Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.
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Additional data supporting this study are available from the corresponding author on reasonable request. The RNA-seq and ribosome-profiling data are available in the Sequence Read Archive with the accession code PRJNA579767: K. oxytoca native nif cluster, RNA-seq (SRX7032059, SRX7032060 and SRX7032061) and ribosome-profiling (SRX7034729, SRX7034730, SRX7034731 and SRX7034732); K. oxytoca refactored nif cluster v2.1, RNA-seq (SRX7036110) and ribosome-profiling (SRX7036099); K. oxytoca refactored nif cluster v3.2, RNA-seq (SRX7035703, SRX7035704 and SRX7035705) and ribosome-profiling (SRX7036113, SRX7036114 and SRX7036115).
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This work was supported by the National Science Foundation (grant no. NSF-1331098), the Abdul Latif Jameel Water and Food Security Lab (J-WAFS) at the Massachusetts Institute of Technology, the US National Science Foundation Synthetic Biology Engineering Research Center (grant no. SynBERC EEC0540879) and the Office of Naval Research Multidisciplinary University Research Initiative (MURI grant no. N00014-13-1-0074). We thank G. O’Toole of Dartmouth College for the yeast shuttle vectors.
M.-H.R. and C.A.V. have filed a patent application (US provisional application no. 62/820,765) on this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Nitrogenase activity in wild-type R. sp. IRBG74 and a Δnif mutant strain, in which the native nif clusters are deleted.
(a) The nif clusters in R. sp. IRBG74. The deleted regions generated by the suicide plasmids pMR45–46 are marked (Methods). (b) Transfer of native nif constructs into R. sp. IRBG74. Nitrogenase activity was detected only from the transfer of the R. sphaeroides nif cluster into R. sp. IRBG74 MR17 (ΔhsdR, recA) but not into R. sp. IRBG74 MR19 (ΔhsdR, recA Δnif). Expression of A. caulinodans nifV on the plasmid pMR49 in R. sp. IRBG74 MR17 was induced by 0.5 mM IPTG. The co-transfer of the complete A. caulinodans nif cluster on the two plasmids pMR19 and pMR20 did not yield activity in R. sp. IRBG74 MR17. Error bars represent standard deviation from three independent experiments on different days. Asterisk indicate ethylene production below the detection limit. Rsp, R. sphaeroides; Aca, A. caulinodans. (c) Plasmid maps used in (b).
Ribosome profiling data for the K. oxytoca nif cluster in its native host (top) and when transferred into different strains are shown.
Extended Data Fig. 3 The effect of NifA overexpression on the nifH promoter activity in R. sp. IRBG74.
(a) The design of the reporter constructs used to measure nifH promoter activity shown. The nifH promoter activity was analysed in R. sp. IRBG74 using flow cytometry. Overexpression of R. sp. IRBG74 NifA increased the activity of the R. sp. IRBG74 nifH promoter but failed to complement or enhance the activities of the other nifH promoters including K. oxytoca, P. stutzeri and A. caulinodans. Error bars represent standard deviation from three independent experiments on different days. WT, wild-type; Rsp, R. sp. IRBG74; Kox, K. oxytoca M5al; Pst, P. stutzeri A1501; Aca, A. caulinodans ORS571 (b) Plasmid maps used to assess the effect of nifA overexpression in R. sp. IRBG74.
Extended Data Fig. 4 Nitrogenase activity when different inducible nif clusters are transferred to E. coli MG1655.
