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.
Bibliographical noteFunding Information:
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.
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't
- Research Support, U.S. Gov't, Non-P.H.S.