title: Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae.
abstract: The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N2and CO2from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N2from the air into crops.
authors.affiliation: Instituto de Investigaciones en Biodiversidad y Biotecnología, Consejo Nacional de Investigaciones Científicas y Técnicas, Mar del Plata, Buenos Aires, Argentina; Fundación para Investigaciones Biológicas Aplicadas, Argentina.
authors.forename: Rafael
authors.initials: R
authors.lastname: Ambrosio
authors.affiliation: Instituto de Investigaciones en Biodiversidad y Biotecnología, Consejo Nacional de Investigaciones Científicas y Técnicas, Mar del Plata, Buenos Aires, Argentina; Fundación para Investigaciones Biológicas Aplicadas, Argentina.
authors.forename: Juan Cesar Federico
authors.initials: JCF
authors.lastname: Ortiz-Marquez
authors.affiliation: Instituto de Investigaciones en Biodiversidad y Biotecnología, Consejo Nacional de Investigaciones Científicas y Técnicas, Mar del Plata, Buenos Aires, Argentina; Fundación para Investigaciones Biológicas Aplicadas, Argentina. Electronic address: lcuratti@fiba.org.ar.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Curatti
mesh_terms: D064751:Ammonium Compounds / Q000302:isolation & purification / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000502:physiology*; D053898:Biosynthetic Pathways / Q000235:genetics; D005308:Fertilizers / Q000382:microbiology*; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D058086:Microalgae / Q000254:growth & development* / Q000382:microbiology; D009586:Nitrogen Fixation / Q000502:physiology; D063245:Plant Development / Q000502:physiology*
chemical_list: D064751:Ammonium Compounds; D005308:Fertilizers
keywords: Ammonium excretion; Fertilization; Microalgae; Nitrogen assimilation; Nitrogen fixation; Plants