Abstract: Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

Abstract: Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

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 consortia of microbes that are functionally optimized for nitrogen fixation and deliver such to plants in a targeted, efficient, and environmentally sustainable manner. The microbes within the consortium differ in nutrient utilization, temporal occupation, oxygen adaptability, and/or spatial occupation, which enables the microbes to deliver nitrogen to a cereal plant in a spatially targeted (e.g. rhizospheric) and temporally targeted (e.g. during advantageous stages of plant's life cycle) manner. The present disclosure also provides methods of creating a synthetic composition of microbes and methods of using compositions of microbes to fix atmospheric nitrogen and deliver such to a crop.

Abstract: Methods and systems are provided for generating and utilizing a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.

Abstract: The present disclosure provides non-intergeneric remodeled 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, by mitigating or eliminating the need for exogenous nitrogen-containing fertilizers. The remodeled microbes have unique colonization and nitrogen fixation abilities, which enable the microbes to deliver nitrogen to a cereal plant in a spatially targeted (e.g. rhizospheric) and temporally targeted (e.g. during advantageous stages of plants life cycle) manner. The microbes are able to replace the standard agricultural practice of sidedressing and enable a more environmentally sustainable form of farming.

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.

Abstract: The present disclosure provides guided microbial remodeling (GMR) methods for the rational improvement of plant-associated microbes to perform plant-beneficial functions. The GMR methods described herein allow for non-intergeneric genetic optimization of key regulatory networks within the microbes, which improve plant-beneficial functions over wild-type microbes but don't have the risks associated with transgenic approaches (e.g., unpredictable gene function, public and regulatory concerns, etc.). The present disclosure also provides remodeled microbes and compositions thereof. The utilization of remodeled microbes and compositions thereof will enable farmers to realize more productive and predictable crop yields without the nutrient degradation, leaching, or toxic runoff associated with traditional synthetically derived fertilizers.

Abstract: Methods of predicting in planta phenotypes of microbial strains are provided. The methods can include culturing microbial strains in a plant exudate medium and assaying in vitro phenotypes of the microbial strains. The methods may also include using the in vitro phenotypes to predict in planta phenotypes of the microbial strains. Methods of using the microbial strains in field trials are also provided.

Abstract: Methods and systems are provided for increasing the phosphate solubilization activity of a microbe. Microbes identified through the use of the disclosed methods and systems are also provided. The methods include the steps of and the systems include instructions for identifying a coding sequence associated with phosphate solubilization, altering the coding sequence associated with phosphate solubilization, functionally linking the altered coding sequence associated with phosphate solubilization to a promoter, and introducing the altered coding sequence associated with phosphate solubilization under the control of the promoter into the microbe. Alterations to the coding sequence can include, e.g., codon randomization of native codons, deletion of coding sequence, and insertion of regulatory sequences.

Abstract: Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

Abstract: The present disclosure provides a bacterial composition, comprising: at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system, wherein the bacterial strain comprises a modification in or one or more genes selected from the group consisting of bcsll, bcslll, yjbE, fhaB, pehA, glgA, otsB, treZ, and cysZ. The present disclosure further provides a bacterial composition and method for increasing the colonization of a plant growth promoting bacterial strain on a plant, wherein the plant growth promoting bacterial strain has been remodeled to increase colonization of said plant, In a further aspect, the present disclosure provides methods of increasing nitrogen or nitrogen fixation available to a plant.

Abstract: Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

Abstract: Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

Abstract: Methods and systems are provided for generating and utilizing a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.

Abstract: Methods and systems are provided for generating and utilizing a genetically engineered bacterium comprising a modification in glnD, wherein said modification is selected from the group consisting of: deletion of the entire gene, deletion of substantially the entire gene, deletion of an ACT domain, deletion of more than 50% of an ACT domain, deactivation of an ACT domain, and deactivation of an UTase domain.

Abstract: Methods and systems are provided for generating and utilizing a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.

Abstract: Methods for design of genetic circuits are provided.