Screening for nitrogen fixation

Abstract

An essentially glutamine-free medium comprises a glutamine biosensor for detecting glutamine in an analyte. The medium is useful in methods of, for example, detecting, screening for, identifying, and selecting nitrogen-fixing microbes. The medium is also useful in methods of, for example, determining the nitrogen status of plants, plant products, and soil.

1 . An essentially glutamine-free medium comprising a glutamine biosensor for detecting glutamine in an analyte. 
     
     
         2 . The medium of  claim 1 , wherein the glutamine biosensor comprises a bacterial cell. 
     
     
         3 . The medium of  claim 2 , wherein the bacterial cell is  E. coli.    
     
     
         4 . The medium of  claim 2  or  3 , wherein the bacterial cell is a glutamine auxotroph. 
     
     
         5 . The medium of any one of  claims 2  to  4 , wherein glutamine synthetase is down-regulated in the bacterial cell as compared to a wild-type bacterial cell. 
     
     
         6 . The medium of any one of  claims 2  to  5 , wherein the bacterial cell comprises a reporter gene that is expressed in the presence of glutamine. 
     
     
         7 . The medium of  claim 6 , wherein the reporter gene is lux. 
     
     
         8 . The medium of any one of  claims 1  to  7 , wherein the glutamine biosensor is the  E. coli  strain GlnLux. 
     
     
         9 . The medium of any one of  claims 1  to  8 , wherein the growth medium is Lysogeny Broth (LB), M9 medium, agar, or combinations thereof. 
     
     
         10 . The medium of any one of  claims 1  to  9 , further comprising casamino acids. 
     
     
         11 . The medium of  claim 10 , wherein the casamino acids are present in an amount of about 0.5% v/v. 
     
     
         12 . The medium of any one of  claims 1  to  11 , further comprising a protease inhibitor. 
     
     
         13 . The medium of  claim 12 , comprising a cocktail of protease inhibitors. 
     
     
         14 . The medium of  claim 13 , wherein the cocktail of protease inhibitors is present in an amount of about 1% v/v. 
     
     
         15 . The medium of any one of  claims 1  to  14 , wherein the medium is liquid, solid, or semi-solid. 
     
     
         16 . The medium of any one of  claims 1  to  15 , wherein the glutamine biosensor is diluted up to about 1000-fold. 
     
     
         17 . Use of the medium of any one of  claims 1  to  16 , for culturing, screening, detecting, or selecting a nitrogen-fixing microbe or an inoculant nitrogen-fixing microbe. 
     
     
         18 . Use of the medium of any one of  claims 1  to  16  for identifying, screening for, detecting, or quantifying glutamine content in an analyte. 
     
     
         19 . The use of  claim 18 , wherein the analyte is a plant, plant organ, seed, soil, or an extract thereof. 
     
     
         20 . The use of  claim 19 , wherein the plant, plant organ, or seed is a legume or a cereal or the extract is derived from a legume or a cereal. 
     
     
         21 . The use of  claim 20 , wherein the cereal is maize. 
     
     
         22 . The use of  claim 20 , wherein the plant, plant organ, or seed is a green manure or the extract is derived from a green manure. 
     
     
         23 . The use of any one of  claims 18  to  21 , wherein the analyte is a microbe or an extract thereof. 
     
     
         24 . The use of  claim 23 , wherein the microbe is a bacterium or a fungus or the extract is derived from a bacterium or fungus. 
     
     
         25 . The use of  claim 24 , wherein the bacterium is  Rhizobium.    
     
     
         26 . The use of  claim 23 , wherein the microbe is an endophyte. 
     
     
         27 . The use of  claim 26 , wherein the endophyte is a maize endophyte. 
     
     
         28 . A method for identifying, screening for, detecting or selecting for a nitrogen-fixing microbe or an inoculant nitrogen-fixing microbe, the method comprising the steps of 1) culturing the microbe in the medium of any one of  claims 1  to  16 ; and 2) visualizing and/or measuring a signal from the glutamine biosensor to identify, screen for, detect or select the microbe. 
     
     
         29 . The method of  claim 28 , for selecting for nitrogen-fixing microbes or inoculant nitrogen-fixing microbes in a high-throughput assay. 
     
     
         30 . A method for identifying, screening for, or detecting nitrogen fixation in a plant, plant organ, seed, soil, or extract thereof, the method comprising the steps of 1) placing at least a portion of the plant, plant organ, seed, soil, or extract thereof on or in the medium of any one of  claims 1  to  16 ; and 2) visualizing and/or measuring a signal from the glutamine biosensor as indicative of nitrogen fixation. 
     
     
         31 . The method of  claim 30  for detecting nitrogen fixation in a plant, plant organ, seed, soil, or extract thereof so as to determine the effects of different environmental conditions and/or plant genotypes on nitrogen fixation in the plant, plant organ, seed, soil, or extract thereof. 
     