(a) The same universal controller system based on K. oxytoca nifA was optimized and used for all three clusters (Supplementary Figure 15, 17b). The controller plasmid pMR104 and genetic parts are provided in Supplementary Table 3 and 4. (b) The nif clusters from K. oxytoca, P. stutzeri, and A. vinelandii are shown. The deleted regions corresponding the NifLA regulators are marked, and their corresponding genomic locations are provided in Supplementary Table 3. The dotted lines indicate that multiple regions from the genome were cloned and combined to form the nif cluster. The clusters were carried on the plasmids pMR23–25 (Supplementary Table 3). (c) The induction of the nifH promoters from each species by the controller are shown (+, 50 μM IPTG). (d) The nitrogenase activities of the native cluster (intact nifLA) are compared to the inducible clusters in the presence and absence of 50 μM IPTG. The dashed lines indicate the activity of the native clusters in the wild-type context (top to bottom, K. oxytoca M5al, P. stutzeri A1501 and A. vinelandii DJ). (e) Regulation of nitrogenase activity by ammonium. Ammonium tolerance of nitrogenase from the native (black bar) and inducible (grey bar) systems was tested in the presence of 17.1 mM ammonium acetate and 50 μM IPTG (inducible). Asterisks indicate ethylene production below the detection limit. (f) Regulation of nitrogenase activity by oxygen. The native nif cluster is compared to the inducible version including the controller plasmid and 50 μM IPTG. Nitrogenase activities were measured after 3 h of incubation at constant oxygen concentrations (0 to 3%) in the headspace (Methods). Error bars represent standard deviation from three independent experiments on different days.
(a) Controllers whose output is T7 RNAP integrated on the genome of P. protegens Pf-5 are described. Substituted genetic parts for the controller optimization compared to the controller module pKT249 in E. coli MG1655 are highlighted in red. The response functions for the controllers with the reporter plasmid pMR81 was measured in the P. protegens Pf- 5 controller strain MR7. The controller driving the expression of GFP by the T7 promoter led to 96-fold induction by IPTG. (b) The genetic parts used to build the refactored v3.2 nif gene cluster are shown (provided in Supplementary Table 4). (c) The activity of the refactored nif cluster v3.2. Nitrogenase expression was induced by 1 mM IPTG. (d) The function of the transcriptional parts of the cluster v3.2 was analysed by RNA- seq (Supplementary Figure 18). The performance of the promoters (left) and terminators (right) was calculated (Methods). (e) The translation efficiency of the nif genes v3.2 as calculated using ribosome profiling and RNA-seq. Lines connect points that occur in the same operon. (f) The ribosome density (RD) is compared for the refactored v3.2 nif genes in P. protegens Pf-5 versus that measured for the nif genes from the native K. oxytoca cluster in K. oxytoca (→Klebsiella: R2 = 0.68). Error bars represent standard deviation from three independent experiments on different days.
Extended Data Fig. 6 Control of nitrogenase fixation in A. caulinodans ORS571 under changing environmental conditions.
(a) The effect of the absence or presence of 10 mM ammonium chloride is shown. The WT NifA from A. caulinodans ORS571 is compared to different combinations of amino acid substitutions. NifA/RpoN expression is induced by 1 mM IPTG (+) for A. caulinodans ΔnifA containing the controller plasmid pMR124–127 (+). An asterisk indicates ethylene production below the detection limit. (b) The nitrogenase activity is shown as a function of the oxygen concentration in the headspace (Methods). The native nif cluster (wild-type A. caulinodans ORS571, black) is compared to the inducible version (grey) including the controller plasmid and 1 mM IPTG. Error bars represent standard deviation from three independent experiments on different days.
Extended Data Fig. 7 Ammonium repression of the transferred nif clusters in E. coli MG1655 and P. protegens Pf-5.
Nitrogenase sensitivity to ammonium was measured by acetylene reduction assay in the absence (-) or presence (+) of 17.1 mM ammonium acetate. The sensitivity of the native and inducible nif clusters in E. coli MG1655 (a) and P. protegens Pf- 5 (b). Note that the data are from Fig. 4 and Supplementary Figure 8. (c) The specific nitrogenase activities of the native A. vinelandii nif cluster are compared to the inducible A. vinelandii cluster in the presence (+) and absence (-) of 17.1 mM ammonium acetate in P. protegens Pf-5. The nif clusters from the inducible version were induced by 50 μM and 0.5 mM IPTG in E. coli MG1655 and P. protegens Pf-5, respectively. Asterisks indicate ethylene production below the detection limit. Error bars represent standard deviation from three independent experiments on different days.
