Control of nitrogen fixation in rhizobia that associate with cereals

Abstract

Disclosed herein are engineered rhizobia having nif clusters that enable the fixation of nitrogen under free-living conditions, as well as ammonium and oxygen tolerant nitrogen fixation under free-living conditions. Also provided are methods for producing nitrogen for consumption by a cereal crop using these engineered rhizobia.

1 . A rhizobium that can fix nitrogen under aerobic free-living conditions, comprising a symbiotic rhizobium having an exogenous nif cluster, wherein the exogenous nif cluster confers nitrogen fixation capability on the symbiotic rhizobium under aerobic free-living conditions, and wherein the rhizobium is not  Azorhizobium caulinodans.    
     
     
         2 . The rhizobium of  claim 1 , wherein the exogenous nif cluster is selected from a group consisting of a free-living diazotroph, a symbiotic diazotroph, a photosynthetic Alphaproteobacteria, a Gammaproteobacteria, a cyanobacteria, a firmicutes, a  Rhodobacter sphaeroides , and a  Rhodopseudomonas palustris.    
     
     
         3 . The rhizobium of  claim 1 , wherein the exogenous nif cluster is an inducible refactored nif cluster. 
     
     
         4 . The rhizobium of  claim 3 , wherein the inducible refactored nif cluster is an inducible refactored  Klebsiella  nif cluster. 
     
     
         5 . The rhizobium of  claim 1 , wherein the rhizobium is IRBG74. 
     
     
         6 . The rhizobium of  claim 1 , wherein the exogenous nif cluster comprises 6 nif genes or operons. 
     
     
         7 . The rhizobium of  claim 6 , wherein the 6 nif genes or operons are nifHDK(T)Y, nifEN(X), nifJ, nifBQ, nifF, and nifUSVWZM. 
     
     
         8 . The rhizobium of  claim 6 , wherein each nif gene or operon of the exogenous nif cluster is preceded by a T7 promoter. 
     
     
         9 . The rhizobium of  claim 1 , further comprising an endogenous nif cluster. 
     
     
         10 . The rhizobium of  claim 1 , wherein the exogenous nif cluster further comprises a terminator. 
     
     
         11 . The rhizobium of  claim 8 , wherein the T7 promoter has a terminator and wherein the terminator is downstream from the T7 promoter. 
     
     
         12 . The rhizobium of  claim 11 , wherein the exogenous nif cluster is a refactored rhizobium IRBG74 nif cluster. 
     
     
         13 - 33 . (canceled) 
     
     
         34 . A method for making a nitrogen-fixing bacterium, the method comprising:
 a) identifying a host bacterium;   b) selecting a donor bacterium having a nif cluster based on evolutionary distance between the host bacterium and the donor bacterium;   c) inserting the nif cluster of the donor bacterium to the host bacterium, thereby making a nitrogen-fixing bacterium.   
     
     
         35 . The method of  claim 34 , wherein the evolutionary distance between the host bacterium and the donor bacterium is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% substitutions per site in 16S ribosomal RNA gene sequence. 
     
     
         36 . The method of  claim 34 , wherein the host bacterium and the donor bacterium are in the same genus, family, order, or class. 
     
     
         37 - 39 . (canceled) 
     
     
         40 . The method of  claim 34 , wherein the host bacterium is  E. coli  and the donor bacterium is  K. oxytoca.    
     
     
         41 . (canceled) 
     
     
         42 . The method of  claim 34 , wherein the host bacterium is  Rhizobium  IRBG74, and the donor bacterium is  R. sphaeroides.    
     
     
         43 . The method of  claim 34 , wherein the host bacterium is a nonsymbiotic bacterium, e.g.,  Azotobacter, Beijerinckia , or  Clostridium  bacterium. 
     
     
         44 - 45 . (canceled) 
     
     
         46 . The method of  claim 34 , wherein the inserted nif cluster is under inducible control. 
     
     
         47 - 59 . (canceled)