Engineering Pseudomonas protegens Pf-5 for Nitrogen Fixation and its Application to Improve Plant Growth under Nitrogen-Deficient Conditions
et al. (2013) Engineering Pseudomonas protegens Pf-5 for Nitrogen Fixation and its Application to
Improve Plant Growth under Nitrogen-Deficient Conditions. PLoS ONE 8(5): e63666. doi:10.1371/journal.pone.0063666
Engineering Pseudomonas protegens Pf-5 for Nitrogen Fixation and its Application to Improve Plant Growth under Nitrogen-Deficient Conditions
Lorena Setten 0
Gabriela Soto 0
Matteo Mozzicafreddo 0
Ana Romina Fox 0
Christian Lisi 0
Massimiliano Cuccioloni 0
Mauro Angeletti 0
Elba Pagano 0
Antonio Daz-Paleo 0
Nicola s Daniel Ayub 0
Marie-Joelle Virolle, University Paris South, France
0 1 Instituto de Gene tica Ewald A. Favret (CICVyA-INTA) , Castelar, Buenos Aires , Argentina , 2 Consejo Nacional de Investigaciones Cient ficas y Te cnicas (CONICET), Cuidad Auto noma de Buenos Aires, Argentina, 3 School of Biosciences and Biotechnology, University of Camerino , Camerino (MC) , Italy
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.
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Funding: This work was supported by Instituto Nacional de Tecnologa Agropecuaria (grant AEGR#3425 to ADP) and Agencia Nacional de Promocio n Cientfica
y Tecnol ogica (grant PICT 2011-1325 to NA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Nitrogen fertilizer application is an essential input for crop
productivity in most regions of the world [1]. Reduction or
elimination of this application will attenuate our dependence on
fossil fuels as they are necessary for the production of nitrogen
fertilizer. Both from health and environmental perspectives, the
quality of ground water is adversely affected by nitrogen
fertilization [2]. The interest in these problems has prompted
the study of biological nitrogen fixation.
Nitrogen fixation is widespread among the Eubacteria and
Archae domains [3]. These metabolically divergent and
nonphylogenetic groups of microorganisms are collectively known as
diazotophs; all of them possess a metallo-enzyme complex, the
nitrogenase, which catalyzes one of the most remarkable chemical
transformations in biological systems: the ATP-dependent
reduction of atmospheric dinitrogen (N2) to bioavailable ammonia [4].
In concordance with their exceptionally genetic diversity,
diazotrophs have diverse lifestyles and several mechanisms to prevent
O2 from damaging their nitrogenase. There are numerous
freeliving strains of bacteria and archaea that are able to fix N2 under
aerobic (e.g. Azotobacter vinelandii and Anabaena sp. strain PCC 7120)
and anaerobic (e.g. Klebsiella pneumonia and Methanosarcina barkeri)
conditions [5], [6], [7]. In addition, many strains of nitrogen-fixing
Proteobacteria (e.g. Ensifer meliloti and Cupriavidus taiwanensis),
Actinobacteria (e.g. Frankia alni and Auraticoccus monumenti) and
Cyanobacteria (e.g. Nostoc punctiforme) can form symbiotic associations
with legumes (e.g. soybean and alfalfa) and non-leguminous plants
(e.g. shrubs and trees), providing the host with nitrogen-rich
compounds [8], [9], [10], [11], [12], [13]. Unfortunately, this type
of symbiosis is not observed in the most economically important
crops (e.g. maize, rice and wheat) [14].
In this context, finding nitrogen-fixing inoculants to improve the
production of important non-leguminous crops has often been
touted as one of the ultimate goals of nitrogen fixation research
[2], [15]. An approach to mitigate this difficulty is the use of
endophytic diazotrophs. For example, Wood and coworkers have
shown that 20% of wheat shoots nitrogen has been derived from
Azospirillum brasilense nitrogen fixation under l (...truncated)