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A haem-sequestering plant peptide promotes iron uptake in symbiotic bacteria

Nature Microbiology volume 7, pages 1453–1465 (2022)Cite this article

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Abstract

Symbiotic partnerships with rhizobial bacteria enable legumes to grow without nitrogen fertilizer because rhizobia convert atmospheric nitrogen gas into ammonia via nitrogenase. After Sinorhizobium meliloti penetrate the root nodules that they have elicited in Medicago truncatula, the plant produces a family of about 700 nodule cysteine-rich (NCR) peptides that guide the differentiation of endocytosed bacteria into nitrogen-fixing bacteroids. The sequences of the NCR peptides are related to the defensin class of antimicrobial peptides, but have been adapted to play symbiotic roles. Using a variety of spectroscopic, biophysical and biochemical techniques, we show here that the most extensively characterized NCR peptide, 24 amino acid NCR247, binds haem with nanomolar affinity. Bound haem molecules and their iron are initially made biologically inaccessible through the formation of hexamers (6 haem/6 NCR247) and then higher-order complexes. We present evidence that NCR247 is crucial for effective nitrogen-fixing symbiosis. We propose that by sequestering haem and its bound iron, NCR247 creates a physiological state of haem deprivation. This in turn induces an iron-starvation response in rhizobia that results in iron import, which itself is required for nitrogenase activity. Using the same methods as for l-NCR247, we show that the d-enantiomer of NCR247 can bind and sequester haem in an equivalent manner. The special abilities of NCR247 and its d-enantiomer to sequester haem suggest a broad range of potential applications related to human health.

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Fig. 1: NCR247 induces an iron-starvation response and drives iron import into S. meliloti.
Fig. 2: NCR247 binds and sequesters haem.
Fig. 3: NCR247 binds intracellular haem and drives iron uptake by modulating Irr-mediated iron regulation.
Fig. 4: Potential therapeutic applications of NCR247–haem binding.
Fig. 5: NCR247 sequesters haem and overrides bacterial iron regulation to aid symbiosis.

Data availability

All data in this manuscript are available. Source data are provided with this paper.

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Acknowledgements

This work was supported by NIH grants (R01 GM031030 to G.C.W., R01 AI158501 to S.L., ES028374 and HL120877 to M.B.Y., F32 GM129882 to M.C.A., and R35 GM126982 to C.L.D.). D.M.A. is supported by a grant to the MIT from the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study program. A.A. was supported by the KACST-MIT Ibn Khaldun Fellowship for Saudi Arabian Women. G.C.W. is an American Cancer Society Professor. C.L.D. is a Howard Hughes Medical Institute Investigator. This work was completed in part with resources at the MIT Department of Chemistry Instrumentation Facility with the help of J. Grimes and W. Massefski. Staff at the Bio-Instrumentation Facility at the Department of Biology, MIT, and the Octet biolayer interferometry system (NIH S10 OD016326) is greatly acknowledged. We acknowledge the MIT Center for Environmental Health Sciences (NIH P30 ES002109). This work was performed in part at the Harvard University Center for Nanoscale Systems (CNS); a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF award no. ECCS-2025158. We thank A. McClelland for his assistance with the Raman spectroscopy and N. Watson for her assistance with the negative staining and transmission electron microscopy analysis. We thank D. Wang from the University of Massachusetts, Amherst, for providing materials and advice on the CRISPR knockdown experiment. We thank K. Miller for her assistance with EPR interpretations.

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  1. Areej Alhhazmi

    Present address: Medical Laboratory Technology, College of Applied Medical Sciences, Taibah University, Madinah, Saudi Arabia

Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Siva Sankari, Vignesh M. P. Babu, Ke Bian, Areej Alhhazmi, Mary C. Andorfer, Dante M. Avalos, Tyler A. Smith, Catherine L. Drennan, Sebastian Lourido & Graham C. Walker

  2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA

    Mary C. Andorfer, Dante M. Avalos & Catherine L. Drennan

  3. Harvard Graduate Program in Biophysics, Harvard University, Cambridge, MA, USA

    Dante M. Avalos

  4. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Tyler A. Smith & Sebastian Lourido

  5. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Kwan Yoon

  6. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Catherine L. Drennan

  7. Departments of Biology and Biological Engineering, and Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute for Technology, Cambridge, MA, USA

