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Exploring the Intrinsic Limits of Nitrogenase Transfer from Bacteria to Eukaryotes


Biological nitrogen fixation is widespread among the Eubacteria and Archae domains but completely absent in eukaryotes. The lack of lateral transfer of nitrogen-fixation genes from prokaryotes to eukaryotes has been partially attributed to the physiological requirements necessary for the function of the nitrogenase complex. However, symbiotic bacterial nitrogenase activity is protected by the nodule, a plant structure whose organogenesis can be trigged in the absence of bacteria. To explore the intrinsic potentiality of this plant organ, we generated rhizobium-independent nodules in alfalfa by overexpressing the MsDMI3 kinase lacking the autoinhibitory domain. These transgenic nodules showed similar levels of leghemoglobin, free oxygen, ATP, and NADPH to those of efficient Sinorhizobium meliloti B399-infected nodules, suggesting that the rhizobium-independent nodules can provide an optimal microenvironment for nitrogenase activity. Finally, we discuss the intrinsic evolutionary constraints on transfer of nitrogen-fixation genes between bacteria and eukaryotes.

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Correspondence to Nicolás Daniel Ayub.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1.

Method details. Contains the details of the methods followed for the identification and characterization of MsDMI3 gene (DOCX 16 kb)

Online Resource 2

. Complete sequence of 35S-MsDMI3/1-340 vector showing RB: right border, nptII: kanamycin resistance, CaMV35S: constitutive promoter, MsDMI3/1-340 (DMI3 truncated gene from alfalfa, NOS: terminator and LB: left border within t-DNA region (PDF 145 kb)

Online Resource 3.

Similarity of MsDMI3 protein to the DMI3 protein sequence of others plant species. The aminoacid similarity was analyzed and the domain structures of DMI3 protein sequences were identified by HMMPFAM ( The domain graphics follow pfam graphical codes: a green rectangle represents the protein kinase domain (PF00069), green and purple small green rectangles represent EF-hand domains (PF13405 and PF00036) and a violet rectangle represents EF-hand domain pair (PF13499). When the sequence match the full length of the HMM that models Pfam the rectangles borders are round while if it does not match the rectangle has jagged edge (PDF 425 kb)

Online Resource 4.

Nodules induced by Sinorhizobium meliloti B399 (nitrogen-fixing nodule) and truncated DMI3 kinase (DMI3-induced nodule) (PDF 2128 kb)

Online Resource 5.

Gel electrophoresis of PCR products of 35SMsDMI3/1-340, virC, nifH and AAT. SN: spontaneous nodule induced by nitrogen deficient conditions, RN: Sinorhizobium meliloti B399 nodule, TN: transgenic nodule containting 35SMsDMI3/1-340 vector, A: Agrobacterium rhizogenes MSU440, S: Sinorhizobium meliloti B399, RA: recombinant Agrobacterium rhizogenes MSU440 containing 35SMsDMI3/1-340 vector (PDF 95 kb)

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Soto, G., Fox, A.R. & Ayub, N.D. Exploring the Intrinsic Limits of Nitrogenase Transfer from Bacteria to Eukaryotes. J Mol Evol 77, 3–7 (2013).

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  • Nitrogen fixation
  • Evolution
  • Lateral transfer
  • Bacteria
  • Eukaryotes
  • Oxygen