Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions.

Nitrogen is the second most critical factor for crop production after water. In this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was genetically modified to fix nitrogen using the genes encoding the nitrogenase of Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L medium without nitrogen, displayed high nitrogenase activity and released significant quantities of ammonium to the medium. Pf-5 X940 also showed constitutive expression and enzymatic activity of nitrogenase in ammonium medium or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri transformed with cosmid X940 showed constitutive nitrogenase activity and high ammonium production, suggesting that this phenotype depends on the genome context and that this technology to obtain nitrogen-fixing bacteria is not restricted to Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-deficient conditions. In conclusion, these results open the way to the production of effective recombinant inoculants for nitrogen fixation on a wide range of crops.

Two infA gene mutations independently originated from a mutator genotype in barley.

Cytoplasmic line 2 (CL2) is a chlorophyll mutant that was selected from a plastid mutator genotype in barley. The dynamics of greening and plastid development of CL2 first-leaf blade contrasts with that of monocots. Previous characterizations of CL2 suggested that this mutant has a delay of plastid gene translation during embryogenesis. We hypothesize that CL2 is a mutant in the infA gene, which encodes translation initiation factor 1 (IF1). Wild-type barley infA gene differs in some nucleotides from that in wheat, but the corresponding IF1 proteins are identical. However, infA from CL2 carries a point mutation, which leads to an amino acid change in IF1 residue 52. One CL2-like seedling selected from a new mutator pool also carries a point mutation in infA gene, this time leading to a change of the universally conserved amino acid residue 32. Both point mutations were T --> C substitutions. We sequenced the complementary DNA of the infA transcripts from the wild type and CL2 and found that the mutation was conserved at the mRNA level. Results strongly suggest that CL2 and CL2-like are infA gene mutants, this being the first time that a mutant phenotype is attributed to infA gene in a higher plant.