Abstract:

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.

Author affiliation: Setten, Lorena. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Mozzicafreddo, Matteo. Università di Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Lisi, Christian Daniel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Cuccioloni, Massimiliano. Università di Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: Angeletti, Mauro. Università di Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: Pagano, Elba Maria. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Diaz Paleo, Antonio Horacio. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Ayub, Nicolás Daniel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

Snakin-1, a peptide produced by higher plants, has broad-spectrum antibiotic activity, inhibiting organisms ranging from Bacteria to Eukaryotes. However, the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a mutation library of Pseudomonas fluorescens Pf-5 in LB and agar medium supplemented with alfalfa snakin-1 (MsSN1). We identified three biofilm formation-related Pseudomonas mutants that showed increased resistance to MsSN1. Genetic, physiological and bioinformatics analysis validated the results of the mutant screens, indicating that bacterial adhesion protein lapA is probably the target of MsSN1. Collectively, these findings suggest that snakin-1 acts on microbial adhesion properties.

Author affiliation: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: García, Araceli Nora. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Mozzicafreddo, Matteo. Universita Degli Di Camerino; Italia

Author affiliation: Cuccioloni, Massimiliano. Universita Degli Di Camerino; Italia

Author affiliation: Angeletti, Mauro. Universita Degli Di Camerino; Italia

Author affiliation: Pagano, Elba Maria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.

Author affiliation: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina

Author affiliation: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina

Author affiliation: Mozzicafreddo, Matteo. University of Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: García, Araceli Nora. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Cuccioloni, Massimiliano. University of Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: Angeletti, Mauro. University of Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: Salerno, Juan Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Ayub, Nicolas Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina