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Interspecies Transfer and Regulation of Pseudomonas stutzeri A1501 Nitrogen Fixation Island in Escherichia coli
Interspecies Transfer and Regulation of Pseudomonas stutzeri A1501 Nitrogen Fixation Island in Escherichia coli
Journal of Microbiology and Biotechnology. 2015. Sep, 25(8): 1339-1348
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : February 11, 2015
  • Accepted : March 30, 2015
  • Published : September 28, 2015
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About the Authors
Yunlei Han
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Na Lu
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China
Qinghua Chen
CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, P.R. China
Yuhua Zhan
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Wei Liu Liu
Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Zhejiang 310021, P.R. China
Wei Lu
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Baoli Zhu
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China
Min Lin
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Zhirong Yang
Key Laboratory of Bio-resources and Eco-environment Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, P.R. China
Yongliang Yan
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
yanyongliang@caas.cn

Abstract
Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif -specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.
Keywords
Introduction
The biological nitrogen fixation performed by a limited number of bacterial and archaeal species directly or indirectly supports all plant growth on Earth [14] . The wellknown Mo-Fe-containing protein catalyst Mo-nitrogenase carries out the reduction process of converting dinitrogen to ammonia. Highly conserved among diazotrophs and typically located contiguously in the genome, nitrogen fixation systems may originate from the most recent common ancestor, providing the possibility that the capability for nitrogen fixation could be acquired through horizontal gene transfer in natural environments or under laboratory conditions [20] .
The ability to fix nitrogen is widely distributed among bacteria and archaea but is an extremely rare feature in the genus Escherichia . Over the past decades, considerable effort has been made to engineer novel nitrogen-fixing organisms via transfer of nif genes from various diazotrophs [11 , 18 , 21 , 27] , and the genetic transfer of nitrogen fixation genes to enteric bacteria has received much attention. In the early 1970s, pioneering work proved that a functional nitrogenase could be transferred between two Enterobacter strains; typically, E. coli was shown to express an active nitrogenase from Klebsiella pneumoniae [5 , 6] . Most recently, a nif cluster consisting of nine genes from Paenibacillus sp. WLY78 was transferred to E. coli , and the nif cluster enabled the synthesis of catalytically active nitrogenase [21] . However, such a horizontally acquired ability is not sufficient to enable diazotrophic growth on nitrogen-free medium of E. coli. Klebsiella and Azotobacter can enable associative nitrogen fixation with non-legume plants such as rice, corn, and sorghum, thereby enhancing the growth of these plants. Unlike most other strains, the nitrogen-fixing P. stutzeri A1501, isolated from the root of rice, harbors a presumptive nitrogen fixation island (NFI) [26] . Inoculation of Arabidopsis , alfalfa, tall fescue, and maize with Pseudomonas protegens Pf-5 carrying the NFI from P. stutzeri A1501 increased both the ammonium concentration in the soil and plant productivity under nitrogen-deficient conditions [18] .
As described above, the physiological roles of the transferred nif clusters/systems in the recipient bacteria are clear; however, the regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, the NFI of P. stutzeri A1501 was transferred into E. coli via a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. Subsequently, expression of the nif genes in the recombinant E. coli strain EN-01 was characterized under different growth conditions, and the regulatory mechanisms of the heterologous NFI in E. coli were also investigated.
To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of horizontal gene transfer and the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, gene transfer of the nitrogen fixation pathway into well-developed hosts such as E. coli represents not only an attractive alternative to the production of the natural parent product itself but also a useful tool in the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.
Materials and Methods
- Bacterial Strains and Growth Conditions
P. stutzeri A1501 and its mutant derivatives were grown at 30℃ in LB medium or in minimal lactate-containing medium (medium K) as described previously [9] . E. coli DH10B T1-phage resistant cells (Invitrogen, China) were used as the recipient strain for constructing the engineered E. coli strains that carry nitrogen fixation genes. E. coli and the recombinant strains were grown in LB medium or SOC medium at 37℃ with shaking.
- Construction of the ∆cobS/gshPDouble Mutant
The strategy for constructing a ∆ cobS/gshP mutant was to knock out cobS with a tetracycline (Tc) resistance gene and insert a hygromycin (Hyg) resistance gene in the middle of the gshP region (see Table S1 for a list of the primers used and their sequences). In-frame deletion mutants were generated via homologous recombination using the pKnockout vector pK18mob and previously described methods [23] . Crossover events were first screened using Minimal K medium containing Tc, followed by screening using the same medium containing kanamycin (Km). Single crossover events confer resistance to both Tc and Km. Double crossover events confer resistance to tetracycline only, which enabled the identification of the desired P. stutzeri A1501 mutant, named ∆ cobS , with successful conjugal transfer. Recombination at the correct location was confirmed by PCR. The success rate of transfer was approximately 1 out of 55 mutants.
The same procedure was used to transfer Hyg resistance to the ∆ cobS mutant via inactivation of gshP by the insertion of a hygromycin resistance gene. The resulting P. stutzeri mutant was named A1501B. The resulting double-resistant mutant strain that lacked a reduction in nitrogen-fixing ability was named A1501R2.
- Transfer of NIF toE. coliUsing BAC Resources.
The BAC library was constructed using protocols similar to those found in Osoegawa et al. [16] . Eco RI and Eco RI methylase were used to partially digest the DNA of A1501R2 and to ligate the partially digested DNA into the vector pTARBAC2.1. Vectors were transferred to electrocompetent E. coli DH10B T1 phageresistant cells (Invitrogen, USA) for expression. The library was designated EN and comprised approximately 6,000 clones arrayed in 624 384-well microtiter dishes. High-density replica filters were prepared as previously described [16] .
- High Molecular Weight Genomic DNA Preparation.
Isolating chromosomal DNA is a critical step in the construction of a genomic DNA library. To construct a large-insert (>50 kb) library, high molecular weight DNA must be isolated from cells. Detailed procedures for the preparation of high molecular weight DNA have been described previously [16] . A1501R2 cells were embedded in agarose plugs at a final concentration of 3 × 10 8 cells/ml, followed by a sequence of treatments. DNA used in the construction of the library was partially digested with Eco RI in the presence of Eco RI methylase and size-fractionated by pulsed-field gel electrophoresis using a CHEF apparatus (BioRad, USA). The size-fractionated agarose gels were stored in 0.5 M EDTA until use. Electroelution procedures were used to obtain the partially digested DNA from the gel slices [16] .
- BAC Vector Preparation
The pTARBAC2.1 plasmid DNA was isolated using cesium chloride gradient purification, digested with Eco RI and treated with calf intestine phosphatase (New England Biolabs, USA), and then separated on a 1.0% agarose CHEF gel. The vector fragment was purified from the gel as previously described [16] .
- Construction of the BAC Library
The BAC library was constructed following standard protocols using the pTARBAC2.1 vector [16] . The ligation products were transformed into electrocompetent E. coli DH10B T1 phage-resistant cells (Invitrogen, USA). High-density replica filters were prepared.
- Single Colony Isolation of RecombinantE. coliBearing the Nitrogen Fixation Island
Nitrogen-fixing strains of recombinant E. coli DH10B were selected on LB agar plates containing sucrose and the antibiotics 20 µg/ml chloramphenicol (Cm), 100 µg/ml Hyg, and 10 µg/ml Tc.
- PCR Verification of Positive Clones
PCR primers were constructed based on the genomic sequence of A1501R2 (see Table S1 for a list of the primers used and their sequences). LB plates contained 100 µg/ml Hyg and 10 µg/ml Tc, and positive clones were transferred to a new tube containing the PCR mix. PCRs were carried out in a total volume of 20 µl. After thermocycling, 2 µl of each PCR was analyzed on a 1.5% agarose gel (ReadyGel, Amersham Biosciences, USA).
- BAC End Sequence Analysis
All acquired clones were end-sequenced with a pair of universal primers, the T7 primer and SP6 primer (see Table S1 for a list of the primers used and their sequences).
- Nitrogenase Activity Assays
Nitrogenase activity is measured by the reduction of acetylene relative to the increase in ethylene production using gas chromatography analysis. The nitrogenase activity of P. stutzeri was determined according to the protocol described by Desnoues et al. [9] . The nitrogenase activity of E. coli was determined according to the protocol described by Cannon et al. [6] . Nitrogenase-specific activity is expressed as nmol ethylene/mg protein/h. Each experiment was repeated at least three times.