Expression from the nifH promoters was analysed in E. coli MG1655 containing the controller plasmid pMR104, P. protegens Pf-5 MR10 (for K. oxytoca) and MR9 (for P. stutzeri and A. vinelandii) at varying initial oxygen levels in the headspace. The three nifH promoters were induced with 0.05 mM IPTG and 0.5 mM IPTG in E. coli MG1655 and P. protegens Pf-5, respectively, and incubated at varying initial oxygen concentrations. Error bars represent standard deviation from three independent experiments on different days.
The modified nif clusters of A. vinelandii on the plasmids pMR25–28 were analysed in the controller strain P. protegens Pf-5 MR9. The deleted regions from the clusters were provided in Supplementary Table 3. Nitrogenase was induced with 0.5 mM IPTG. Dots in the DNA line indicate where multiple regions were cloned from genomic DNA and combined to form one large plasmid-borne nif cluster. An asterisk indicates ethylene production below the detection limit. Error bars represent standard deviation from three independent experiments on different days.
(a) Controller plasmids used to drive expression of T7 promoters. (b) Inducibility of the T7 promoter by the controller plasmids encoding T7 RNAP under the regulation of the 12 sensors was tested with a reporter plasmid pMR123 (right). (c) Inducible control of nitrogenase activity in response to 12 inducers was tested with each of 12 controller plasmid and the plasmid pMR138 (right) carrying the refactored nif cluster v2.1 on pBBR1 origin. The choline-Cl inducible system was omitted for activity assay as the system was not inducible. For the DAPG-, DHBA-, and vanillic acid-inducible system, the refactored cluster v2.1 was carried on a lower copy number plasmid pMR31 (right) as there was no colony formation from the transformation of the plasmid pMR138. The inducer concentrations are: 400 μM arabinose, 1 mM choline-Cl, 500 nM 3OC14HSL, 50 μM cuminic acid, 25 nM 3OC6HSL, 25 μM DAPG, 500 μM DHBA, 1 mM IPTG, 100 nM aTc, 250 μM naringenin, 50 μM vanillic acid, and 250 μM salicylic acid. Plasmid and genetic parts are provided in Supplementary Table 3 and 4. Error bars represent standard deviation from three independent experiments on different days.
Supplementary Figs. 1–22 and Supplementary Tables 1–6.
Plasmid pMR3_Native nif cluster of Klebsiella oxytoca M5al.
Plasmid pMR5_Native nif cluster of Pseudomonas stutzeri A1501.
Plasmid pMR7_Native nif cluster of Azotobacter vinelandii DJ.
Plasmid pMR9_Native nif cluster of Cyanothece ATCC51142.
Plasmid pMR11_Native nif cluster of Paenibacillus polymyxa WLY78.
Plasmid pMR13_Native nif cluster of Azospirillium brasilense Sp7.
Plasmid pMR15_Native nif cluster of Rhodobacter sphaeroides 2.4.1.
Plasmid pMR17_Native nif cluster of Rhodopseudomonas palustris CGA009.
Plasmid pMR19_Native nif cluster of Azorhizobium caulinodans ORS571 (Part 1 of 2).
Plasmid pMR20_Native nif cluster of Azorhizobium caulinodans ORS571 (Part 2 of 2).
Plasmid pMR21_Native nif cluster of Gluconacetobacter diazotrophicus PA1 5.
Plasmid pMR29_Refactored nif cluster v2.1.
Plasmid pMR38_Refactored nif cluster v3.2.
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Ryu, MH., Zhang, J., Toth, T. et al. Control of nitrogen fixation in bacteria that associate with cereals. Nat Microbiol 5, 314–330 (2020). https://doi.org/10.1038/s41564-019-0631-2
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