     
         32 . The method of  claim 31 , wherein about 1 mg of the plant, plant organ, seed, soil, or extract thereof is placed on or in the medium. 
     
     
         33 . The method of  claim 31  or  32 , wherein the plant, plant organ, seed, soil, or extract thereof is diluted up to about 1000-fold prior to placing on or in the medium. 
     
     
         34 . The method of any one of  claims 31  to  33 , wherein the medium is solid and the plant, plant organ, seed, soil, or extract thereof is placed on the medium. 
     
     
         35 . The method of any one of  claims 31  to  34 , wherein the medium is solid and the plant, plant organ, seed, soil, or extract thereof is embedded within the medium. 
     
     
         36 . The method of any one of  claims 31  to  35 , wherein the at least a portion of the plant, plant organ, seed, soil, or extract thereof is a leaf punch. 
     
     
         37 . A glutamine biosensor, the glutamine biosensor comprising an essentially glutamine-free culture of the GlnLux strain of  E. coli  cells. 
     
     
         38 . A method for determining whether soil requires fertilization to support growth of a plant, the method comprising the steps of 1) placing at least a portion of the plant in or on the medium of any one of  claims 1  to  16 ; and 2) visualizing and/or measuring a signal from the glutamine biosensor, wherein an absent signal or a signal below a predetermined level indicates that the soil requires fertilization. 
     
     
         39 . A method for determining whether a microbe is a nitrogen-fixing microbe, the method comprising the steps of 1) placing a culture of the microbe in or on the medium of any one of  claims 1  to  16 ; and 2) visualizing and/or measuring a signal from the glutamine biosensor; wherein the presence of a signal indicates that the microbe is a nitrogen-fixing microbe. 
     
     
         40 . The method of  claim 39 , wherein the microbe is a bacterium or fungus. 
     
     
         41 . The method of  claim 40 , wherein the bacterium is  Rhizobium.    
     
     
         42 . The method of  claim 40 , wherein the microbe is an endophyte. 
     
     
         43 . The method of  claim 42 , wherein the endophyte is a maize endophyte. 
     
     
         44 . A method for identifying, screening for, detecting, or quantifying glutamine content in an analyte, the method comprising the steps of 1) placing the analyte on or in the medium of any one of  claims 1  to  16 ; and 2) visualizing and/or measuring a signal from the glutamine biosensor as indicative of glutamine content in the analyte. 
     
     
         45 . The method of  claim 44 , wherein the analyte is a plant, plant organ, seed, soil, or an extract thereof. 
     
     
         46 . The method of  claim 45 , wherein the plant, plant organ, or seed is a legume or a cereal or the extract is derived from a legume or a cereal. 
     
     
         47 . The method of  claim 46 , wherein the cereal is maize. 
     
     
         48 . The method of  claim 45 , wherein the plant, plant organ, or seed is a green manure or the extract is derived from a green manure. 
     
     
         49 . The method of  claim 44 , wherein the analyte is a microbe or an extract thereof. 
     
     
         50 . The method of  claim 49 , wherein the microbe is a bacterium or a fungus or the extract is derived from a bacterium or fungus. 
     
     
         51 . The method of  claim 50 , wherein the bacterium is  Rhizobium.    
     
     
         52 . The method of  claim 49 , wherein the microbe is an endophyte. 
     
     
         53 . The method of  claim 52 , wherein the endophyte is a maize endophyte. 
     
     
         54 . A method for predicting plant efficiency in absorbing and assimilating soil nitrogen and/or in scavenging nitrogen from senescing plant cells, the method comprising the steps of 1) placing at least a portion of the plant or an extract thereof in or on the medium of any one of  claims 1  to  16 ; and 2) visualizing and/or measuring a signal from the glutamine biosensor, wherein the presence of a signal indicates predicted efficiency in absorbing and assimilating soil nitrogen and/or scavenging nitrogen from senescing plant cells. 
     
     
         55 . The method of  claim 54 , wherein the presence of a signal at or above a predetermined level indicates predicted efficiency in absorbing and assimilating soil nitrogen and/or scavenging nitrogen from senescing plant cells. 
     
     
         56 . The method of  claim 54  or  55 , wherein intensity of the signal is positively correlated with predicted efficiency in absorbing and assimilating soil nitrogen and/or scavenging nitrogen from senescing plant cells. 
     
     
         57 . The method of any one of  claims 28  to  56 , wherein the signal is visualized and/or measured using a CCD camera or a luminometer. 
     
     
         58 . An assay for biological nitrogen fixation, the assay comprising the medium of any one of  claims 1  to  16 . 
     
     
         59 . The assay of  claim 58 , further comprising a CCD camera or a luminometer for detecting a signal from the medium in the presence of an analyte that exhibits nitrogen fixation. 
     
     
         60 . The assay of  claim 58  or  59 , further comprising instructions for use. 
     
     
         61 . The assay of any one of  claims 58  to  60 , further comprising a mail-in package for shipping the assay to a secondary location for determining the result of the assay.