- Biolog Phenotype Profiling
Colonies from the test strains were inoculated from pre-growth LB agar plates into Biolog inoculating fluid (Biolog, USA), and the optical density of the inoculating suspension was adjusted to 60% transmittance using a turbidimeter. Cell suspensions were inoculated (150 µl of suspension per well) into 96-well Biolog gram-negative (GN2) MicroPlates, which were then incubated at 37℃ for 24 h. Each strain was assayed in duplicate plates. The OD 595 was measured with a microtiter plate reader (model Vmax; Molecular Devices Corp., USA) after 6 and 24 h of incubation.
- Quantitative Real-Time PCR
Gene expression was examined by quantitative real-time PCR. For RT-PCR, EN-01 strains were grown under nitrogen-fixing conditions (without (NH 4 ) 2 SO 4 and O 2 ) or excess-nitrogen conditions (10 mM (NH 4 ) 2 SO 4 without O 2 ). The cultures were harvested by centrifugation at 4℃, and total RNA was isolated using the SV Total RNA Isolation System (Promega, USA) according to the manufacturer’s instructions. RT-PCR experiments were performed with three independent RNA preparations using the ABI 7500 Real-Time PCR System (Applied Biosystems, USA) and SYBR Green PCR Master Mix (Applied Biosystems, USA) according to the manufacturer’s recommendations. First-strand cDNAs were synthesized from 2 µg of total RNA in a 20 µl reaction volume, using the ProtoScript First Strand cDNA Synthesis Kit (New England Biolabs, USA). The 16S RNA gene was used as a reference for the normalization of samples. Primers were generated in a previous study [26] .
- Phage Transduction
To move the glnG mutation into the EN-01 background for characterization, phage transduction using the P1 phage was used. The glnG mutation from Baba et al. [2] was used as a donor to prepare the P1 phage lysate that was subsequently used to transduce EN-01 as per the Court Lab Protocol [19] .Selection for transductants was performed on LB agar plates containing kanamycin. The resulting EN-01 mutant was named EN-01M.
- Construction of the GlnG-P Expression Vector
The D54E,S160F mutation was generated by overlap extension PCR as described previously [4] . The final PCR products were double-digested with Nde I and Eco RI and cloned into a pTWIN1 vector that had been digested with the same enzymes to yield pTW-GlnG-P (see Table S1 for a list of the primers used and their sequences).
- Optimization of Protein Expression and Purification
Protein expression and purification were performed as described by Xie et al. [24] . Briefly, a 2 L culture of E. coli BL21 harboring pTW-GlnG-P was grown to an optical density at OD 600 of 0.3 to 0.5 at 37℃ and then transferred to a 30℃ shaker. After 20 min, isopropyl-β- D -thiogalactoside was added to a final concentration of 0.1 mM, and the culture was incubated at 30℃ for 3 h. Cells were then harvested by centrifugation, resuspended in 10 ml of E-buffer (5 mM Tris-HCl, pH 7.8, 1 mM EDTA, and 1 mM dithiothreitol (DTT)), and broken by sonication. After centrifugation at 5,000 × g for 30 min at 4℃, the supernatant was loaded onto a 2 ml chitin (NEB, USA) column equilibrated with Buffer B1 (20 mM Tris-HCl, pH 8.5, 500 mM NaCl, and 1 mM EDTA). Nonspecifically bound proteins were removed by washing with 20 ml of washing buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 1 mM EDTA). The cleavage of GlnG-P-intein was induced with 5 ml cleavage buffer B3 (20 mM Tris-HCl pH 8.5, 500 mM NaCl, 40 mM DTT, and 1 mM EDTA), and the solution was subsequently incubated at 4℃ overnight. Protein concentration was determined by the Bradford protein assay and is expressed in nM. Protein samples were stored at -80℃.
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Overview of the protocol developed to obtain recombinant E .coli bearing the NFI derived from P. stutzeri A1501. Abbreviation: A1501, P. stutzeri A1501; A1501R2, acdS and gshP double mutant of P. stutzeri A1501; NFI, nitrogen fixation island; Tc, tetracycline resistance gene; Hyg, hygromycin resistance gene; Cm, chloramphenicol resistance gene; B, BamHI; H, HindIII; P, PstI; X, XbaI; Xh, XhoI; E, EcoRI, and Em, EcoRI methylase.