    Michael B. Yaffe

  8. Divisions of Acute Care Surgery, Trauma, and Surgical Critical Care, and Surgical Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Michael B. Yaffe

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Contributions

S.S. and G.C.W. conceived the project, designed research and analysed data. S.S. performed all the bacterial and plant experiments. V.M.P.B. and S.S. performed protein purification, size separation and ICP-MS analysis. K.B. performed MS analysis. A.A. performed the cytotoxicity and haemolysis experiments. M.C.A. performed the EPR experiments. D.M.A. performed the mass photometry analysis. T.A.S. performed experiments related to T. gondii. S.S. performed all other biochemical characterizations and other experiments. K.Y. helped with the CRISPR experiment. C.L.D., M.B.Y. and S.L. provided advice and reagents. S.S. and G.C.W. wrote the manuscript and integrated comments from the other authors. V.M.P.B. provided help making the models and figures.

Corresponding author

Correspondence to Graham C. Walker.

Ethics declarations

Competing interests

A provisional patent application related to haem-binding small peptides has been filed by the MIT with G.C.W., S.S. and V.M.P.B. as named inventors. All the other authors do not have any competing interests.

Peer review

Peer review information

Nature Microbiology thanks Isidro Abreu, Peter Mergaert, Philip Poole and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 NCR247 treatment induces an increase in expression of iron uptake genes.

a-d, qRT PCR analysis shows an increase in expression of genes involved in iron uptake (foxA (a), fhuP (b), rhrA (c), and fbpA (d)) upon treatment with 2 µM NCR247 for 30 mins. Cells were grown in iron sufficient medium (5 µM). e-i, qRT PCR analysis show increase in transcript levels of genes involved in iron uptake (hmuP(e), foxA (f), fhup (g), rhrA (h) and fbpA (i)) when grown in iron-replete medium (30 µM) upon NCR247 treatment for 30 mins. In a-i, the data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene SMc00128 and are presented as average of three technical replicates ± s.d. j, Growth pattern of 2 µM NCR247 treated cells when grown in minimal medium lacking Fe, Mn, and Zn or medium supplemented with 30 µM of either FeSO4, MnCl2 or ZnSO4. Data are presented as mean of three biological replicates ± s.d. k, Change in Mn, Co and Zn content of 2 µM NCR247 treated S. meliloti when compared to untreated cells as measured by ICP-MS analysis. Data are presented as mean of two biological replicates ± s.d. In a **P < 0.004, b *** P = 0.0005,d***P = 0.0006**, e*P = 0.0463, f*P = 0.0478, h*P = .0452, k** P = 0.001, *** P = 0.0006 and in c,g,i****P < 0.0001 vs untreated sample; two-way analysis of variance (ANOVA) with multiple comparisons.

Source data

Extended Data Fig. 2 NCR247 binds haem and NSR247 lacks haem binding ability.

a, Purified MBP-NCR247 is a reddish colored protein. b, UV-Vis spectra of MBP-NCR247 showing peaks at 362 nm, 418 nm, and 540 nm with a slight shoulder at 580 nm. c, LC-MS spectrum from MBP-NCR247 (top) when compared to heme standard (Bottom). d, Metal content of purified MBP and MBP-NCR247 (measured using ICP-MS analysis) showing the presence of iron and absence of any other metal. Data are presented as mean of three biological replicates ± s.d. e, EPR spectrum of NSR247 with heme (g values =5.56) indicating a high spin ferric heme (spectrum similar to free heme). f, Resonance –Raman spectrum of NSR247 with heme shows prominent ν peaks indicative of a Fe3+, five-coordinate, high spin (5cHS) b-type heme. g and h, Lack of change in expression of iron uptake gene (hmuP) upon treatment with 2 µM (g) or 15 µM (h) NSR247. Data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene SMc00128 and are presented as average of three technical replicates ± s.d. ****P < 0.0001 NCR247 treated WT vs Untreated; two-way analysis of variance (ANOVA) with multiple comparisons. i, UV-Vis spectra of NCR247- Ferrous heme complex, after reduction by excess Sodium dithionite in an anaerobic chamber, indicating peaks at 420 nm and 550 nm. j, Representative raw image of association and dissociation steps (before and after diving red line) in an Octet bio-layer interferometry experiment (detailed in methods). Biotinylated heme was used as ligand and NCR247 was used as analyte. In a, b, c, e, f, and i, representative data from three independent experiments is shown.

