Abstract: The present disclosure provides engineered gram-positive microbes that are able to fix atmospheric nitrogen and deliver such to plants in a targeted, efficient, and environmentally sustainable manner. The utilization of the taught microbial products will enable farmers to realize more productive and predictable crop yields without the nutrient degradation, leaching, or toxic runoff associated with traditional synthetically derived nitrogen fertilizer.

Abstract: Methods and systems are provided for generating and utilizing a genetically engineered bacterium comprising a modification in a gene regulating nitrogen fixation or assimilation, wherein the modification in the gene regulating nitrogen fixation or assimilation results in one or more of: constitutive expression of a nifA gene in nitrogen limiting and non-nitrogen limiting conditions, activity of nifA in non-nitrogen limiting conditions, decreased uridylyl-transferase activity of GlnD, decreased adenylyl-removing activity of GlnE, and increased ammonium excretion.

Abstract: Systems for plant culture include a chamber featuring one or more walls enclosing a spatial volume internal to the chamber, where the one or more walls include a surface for supporting a plant within the enclosed spatial volume, a gas delivery apparatus with at least one gas source, a nutrient delivery apparatus with a reservoir, a sampling apparatus connected to a port formed in the one or more walls, and a controller configured so that during operation of the system, the controller activates the nutrient delivery apparatus to deliver an aqueous growth medium to the plant, and activates the gas delivery apparatus to deliver into the enclosed spatial volume a mixture of isotopically-substituted gases. Also provided are methods of use of the system for measuring nitrogen in a plant and for identifying microbes capable of providing fixed nitrogen to a plant.

Abstract: The present disclosure provides high-throughput methods for rapidly mutagenizing, screening, and targeting candidate microbes that are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The methods utilize a microbial biosensor capable of detecting the presence/absence of ammonium and/or glutamine in a composition and signaling with a fluorescent reporter. The present disclosure further utilizes rapid visual detection assays capable of processing thousands of candidate microbes. The disclosed methods and biosensor can be used to identify mutant bacteria with improved nitrogen fixing capabilities. Mutant bacteria with improved nitrogen fixing capabilities are also disclosed, as well as methods of utilizing these novel bacteria to provide fixed nitrogen to a plant.

Abstract: A genetically engineered bacterium with a modification in one or more genes selected from: NAC, ptsH, iaaA, gltA, pga, sdiA, fimA1, fimA2, fimA3, fimA4, wzxE, bolA, iscR, fhuF, sodA, sodB, sodC, FNR, arcA, arcB, rpoS, sbnA, treA, treB, phoP, phoQ, yjjPB, ychM, dauA, actP, yusV1, yieL1, yieL2, yieL3, yieL4, pgaB, rafA, melA, uidA, manA, abfA, abnA, lacZ, and yusV2 is disclosed. Methods of use of the genetically engineered bacterium to provide fixed nitrogen to plants, and compositions including the bacterium are also provided.