SCREENING FOR NITROGEN FIXATION

• US 20140329271A1
• Filed: 06/20/2012
• Published: 11/06/2014
• Est. Priority Date: 06/21/2011
• Status: Active Grant

• Current Assignee: University of Guelph
• Original Assignee: University of Guelph
• Inventors: Raizada, Manish, Tessaro, Michael, Shehata, Hanan Reda Hassan Elsayed
• Granted Patent: US 9,279,139 B2
• Time in Patent Office: Days
• US Class Current: 435/34
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/045 Culture media therefor
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
• CPC Class Codes: C12Y 603/01002 Glutamate-ammonia ligase (6.3.1.2)
• CPC Class Codes: G01N 33/6812 Assays for specific amino acids

Abstract

An essentially glutamine-free medium comprising a whole-cell glutamine biosensor, comprising a glutamine auxotrophic E. coli and a Lux reporter gene, for detecting glutamine in an analyte. The medium is useful in methods to detecting, screening, identifying and selecting nitrogen-fixing microbes and in methods of determining the nitrogen state of plants, plant products and soil.

Claims

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 that is a glutamine auxotroph.

3. The medium of claim 2, wherein the bacterial cell is E. coli.

5. The medium of claim 2, wherein glutamine synthetase is down-regulated in the bacterial cell as compared to a wild-type bacterial cell.

6. The medium of claim 2, 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 claim 1, wherein the glutamine biosensor is the E. coli strain GlnLux.

9. The medium of claim 1, wherein the growth medium is Lysogeny Broth (LB), M9 medium, agar, or combinations thereof, and optionally comprises casamino acids and/or one or more protease inhibitors.

11. The medium of claim 9, wherein the medium comprises casamino acids in an amount of about 0.5% v/v, and protease inhibitors in an amount of about 1% v/v.

28. A method for identifying, screening for, detecting or selecting for a nitrogen-fixing microbe or an inoculant nitrogen-fixing microbe, or 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) culturing the microbe in the medium of claim 1; and

2) visualizing and/or measuring a signal from the glutamine biosensor to identify, screen for, detect or select the microbe;

or

1) placing at least a portion of the plant, plant organ, seed, soil, or extract thereof on or in the medium of claim 1; and

2) visualizing and/or measuring a signal from the glutamine biosensor as indicative of nitrogen fixation.

32. The method of claim 28, wherein about 1 mg of the plant, plant organ, seed, soil, or extract thereof is placed on or in the medium, and optionally wherein the plant, plant organ, seed, soil, or extract thereof is diluted up to about 1000-fold prior to placing in or on the medium.

34. The method of claim 28, wherein the medium is solid and the plant, plant organ, seed, soil, or extract thereof is placed on the medium or embedded within the medium.

36. The method of claim 28, wherein the at least a portion of the plant, plant organ, seed, soil, or extract thereof is a leaf punch.

39. A method for determining whether a microbe selected from a bacterium or fungus is a nitrogen-fixing microbe, the method comprising the steps ofplacing a culture of the microbe in or on the medium of claim 1;

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.

41. The method of claim 39, wherein the microbe is Rhizobium or an endophyte.

44. A method for identifying, screening for, detecting, or quantifying glutamine content in an analyte, the method comprising the steps ofplacing the analyte on or in the medium of claim 1;

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 wherein the extract is derived from a legume or a cereal.

50. The method of claim 44, wherein the analyte is a bacterium or a fungus or an extract derived from a bacterium or fungus.

51. The method of claim 50, wherein the bacterium or fungus is Rhizobium or an 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 ofplacing at least a portion of a plant or an extract thereof in or on the medium of claim 1;

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,

wherein (a) 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, and/or (b) intensity of the signal is positively correlated with predicted efficiency in absorbing and assimilating soil nitrogen and/or scavenging nitrogen from senescing plant cells.

4. (canceled)

10. (canceled)

12-14. -14. (canceled)

15. The medium of 1, wherein the medium is liquid, solid, or semi-solid, and optionally wherein the glutamine biosensor is diluted up to about 1000-fold.

16-27. -27. (canceled)

29-31. -31. (canceled)

33. (canceled)

35. (canceled)

37. A glutamine biosensor, the glutamine biosensor comprising an essentially glutamine-free culture of the GlnLux strain of E. coli cells.

38. (canceled)

40. (canceled)

42-43. -43. (canceled)

47-49. -49. (canceled)

52-53. -53. (canceled)

55-61. -61. (canceled)