobic conditions but only executes nitrogen-fixing activity anaerobically12. Given the importance of oxygen in nitrogen fixation, we explored the potential mechanism for oxygen protection in E. coli EN-01 under anaerobic nitrogen-fixing conditions.
In the present study, the scavenging capacity of E. coli EN-01 for superoxide radicals, hydrogen peroxide, and hydroxyl radicals appeared to increase under nitrogen-fixation conditions. The genes sodC and katE, which encode superoxide dismutase and the hydroperoxidase HPII, were up-regulated 2.94-fold and 15.47-fold, respectively, under nitrogen-fixation conditions (Supplementary Table S3). Furthermore, proteomic analysis revealed these two gene products were increased as well (Supplementary Table S2). Other genes such as the hydrogenase 1-coding genes hyaABCDEF, cytochrome bd-II synthetic-related genes, which have higher oxygen affinity than ATP terminal oxidase, and all cytochrome c synthetic genes, were also up-regulated. The osmC gene, whose product is involved in the detoxification of organic hydroperoxides49,50, was also up-regulated 5.81-fold under nitrogen-fixation conditions (Supplementary Table S3). In addition, the yhbO gene was up-regulated 7.5-fold, and its product was only detected under nitrogen-fixation conditions (Supplementary Table S2). YhbO has been shown to function in the response to various stresses. Mutating the yhbO gene in E. coli leads to increased sensitivity to heat, oxidative, hyperosmotic, pH and UV stresses51.
Although no significant changes were detected at the mRNA level, proteomic analysis revealed that Dps was induced 4.06-fold under nitrogen-fixation conditions (Supplementary Table S2). The Dps protein, a nonspecific DNA-binding protein, is reported to reduce the production of oxidative radicals through ferroxidase activity and therefore to protect the cell from oxidative stress, UV irradiation, iron and copper toxicity, among others52,53.
Taken together, the genes or proteins mentioned above, despite their unclear functions, are thought to be involved in the oxygen stress response under nitrogen-fixation conditions and to simultaneously protect the nitrogenase complex from O2 inhibition by providing a tolerable oxygen environment in E. coli EN-01.
Discussion
To date, the ability to fix nitrogen is exclusively found among bacteria, including green sulfur bacteria, firmibacteria, actinomycetes, cyanobacteria and all subdivisions of proteobacteria54,55. Genomic analyses have revealed that nitrogen-fixation genes are clustered in most diazotrophs56. The nif genes were further presumed to have been lost in most bacterial and archaeal lineages during evolution and that horizontal gene transfer played a pivotal role in the recent acquisition of nif genes in some species55,56,57. In agreement with this hypothesis, several groups have found that transferring nif genes to non-nitrogen fixers successfully yields functional recombinant nitrogen-fixing strains, albeit with far lower nitrogenase activity compared with the donor strains5,11,12,18.
The structural and regulatory systems that control nitrogen fixation in different diazotrophs are extremely well conserved, whereas the regulatory mechanisms of nif genes differ from one organism to another28,54. Therefore, for recombinant nitrogen-fixing strains harboring a heterologous gene system, a variety of metabolic penalties and regulatory barriers may exist between the two systems. Understanding how to overcome and escape the metabolic penalties and regulatory barriers is essential to fine-tune acquired nitrogen-fixing capacity of the non-diazotrophic host.
In this work, transcriptomic and proteomic analysis separately identified 1156 genes and 138 proteins whose expression was significantly altered when E. coli EN-01 was incubated under nitrogen-fixation conditions. Our results highlight the metabolic penalties and regulatory barriers for nitrogen signal transduction and metabolism brought by excess fixed nitrogen in the medium. These genes and proteins, particularly the 24 genes that overlapped between the two transcriptomic analyses (the previous transcriptomic analysis of P. stutzeri A1501 and the E. coli analysis presented in this work), are potential targets for subsequent modification in E. coli EN-01 to optimize nitrogen-fixing capacity.