- Electrophoretic Mobility Shift Assays (EMSAs)
EMSAs were carried out according to previously described methods [28] . Using a pGEM-T bearing the promoter regions of nifA or nifH as a template, the promoter regions of nifA and nifH were successively amplified by PCR with IRDye-800 fluorescent DNA primers (Li-Cor Biosciences, USA). Labeled DNA fragments were independently mixed with purified GlnG-P proteins using the Odyssey Infrared EMSA Kit (Li-Cor Biosciences, USA) according to the manufacturer’s instructions (see Table S1 for a list of the primers used and their sequences). For competitive inhibition of the binding reaction, 50- or 100-fold unlabeled DNA fragments were added to that reaction system. After incubation on ice for 10 min, the reactants were run on a 5% TBE polyacrylamide gel (BioRad, USA) at 110 V for 1 h, with 0.5× TBE as the running buffer.
Results
- Construction of RecombinantE. coliClones Containing the EntireP. stutzeriA1501 NFI
The complete nucleotide sequence of the P. stutzeri A1501 genome has been determined, which led to the identification of a 59-gene, 49 kb island that comprises the largest group of nif genes identified to date. This discovery was also confirmed by transcriptomic analysis showing the characteristics of an “expression island” under nitrogenfixing conditions [26] . In this study, we used a novel and efficient transformation strategy to transfer the 49 kb island into E. coli.
First, the A1501R2 with two antibiotic resistance genes to tetracycline and hygromycin inserted into both sides of the NFI was constructed. Under nitrogen-fixing conditions, A1501R2 had the same nitrogenase activity as the wild type ( Fig. 2 ). Growth was also identical to the parent while grown aerobically in LB. This confirmed that the genes cobS and gshP were not required for the survival and nitrogenfixing capability of P. stutzeri A1501.
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Nitrogenase activity of P. stutzeri A1501, ∆cobS, ∆gshP, and A1501R2.
Second, the BAC library was constructed based on the genomic DNA of A1501R2. High molecular weight DNA fragments (about 50-100 kb) of A1501R2 were cloned into the pTARBAC2.1 vector and then introduced into the nonrestricting E. coli DH10B strain. BAC clones containing the complete P. stutzeri A1501 NFI could then be isolated easily by selecting for both resistance genes. Altogether, 17 recombinants carrying the complete NFI were obtained by plating on LB agar containing 100 µg/ml Hyg and 10 µg/ml Tc. BAC plasmids were then end-sequenced to determine whether the sequences and sizes of the inserts were correct. Among the 17 strains, the insert sizes ranged from 59-124 kb; the insert sites were also slightly different. We tested various growth conditions using a series of carbon sources and found that optimal conditions consisted of anaerobic conditions with a supply of glucose. Therefore, for the 17 recombinant strains, all of the nitrogenase activity assays were carried out under anaerobic conditions using glucose as the sole carbon source. The nitrogenase activity assays proved that all 17 recombinants could reduce acetylene to ethylene ( Table 1 ). Compared with P. stutzeri A1501, the recombinant E. coli strains showed approximately 10% of the nitrogenase activity for acetylene reduction. The recombinant strain with the highest nitrogenase activity and the smallest insert size (59.3 kb) was used for further study and named E. coli EN-01.
Nitrogenase activity of the recombinantE. colistrains.
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Nitrogenase activity of the recombinant E. coli strains.
- Physiological Alteration ofE. coliTransformed with the EntireP. stutzeriNFI
As a large fragment of foreign DNA carried by a BAC vector, the entire NFI would presumably affect host gene replication, energy distribution, etc. , especially because the NFI represents a “functional entity” as a nitrogen-fixing unit and likely reconstructs a global host network for metabolism and energy utilization. We compared the growth curves of wild-type E. coli and E. coli EN-01 under rich medium (LB) conditions; the results indicated a reduced growth rate ( Fig. 3 A). We also compared them in a Biolog GN2 carbon metabolism characterization system using 95 common carbon sources. The results showed rapid changes in the utilization of 35 of the carbon sources in EN-01 . Decreases of over 2-fold were found for 19 of the carbon sources, including maltose, D -sorbitol, glycerol, D, L -lactose, and L -alanine; however, an increase of 1-fold was found for L -arabinose (data not shown). Therefore, it seems that the metabolism of the host E. coli bearing the introduced NFI was altered, indicating a coupling of the networks for nitrogen and carbon metabolism. Such coupling may have strong effects on the reconstruction of metabolic flux in the recombinant strain EN-01, which displayed a novel physiological phenotype compared with the wild-type E. coli strain.