Source data

Extended Data Fig. 3 Haem-induced multimerization of NCR247.

a, UV-Vis spectrum of NCR247-heme complex upon addition of increasing concentrations of heme (0.2 to 20 molar equivalents). Peak at 366 nm visibly increases in height even after peaks at 450 nm and 580 nm are saturated. b, Size-exclusion chromatogram of native MBP-NSR247 from E. coli grown with ALA (predominant monomer) c, Mass photometry analysis indicating the average molecular weight of the species that existed after addition of half molar equivalent, equimolar, and excess heme to the monomer fraction of purified MBP-NCR247. Data are presented as mean of three independent replicates ± s.d. d, Whole view of the grid used for negative staining made from the hexameric fraction of purified MBP-NCR247 showing multiple daisy like species marked in red. e, Tris tricine SDS gel showing multimerization of NCR247 peptide upon addition of heme (FePPIX) and CoPPIX and crosslinking with formaldehyde. In a, b, d and e representative data from three independent experiments is shown.

Source data

Extended Data Fig. 4 NCR247 drives iron uptake by controlling Irr mediated iron regulation.

a, Fluorescence of FITC-NCR247 quenched by increasing concentrations of heme. Fluorescence of FITC-NSR247 remains unquenched even after addition of excess heme. b-f, Decrease in expression of genes involved in iron uptake (fhuP (b), fbpA (c), rhrA (d), hmuP (e), foxA (f)) in a 2 µM NCR247 treated Δirr when compared to NCR247 treated wild type S. meliloti, when grown in iron-replete medium (30 µM). NCR247 was treated for 30 mins g and h, Growth pattern of untreated(g) and NCR247 treated (h) wildtype and Δirr cells in iron-depleted (-Fe) and iron-replete media (30 µM). i, Derepressed expression of hmuP in an untreated ΔrirA when compared to wildtype S. meliloti as measured by qRT-PCR analysis. j, Increased uptake of 55Fe in untreated ΔrirA when compared to untreated and NCR247 treated wildtype S. meliloti. k and l, Growth pattern of untreated (k) and NCR247 treated (l) wildtype and ΔrirA cells in iron-depleted (-Fe), iron sufficient (5 µM) and iron-replete media (30 µM). In a, g, h, j, k and l data are presented as mean of three independent replicates ± s.d. In b-f and i, The data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene SMc00128 and are presented as average of three technical replicates ± s.d. In b-f, ****P < 0.0001 NCR247 treated WT vs Δirr - samples; In i, ***P = 0.0003 WT untreated vs WT NCR247 treated; two-way analysis of variance (ANOVA) with multiple comparisons.

Source data

Extended Data Fig. 5 Model of regulation of iron metabolism by Irr and RirA in S. meliloti and proposed mechanism of action of NCR247.

In S. meliloti regulation of iron status is controlled by two anti-parallel regulators Irr and RirA. Both Irr and RirA bind to DNA elements upstream of genes and repress gene expression. However, Irr senses iron status through heme94 (Irr loses DNA binding ability upon heme binding28 and RirA through Fe-S cluster formation (functional Fe-S cluster binding on RirA is needed for RirA to bind DNA29. During low intracellular iron concentrations, due to low intracellular heme availability, Irr remains stable and represses genes involved in iron storage, iron export, and also rirA25. Hence, in this condition, the expression of rirA is lowered and the availability of Fe-S cluster is scarce. This lack of RirA leads to an increase in expression of iron uptake genes. At high iron concentration, heme is available to bind Irr and this leads to the inability of Irr to bind DNA for repression. This leads to an increase in transcription of rirA and repression by RirA leads to a decrease in expression of iron uptake genes to prevent further iron uptake. When NCR247 is present during these conditions, it sequesters heme and hence heme is not available to inactivate Irr mediated repression. This leads to unusual availability of active Irr and repression of rirA. This leads to activation of iron uptake genes. Thus, NCR247 treatment leads to an iron starvation response and increase in import of iron even during iron sufficient and replete conditions.

Extended Data Fig. 6 Nodules of M. sativa and M. truncatula inoculated with Δirr S. meliloti are pale and deformed.

a, b, Representative image of 21-day nodules of M. sativa inoculated with wild type (Rm1021) (a) or Δirr S. meliloti (b). c, d, Representative image of 21-day nodules of M. truncatula (A17) inoculated with wild type (Rm1021) (c) or Δirr S. meliloti (d). In a-d, representative image from 7 sets of plants with each pair is shown. e and f, Table representing shoot height of the plant and number of nodules elicited for each pair. A total of 7 plants from each pair were considered for measurements.