The nitrogen regulatory cascade of P. stutzeri A1501 comprises the AmtB–GlnK–NtrBC general nitrogen regulation proteins and the nif-specific regulatory protein NifLA22,24,26,31. E. coli has a similar nitrogen regulatory system to P. stutzeri but harbors an additional PII protein and a cascade regulation by the Nac regulatory protein32,33. In the present work, a similar expression network of NFI genes in E. coli to that operating in P. stutzeri A1501 was predicted based on the expression patterns of rpoN, glnD, glnA, glnK, ntrBC, nifLA and other nitrogen regulation-related genes (Fig. 3). Given that the purified E. coli NtrC protein can bind specifically to the A1501 nifLA operon promoter region12, we postulated the regulatory coupling of these two evolutionary divergent systems through a direct activating interaction. Additionally, the nac gene was also predicted to be involved in nif gene regulation in EN-01 although the mechanism requires further investigation. Interestingly, an expression difference was observed in the two PII counterparts GlnB and GlnK in E. coli. In principle, the functions of GlnK and GlnB overlap in Enterobacteriaceae. However, both proteins exhibited distinct physiological roles in the regulation of nitrogen assimilation over a wide range of environmental conditions, despite having distinct expression patterns. The glnB promoter contains a putative -10 sequence for RpoS activation, while the transcription of glnK is induced by NtrC and RpoN35. We therefore presumed that glnK may play a more important role than GlnB in nitrogen regulation in E. coli under nitrogen-fixation conditions. Notably, the PST1302-1306 cluster within the NFI showed unregulated expression in E. coli EN-01 but was highly up-regulated in P. stutzeri A1501 under nitrogen-fixation conditions24. The weak transcription of these nitrogenase component genes may predict imperfections and the necessity for further rebuilding of nif gene expression regulation in E. coli EN-01.
A core challenge for diazotrophs is the need to generate sufficient energy to drive nitrogen fixation. In the present work a strong shift in the metabolic flux of the EN-01 strain was observed under nitrogen-fixation conditions. The key enzyme of the PPP was up-regulated, but the glyoxylate shunt and Entner-Doudoroff pathway were inhibited. Thus, the PPP pathway was concluded to serve as the primary route for glucose catabolism to support nitrogen fixation. Moreover, anaerobic respiration-related electron transfer systems and substances were induced. These data highlight the energy acquisition pathway of EN-01 to adapt to nitrogen fixation. However, as an anaerobic fermentative bacterium, EN-01 produces a large number of organic acids in the media, which may negatively affect nitrogenase activity, since the donor strain P. stutzeri A1501 is an alkali strain. Changing the intracellular acid environment may thus be a feasible method to increase the nitrogen-fixing ability of E. coli EN-01.
We identified a set of genes that might be involved in protecting nitrogenase from oxygen via scavenging of superoxide radicals, hydrogen peroxide, and hydroxyl radicals. Two proteins, YhbO and Dps, also appeared to be involved in the oxygen protection of nitrogenase by protecting the cell from oxidative stress. These genes or proteins may protect the nitrogenase complex from O2 inhibition by establishing a tolerable oxygen environment in E. coli EN-01. Therefore, we speculate that improving the expression of antioxidant capacity-related pathway genes may satisfy the need for oxygen protection under microaerobic conditions and perhaps drive the metabolic flux shift from anaerobic respiration to aerobic respiration to produce more energy and reduce the energy requirements of the nitrogen fixation.
The nitrogenase activity of E. coli EN-01 was much lower than that in P. stutzeri A1501. According to the data in this work, this difference may be due to the regulatory divergence of nitrogen assimilation, the inactivation of the gene operon PST1302-1306, and the production of organic acids. Another factor may also be involved. In our recent work, a species-specific regulatory ncRNA nfiS identified in P. stutzeri A1501, could directly base paired with the mRNA of the nitrogenase component nifK to enhance translation efficiency and transcript half-life, thereby regulating nitrogenase biosynthesis27. Blast results showed that no sequence homologous to A1501 nfiS was present in the E. coli genome. Introduction of nfiS regulation into E. coli EN-01 may be a useful way to refine NFI gene regulation.
From an agri-biotechnology perspective, the successful engineering of an N2-fixing organism would significantly reduce the need for nitrogen fertilizers15,16,54. Biosynthetic techniques have been used by several groups to improve or optimize nitrogenase activity in metabolically engineered strains. Wang X. et al. replaced the nif regulatory elements in the recombinant E. coli strain with a T7 RNA polymerase–LacI expression system to overcome regulatory barriers, and the newly constructed T7-dependent nif system bypassed the original complex regulatory circuits with minor physiological limitations9. Subsequent work from the same group pinpointed the electron-transfer components from plant organelles that can be used to support nitrogenase activity and reduction of the number of target genes required to engineer nitrogen fixation in plants58. Another group observed that five separate gene clusters, as well as a combined cluster from Paenibacillus sp. WLY78 and Klebsiella oxytoca, increase the nitrogenase activity of the recombinant E. coli 78-7, which harbors a minimal nif gene cluster from Paenibacillus sp. WLY7811,19.