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Phenotypes of the recombinant E. coli EN-01 under different conditions. (A) Growth curve of E. coli EN-01 in LB medium; (B) time course of the nitrogenase activity of EN-01 under different ammonium concentrations; (C) nitrogenase-specific activity of strain EN-01 in NFDM medium with different carbon resources; (D) nitrogenase activity of EN-01 at different temperatures.
- The Expression of NFI in RecombinantE. coliEN-01 is Regulated by Ammonia, Carbon, Oxygen, and Temperature
Although both the host P. stutseri A1501 and the recipient E. coli belong to γ -Proteobacteria, the two species have obvious differences in taxonomic status and in the microenvironments in which they live. As members of Enterobacter iaceae, E. coli can generate energy through anaerobic fermentation. Their optimum growth temperature was 37℃, whereas for P. stutzeri A1501 isolated from rice soil, the optimum growth temperature was 30℃, and the optimum nitrogen-fixing condition was a nitrogen-free, microaerobic environment. Overall, nitrogen fixation activity was dependent on ammonia, carbon, and oxygen availability and on temperature [9] .
In many diazotrophs, the addition of excess ammonium to the growth medium results in immediate repression of nif gene transcription. To examine whether the recombinant strain EN-01 is subject to this regulation, we investigated the effects of ammonium on nitrogenase activity. As shown in Fig. 3 B, (NH 4 ) 2 SO 4 was added to yield final ammonium concentrations of 0, 0.1 and 1 mmol in the medium, and the nitrogenase activity of E. coli EN-01 was highest in the nitrogen-free medium. In addition, nitrogenase activity was inhibited in the medium containing 0.1 mmol ammonium ion, whereas the medium containing 1 mmol ammonium ion completely inhibited the nitrogenase activity of EN-01.
Carbon and nitrogen metabolism are two major cellular processes, and they are usually coupled to reach optimal growth via various strategies. Microbes utilize different carbon sources at various rates; this is reflected by the variation in ultimate energy production that affects nitrogen fixation efficiency. The experiments demonstrated that under anaerobic and nitrogen-deficient conditions, the recombinant E. coli EN-01 strain exhibits the greatest nitrogenase activity by using glucose or mannose as the sole carbon source. Furthermore, it exhibits rapidly decreased activity when using fructose, sucrose, lactose, and sodium pyruvate, and it has almost no detectable activity when using sodium lactate or arabinose ( Fig. 3 C).
Nitrogenase is highly sensitive to oxygen, which can cause it to be irreversibly inactivated. For P. stutzeri A1501, nitrogenase activity was detected at a range of initial oxygen concentrations (from 0.5% to 4%), suggesting that nitrogen fixation occurs at low oxygen tension [9] . To evaluate the effect of oxygen on the expression of nitrogenase in E. coli EN-01, we determined nitrogenase activity in air and under microaerobic and anaerobic conditions. The results showed that EN-01 nitrogenase activity can only be detected through acetylene reduction in the anaerobic environment when using the nitrogen-free medium NFDM in the presence of various carbon sources, including glucose, mannose, fructose, sucrose, lactose, and pyruvate. Among the various carbon sources, glucose was optimal for nitrogen fixation. Furthermore, no activity was detected when the cells grew on lactate, but P. stutzeri A1501 was able to fix nitrogen and grow on lactate as the sole carbon source. The results also showed that, in the EN-01 strain, nitrogenase activity was regulated by the concentration of oxygen in the environment, which was also the case for P. stutzeri A1501.
Temperature could be one of the factors affecting nitrogenase activity. It has been reported that NifA, a positive regulator of nitrogenase activity, is very sensitive to temperature [12 , 22] . On the other hand, nitrogenase is also temperature-sensitive; higher temperatures can decrease its activity or denature it. The optimal temperature for the growth of P. stutzeri A1501 is 30℃, whereas for E. coli , the optimal temperature is 37℃. Therefore, we assayed the nitrogenase activities of EN-01 at 30℃, 33℃, and 37℃. The results showed that the recombinant EN-01 could grow normally at both 37℃ and 30℃, but it had higher nitrogenase activity at 30℃. Increasing the growth temperature to 33℃ or 37℃ reduced the nitrogenase activity 6- and 14-fold, respectively ( Fig. 3 D).