Source data

Extended Data Fig. 7 CRISPR construct and genetic localization of the mutants.

a, Sequence of genetic locus of NCR247. Pale green is the 5’UTR region, dark yellow is the NCR247 coding region (signal sequence and coding region separated by an intron), Grey is part of the 3’UTR region. Location of gRNAs are marked in bold. The homozygous deleted region common in all the mutants is designated in red. b, Sequences of the gRNAs used in two different CRISPR constructs used. c, Location of deletions and substitution mutations in the 8 homozygous hairy roots obtained when compared to the wildtype sequence. Mutant 1-6 was obtained when the CRISPR construct 1 was used and Mutant 7-8 was obtained using CRISPR construct 2. d, Total iron content of the whole nodules isolated from wildtype and NCR247 knockdown roots as measured by ICP-MS analysis. 28 nodules from each type were collected and analysed in two technical replicates. In d, **P = 0.007 WT vs NCR247 knockdown; two tailed unpaired t test.

Source data

Extended Data Fig. 8 Sequences similar to NCR247.

a, UV-Vis spectrum of NCR peptides (NCR169, NCR035, NCR211, and NCR247) with heme indicating that only NCR247 shows a spectrum characteristic of heme binding proteins. b, Sequence alignment of NCR247 from the plants M. sativa and M. truncatula. c and d, Sequence alignment of NCR247 from M. truncatula and C-terminal region of DppD (protein involved in heme transport) of Hemophilus influenzae (c) and E. coli (d). In b, c, and d, alignments were performed using CLUSTAL Omega. NCR247 from M. sativa and C-terminal end of DppD were significantly similar sequences with a e-value less than 5 obtained in a BLAST search. e and f, Sequence similar to NCR247 from C-terminal end of DppD of E. coli, tagged to MBP purifies as a reddish colored protein (e) and shows EPR spectrum (mixture of high and low spin heme) similar to other haem binding proteins95 g and h, Chemically synthesized peptide with sequence similar to NCR247 from C-terminal end of DppD of H. influenzae shows reddish color upon binding heme (g) and exhibits a UV-Vis spectrum (366 nm, 427 nm, and 540 nm) characteristic of haem binding proteins(h). In a and e-h representative data from three independent experiments is shown.

Source data

Extended Data Fig. 9 NCR247 suitability for potential therapeutic applications.

a, Standard cytotoxicity assay (Methods) on HEK293 cell line indicates negligible hemolysis by L and D-NCR247. b, Standard hemolysis assay (Methods) on hRBC indicates negligible hemolysis by L and D-NCR247. In a and b, Triton X-100 was used as a positive lysis control and data was normalized to PBS blank. Data are presented as mean of three independent replicates ± s.d. c, d, Pull down of heme by biotinylated NCR247 from 0-day old (c)and 42-day old plasma(d). Oxalic acid assay was used to measure the total heme content of the pull-down. Data are presented as mean of three independent replicates (with three technical replicates for each) ± s.d. In a, b, **P = 0.0016, ***P = 0.0009 vs TritonX-100 treated sample and in d, and ****P < 0.0001 vs beads only; two-way analysis of variance (ANOVA) with multiple comparisons.

Source data

Extended Data Fig. 10 NCR247 sequestering haeme from haemoproteins in planta is less probable.

a, UV-Vis spectrum showing poor interaction of oxidized regioisomers of NCR247 and a presence of minor Soret band with heme when compared to reduced NCR247. b, UV-Vis spectrum showing inability of NCR247 to sequester heme from Cytochrome c. The absorption spectrum of Cytochrome c remains unaltered even after addition of excess NCR247.

Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1, Supplementary Tables 1 and 2.

Reporting Summary

Peer Review File

Supplementary Table 1

Genes modified in expression following NCR247 treatment that are annotated to be involved in iron.

Supplementary Table 2

Primer, plasmid and strain list.

Supplementary Data

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Sankari, S., Babu, V.M.P., Bian, K. et al. A haem-sequestering plant peptide promotes iron uptake in symbiotic bacteria. Nat Microbiol 7, 1453–1465 (2022). https://doi.org/10.1038/s41564-022-01192-y

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