In summary, the results presented in this work demonstrate the overall expression pattern of E. coli EN-01 genes under nitrogen fixation and revealed the regulatory coupling of host genes and the heterologous NFI that supports functional nitrogenase activity in the engineered E. coli strain. We also uncovered the metabolic penalties and regulatory barriers that lead to the reduced nitrogenase activity in E. coli EN-01 and provided clues for biotechnological applications to generate new hypotheses concerning gene regulation and flux, which may lead to an improved ability to fix nitrogen in E. coli. This work may shed light on the metabolic penalties and coordinated patterns between host and horizontally transferred genes and provide the knowledge necessary to pave the way for engineered nitrogen fixation.
Methods
Bacterial strains and growth conditions
The strains and plasmids used in this study are described in Table S5. The recombinant strain E. coli EN-01 bearing an NFI was grown at 37 °C in LB medium12. Chloramphenicol, tetracycline and hygromycin were added to media at a concentration of 20 μg/mL, 10 μg/mL and 100 μg/mL, respectively, as required. P. stutzeri A1501 was cultured at 30 °C in LB medium or minimal lactate-containing medium (medium K) as described previously20.
Nitrogenase activity assays
To measure nitrogenase activity in E. coli EN-01, cells were cultured overnight to an OD600 of 2.0 at 37 °C in LB, and 0.2 mL cultures were directly added to 6 mL NFDM minimal medium (20 g glucose, 0.7 g MgSO4·7H2O, 25 mg Na2MoO4·H2O, 3.6 g ferric citrate, 68 g KH2PO4, 241 g K2HPO4, L−1, pH 7.4) without nitrogen in 10 mL serum bottles. These cultures were sealed and then incubated at 30 °C for 20 h with constant shaking (200 rpm), after which 10% acetylene without oxygen was injected to determine ethylene production by gas chromatography, as described by Cannon et al.59. The nitrogenase activity of P. stutzeri was determined in N-free minimal medium at an OD600 of 0.1 at 30 °C under an argon atmosphere containing 0.5% oxygen and 10% acetylene, according to the protocol described by Desnoues et al.60. Nitrogenase-specific activity is expressed as nmol ethylene per mg protein per h. Each experiment was repeated at least three times.
Total RNA preparation
E. coli EN-01 cells were cultured overnight to an OD600 of 2.0 at 37 °C in LB, and 0.2 mL cultures were directly added to 6 mL NFDM medium containing 20 mM ammonium (nitrogen-repression conditions) or without ammonium (nitrogen-fixation conditions) in 10 mL serum bottles, respectively. These cultures were sealed and incubated at 30 °C for 20 h with constant shaking (200 rpm). The cell densities of EN-01 under anaerobic nitrogen-fixation or nitrogen-repression conditions after 20 h reached 3.43 × 108 CFU/mL and 5.42 × 108 CFU/mL, respectively, indicating similar growth status. The EN-01 cells were then centrifuged at 12,000 g for 5 min. Subsequently, cell pellets were rapidly frozen in liquid nitrogen and stored at −80 °C. Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA). To avoid possible DNA contamination, an additional DNase I digestion was performed at 37 °C for 30 min, and the samples were chilled on ice. Total RNA was purified using RNeasy columns, resuspended in RNase-free water and quantitated on a NanoVue plus.
Microarray analysis
For microarrays, standard methods were used for cDNA synthesis, fragmentation, and terminal biotin labeling based on Affymetrix protocols. Labeled cDNA was hybridized to the Affymetrix E. coli Genome 2.0 array. Hybridized arrays were stained with streptavidin-phycoerythrin using an Affymetrix Fluidic Station. After staining, arrays were scanned with an Affymetrix GeneChip Scanner 3000 based on the total signal intensity. The resulting microarray data were analyzed using Affymetrix software (MAS 5.0). Consensus “detection p-values”, “change p-values”, and “mean expression ratios” were calculated. All signal intensities with mean expression ratios above 2 were considered significant changes if the p-value was below 0.05. Complete microarray data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE37780.