The above results indicate that expression of the NFI in the recombinant strain E. coli EN-01 is very similar to its expression in P. stutzeri A1501, suggesting that the expression and regulation of nitrogen fixation genes may be affected by the nitrogen and oxygen signaling networks of the recipient bacteria E. coli. Variations in the nitrogen, oxygen, and temperature conditions of the external environment may signal through the induction system of E. coli and somehow be transferred to nifLA of the NFI, regulating the expression of nitrogen fixation genes.
- Expression of NFI Genes inE. coliEN-01
As mentioned above, the recombinant strain EN-01 displays nitrogenase activity, indicating the heterologous production of a functional nitrogenase complex encoded by the nifHDK genes. The nitrogenase activity of EN-01 is regulated by ammonia, oxygen, and temperature, strongly suggesting that the nif -specific transcriptional regulator nifLA is active in the heterologous host strain EN-01. To further test the heterologous expression of nifLA and nifHDK , we determined the induction ratio of these genes when strain EN-01 was grown anaerobically under nitrogenfixing conditions compared with when it was grown under excess-nitrogen conditions using quantitative real-time RT-PCR ( Table 2 ). The results showed that, compared with conditions of excess nitrogen, under nitrogen-fixing conditions, the five genes were sig nif icantly up-regulated ( Table 2 ), and the expression patterns were very similar to those in P. stutzeri A1501 [25] . This finding indicates that in the E. coli EN-01 strain, the expression levels of nifLA and nifHDK are controlled by the E. coli nitrogen metabolism regulatory system.
Expression of thenifgenes inE. coliEN-01 under nitrogen-fixing conditions compared with excess-nitrogen conditions.
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Expression of the nif genes in E. coli EN-01 under nitrogen-fixing conditions compared with excess-nitrogen conditions.
- Transcriptional Regulation of theP. stutzeriNFI byE. coliGlnG ProteinviaNifA
Both E. coli and P. stutzeri belong to the γ -Proteobacteria and have highly similar general nitrogen regulatory systems. The nitrogen regulatory protein NRI plays an essential role in nitrogen regulation in different bacteria [17] . It has been shown that in P. stutzeri A1501, NtrC controls the expression of the alternative PII protein GlnK, as well as of the nif -specific activator NifA [13 , 25] . Mutation of ntrC leads to the loss of nitrogenase activity in P. stutzeri A1501 [9] . In E. coli , the NRI protein is encoded by glnG , which has 65.4% homology to the P. stutzeri NtrC protein. Moreover, qRT-PCR analysis showed that glnG was signif icantly upregulated (over 10-fold) in EN-01 under nitrogen-fixing conditions compared with excess-nitrogen conditions. To investigate the role of E. coli GlnG on the expression of the P. stutzeri A1501 NFI, a glnG mutant derived from the recombinant E. coli EN-01 was constructed and named E. coli EN-01M. No nitrogenase activity was detected in E. coli EN-01M, indicating that GlnG may be involved in the expression of the heterologous P. stutzeri A1501 NFI.
A phosphorylated form of the E. coli GlnG protein was purified and used to investigate the direct interaction of the GlnG protein with the P. stutzeri nifLA promoter using EMSA. As shown in Fig. 4 A, a putative GlnG-binding site (containing GCA and TGC) was located in the nifLA promoter region, which is also a σ 54 -dependent promoter bearing the consensus GG-N10-GC motif of the RpoN protein. These motifs are similar to the corresponding consensus sequences found in the promoter regions of the E. coli glnKamtB, glnALG , and gltIJKL gene cluster [3] . The DNA fragment P nifA , which completely covers the nifLA promoter region from positions -327 to 141 relative to the transcription start site, was cloned and fluorescently labeled. The promoter region of the nifHDK operon (P nifH ) was used as a negative control. The DNA fragment was mixed with the purified GlnG, and after that, the mixtures containing the DNA/GlnG complex and the free DNA were resolved by native PAGE. As shown in Fig. 4 B, the addition of purified phosphorylated GlnG protein to P nifA caused a band shift of P nifA . As the amount of non-labeled P nifA increased, the shifted band disappeared, indicating that the phosphorylated GlnG protein binds specifically to DNA fragments containing the nifLA operon promoter region. This observation provides evidence for a direct interaction of the E. coli phosphorylated GlnG with the heterologous nifLA promoter region, suggesting a regulatory coupling of these two different evolutionary systems through a direct, activating interaction.
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Electrophoretic mobility shift assays (EMSAs) of the promoter regions of nifLA from P. stutzeri A1501 and purified phosphorylated E. coli GlnG protein. (A) Sequence of the nifLA promoter region, showing the putative GlnG-binding sites (open boxes). The σ54 promoter (sequence underlined with GG GC in bold), the transcription initiation site (T in bold), and the translation initiation site (underlined ATG) are also highlighted for comparison; (B) EMSA experiments demonstrating the binding of E. coli phosphorylated GlnG to the nifLA promoter region. Lane 1: labeled nifA promoter DNA fragment; lane 2: labeled nifA promoter DNA fragment incubated with phosphorylated GlnG protein; lanes 3 and 4: phosphorylated GlnG protein incubated with labeled nifA promoter DNA fragment containing 50- and 100-fold non-labeled nifA promoter DNA, respectively; lane 5: labeled nifH promoter DNA fragment; and lane 6: labeled nifH promoter DNA fragment incubated with GlnG-P protein.
Discussion
The ability to fix nitrogen is found exclusively among bacteria. Although there is considerable biodiversity among diazotrophic microorganisms, nitrogen fixation is not found in eukaryotes. Eventually, a successfully engineered N 2 -fixing crop may signif icantly reduce the need for chemical fertilizers, resulting in a cleaner environment and higher yield [7 , 8 , 22] . Currently, understanding the physiological changes that take place in an engineered cell after the insertion of a full set of large fragments of foreign DNA, especially from a distant species, may provide the knowledge necessary to pave the way towards the goal of nitrogen fixation engineering.
Biological nitrogen fixation is an energy-dependent and oxygen-sensitive process that requires ATP and a supply of Fe-S clusters, not only for the nitrogenase component but also for the many other proteins involved in electron transfer, including redox and non-redox catalysis and the sensing of regulatory processes [10] . Genetically, one may succeed in transferring 1-2 functional nif genes into foreign organisms, including eukaryotes; it remains a daunting task to insert an entire nitrogen fixation system, because, for it to be functional in a typical “alien” environment, many biochemical and physiological changes may be required.
An important corollary to the acquisition of new genes is the problem of how to best integrate them into the recipient’s existing gene regulatory circuits so that fitness is not initially compromised and can be enhanced in the future through optimal expression of the new genes. A recent report found that the transfer to E. coli of a nine- nif -gene operon derived from Paenibacillus sp. WLY78 and transcribed from the σ 70 promoter resulted in the successful expression of active nitrogenase, but no transcriptional regulation by either oxygen or fixed nitrogen was detectable in the recombinant E. coli [21] . The P. stutzeri A1501 NFI represents the largest nitrogen fixation assembly characterized in any diazotrophic species to date [1 , 15 , 21 , 26] . In contrast to Paenibacillus sp. WLY78, a total of 11 operons in the P. stutzeri A1501 NFI were activated in a NifA-σ 54 -dependent manner under nitrogen-fixing conditions [25] . Expression of the island is stringently regulated in response to oxygen and fixed nitrogen [9] . In this work, a series of recombinant nitrogen-fixing E. coli strains were obtained, and the regulatory mechanisms were investigated. It was observed that nitrogenase activity in the recombinant E. coli strain was dependent on external ammonia concentration, oxygen tension, and temperature, indicating that the NFI was also highly regulated in the new host, in a manner similar to its regulation in the parent strain. We provided further evidence of a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain.
This work will shed light on the structure and functional integrity of the nif island and increase understanding of how the NFI adapts to new cell circumstances in the natural environment. It will also be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.
Acknowledgements
We would like to thank Prof. Ray Dixon and Claudine Elmerich for many helpful discussions, and Dr. Qi Cheng and Ahmed Gomma for critical reading of the manuscript. This work was supported by grants from the National Basic Research Program of China (No. 2015CB755700), the National Nature Science Foundation of China (No. 31230004, 31470174 and 31400076), the National High Technology Research and Development (863) Program of China (No. 2012AA02A703), and the Guangdong Innovative and Entrepreneurial Research Team Program (No. 2013S033).
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