Quantitative real-time PCR
To investigate the expression of NFI genes in E. coli EN-01, real-time quantitative PCR (qPCR) was performed using an ABI 7500 Real-Time PCR System. Primers pairs used for qPCR were designed using DNAman and are listed in Table S6. RNA was extracted from cells treated under the same conditions described for the microarray analyses. Reverse transcription was performed using a cDNA synthesis kit (Promega, USA). SYBR Green premix was used to detect PCR amplification. The 16S RNA gene was used to normalize the results. Additionally, qPCR was also used to verify microarray results.
Proteome analysis by two-dimensional PAGE
As previously described, E. coli EN-01 cells were cultured under the anaerobic nitrogen-fixation or nitrogen-repression conditions. The cells were harvested via high-speed centrifugation (12,000 g, 5 min and 4 °C). Cell pellets were washed three times with PBS buffer and suspended in lysis buffer (8 M urea, 2 M thiourea, 0.5% w/v CHAPS, 2% v/v carrier ampholyte, 1% w/v DTT and 1 mM PMSF) for sonication on ice. The samples were centrifuged at 14000 g, 4 °C for 30 min, then, the supernatant was taken for the Bradford Protein assay and frozen at −80 °C.
The details of two-dimensional protein gel electrophoresis and 2-DE analysis were performed according to the protocol described by Zhengfu Zhou et al.61. Briefly, an equal amount of total protein extract (1000 µg) was isoelectrically separated on a 17 cm immobilized pH gradient strip (Bio-Rad, USA) with a linear pH gradient from 4 to 7. Separation along the second-dimension was performed using vertical 12% gels, followed by Coomassie Brilliant Blue G-250 staining visualization and image acquisition. Gels were scanned with a PowerLook 1000 (UMAX Technologies Inc., Dallas, TX) and the gel images were analyzed with PDQuest V7.3.0 (Bio-Rad Laboratories) software according to the manufacture’s protocol. The protein spots with intensity levels greater than 2.0 or less than 0.5 were picked and subjected to mass spectrometric (MS) analysis.
For MALDI-TOF MS analysis, the protein spots with significant difference were excised from the gels for tryptic digestion as described61. The peptides were analyzed on a 4700 Proteomics Analyzer (Applied Biosystems, USA). Proteins were identified using GPS Explorer software V3.5 (Applied Biosystems) and the function was quoted from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) database.
Measurement of intercellular organic acid content
E. coli DH10B and its recombinant strain EN-01 were cultured under anaerobic nitrogen-fixation conditions, and cells were collected at 0 h and 45 h. A1501 was grown under microaerobic nitrogen-fixation conditions, and the bacteria were centrifuged at 0 h and 10 h. The cell suspension was centrifuged at 12000 g, 4 °C for 10 min, and the supernatant was used to analyze the organic acid content. Organic acid concentrations were determined using an HPLC (1200 series, Agilent Technologies) equipped with a Bio-Rad HPX-87H column with 5 mM H2SO4 as the mobile phase (0.6 ml/min, 50 °C).
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Acknowledgements
This work was supported by National Natural Science Foundation of China (31230004, 31470205, 31470174 and 31770067), the National Basic Research Program of China (2015CB755700), and Ministry of Agriculture (Transgenic Program, No. 2016ZX08009003-002). This study was also conducted with the support of the Agricultural Science and Technology Innovation Program (2014-2017), Fundamental Research Funds for Central Non-profit Scientific Institution (0392017002) and the Guangdong Innovative and Entrepreneurial Research Team Program (No. 2013S033). We would like to thank Dr. Claudine Elmerich and Dr. Ray Dixon for many helpful discussions.
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Y.Y. and M.L. conceived and designed the experiments. Z.Y., Y.H., Y.M., Q.C. and Y.Z. performed the experiments. Z.Y., Y.Y. and M.L. contributed to the writing of the manuscript. Z.Y., W.L., L.C., M.H., Y.Y. and S.C. contributed to the collection and analysis of data. All authors (Z.Y., Y.H., Y.M., Q.C., Y.Z., W.L., L.C., M.H., S.C., Y.Y. & M.L.) discussed the results, commented and approved the version of final manuscript.
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Yang, Z., Han, Y., Ma, Y. et al. Global investigation of an engineered nitrogen-fixing Escherichia coli strain reveals regulatory coupling between host and heterologous nitrogen-fixation genes. Sci Rep 8, 10928 (2018). https://doi.org/10.1038/s41598-018-29204-0
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DOI: https://doi.org/10.1038/s41598-018-29204-0
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