Method for repairing DNA in nitrogen-fixing bacteria unable to synthesise nitrous oxide reductase, pairs of primers, nucleotide sequences and plasmids for modifying the bacteria, and modified bacteria

Abstract

The present invention relates to the production of a recombinant plasmid as a vector for introducing repair sequences into nitrogen-fixing bacterial cells incapable of expressing nitrous oxide reductase. Successful insertion of the recombinant plasmid leads to restoration of the inactive operon, preventing the release of the greenhouse gas nitrous oxide (N₂O) during the denitrification process. The resulting mutant strain exhibits the same agronomic efficiency as the original strain, but with the added benefit of significantly reducing N₂O emissions, contributing to a more sustainable approach to agriculture and food production. Additional aspects of the present invention relate to primer pairs, nucleotide sequences, modified microorganisms, as well as methods for the preparation thereof.

Description

"DNA REPAIR METHOD IN NITROGEN-FIXING BACTERIA UNABLE TO SYNTHESIZ NITROGEN OXIDE" TROSO REDUCTASE, PRIMER PAIRS, NUCLEOTIDE AND PLASMID SEQUENCES FOR BACTERIAL MODIFICATION, AND MODIFIED BACTERIA Technical Field of the Invention

[0001] The present invention utilizes homologous transposition and / or recombination mechanisms and a recombinant plasmid as a vector to introduce the nos operon into nitrogen-fixing bacterial cells. Successful chromosomal insertion of the nos operon genes leads to the phenotypic restoration of the reduction of the greenhouse gas nitrous oxide (N2O) to molecular nitrogen (N2), preventing the release of this harmful gas into the environment during the denitrification process. The resulting mutant strain exhibits the same agronomic efficiency as the original strain, but with the added benefit of significantly reducing N2O emissions, contributing to a more sustainable approach in agriculture and food production. Description of the State of the Art

[0002] Nitrogen (N) is one of the primary nutrients essential for the survival of all living organisms. It is a necessary component in many biomolecules, including proteins, deoxyribonucleic acid (DNA), and chlorophyll. In food, fiber, and energy production systems, N is considered one of the most limiting elements in production (Stein & Klotz, 2016), especially in weathered soils with low levels of organic matter. This limiting factor in production occurs even when 78% of the gas in the atmosphere is represented by molecular nitrogen gas (N2) (Canfield et al., 2010). N2 in the form of nitrogen is accessible only to some microorganisms capable of fixing atmospheric nitrogen, which are able to convert N2 into ammonia (NH3) or through abiotic processes such as the discharge of lightning or the Haber-Bosch industrial process (Lindstrom & Mousavi, 2019). From this point, nitrogen can assume multiple oxidation states and chemical forms, allowing it to be easily accessible to other organisms, including primary producers such as plants (Gruber & Galloway, 2008).

[0003] During the nitrogen cycle, the main transformations in the oxidation state are nitrogen fixation, nitrification, denitrification, anammox, and ammonification (Bernhard, 2010). Besides the oxidation state, nitrogen exists in many different forms, including inorganic forms (e.g., ammonia, nitrate) and organic forms (e.g., amino acids and nucleic acids). Thus, the transformations of nitrogen in its various oxidation states and forms are fundamental for the proper functioning of the ecosystem and productivity in the biosphere. The nitrogen cycle is highly dependent on the activities of a diverse set of microorganisms, such as bacteria, archaea, fungi, plants, and animals (Aryal et al., 2022). These organisms utilize nitrogen in different oxidation states for growth and, in some cases, for energy production in anoxic environments.Production systems become more efficient when the nitrogen cycle is being carried out completely.

[0004] Since the Industrial Revolution, the expansion of human activities has caused irreversible environmental damage, including climate change, changes in the integrity of the biosphere, changes in biogeochemical cycles, such as the phosphorus and nitrogen cycles, changes in the lithosphere, hydrosphere, and biodiversity of the planet (Richardson et al., 2023). It is estimated that the nitrogen applied in agricultural systems around the world from anthropogenic sources is on the order of 190 Tg of N per year. This value is much higher than the suggested annual planetary limit of 62 Tg of N (Richardson et al., 2023). Part of this nitrogen applied in productive fields is mainly in the form of ammonium. (NH4 +Initially, it undergoes an oxidation process and then, in compacted soils and under anaerobic conditions, it is reduced to nitrous oxide (N2O) when the denitrification process is partial (Figure 1A). N2O is a long-lived greenhouse gas (GHG), with an estimated permanence in the atmosphere of 116 ± 9 years (Prather et al., 2015). Furthermore, N2O has a high global warming potential, approximately 300 times more potent than CO2, and has the capacity to promote the destruction of the stratospheric ozone layer (Ravishankara et al., 2009). Global N2O emissions in the period 2007-2016 were around 17 Tg of N per year, with 11.4 Tg (67.1%) coming from anthropogenic sources. Within the context of human interference, agricultural activities account for 7.3 Tg of N per year, that is, 42.9% of total emissions, or 62.4% of anthropogenic emissions (Tian et al., 2020).The main source of N2O emissions in agricultural activities is the excessive use of soluble fertilizers (Galloway et al., 2008). Biochemically, microbial nitrification and denitrification are the two main processes of N2O generation. During the nitrification process, N2O is produced as a byproduct when ammonia is oxidized to nitrite via hydroxylamine (Hallin et al., 2018). N2O is also generated from NO during incomplete denitrification, which intrinsically involves various soil microorganisms, including bacteria, fungi, and archaea (Hallin et al., 2018). The plant also produces nitric oxide (NO), a gaseous free radical, as a signaling molecule in various intracellular and extracellular processes, and during different stages of the legume-rhizobium interaction, such as recognition, infection, nodule development, and root nodule senescence (Signorelli et al., 2020).To date, only one microbial enzyme, copper-dependent N2O reductase (encoded by genes in the nosRZDFYLX operon), has been identified as participating in the process of reducing N2O to N2 (Kuypers et al., 2018).

[0005] The expression of exogenous genes in bacteria that provide new biochemical capabilities to the cell is commonly carried out in circular extrachromosomal DNA molecules called plasmids. However, their inherent instability in segregation across generations and the use of selective pressure markers such as antibiotics hinder the obtaining of new isogenic strains with the desired genotype and phenotype. Therefore, genetic tools that allow the expression of these genes of interest on the bacterial chromosome are strongly preferred in gene editing experiments due to the genetic stability and permanence of the genes in future bacterial populations without the need for selection markers (McKenzie & Craig, 2006a). In this context, transposons, mobile elements that mobilize large fragments of DNA from one region to another in the genome with the help of recombinase enzymes called transposases, are considered.These actively contribute to genetic variability in nature due to their mechanism of chromosomal insertion of exogenous genes. Therefore, transposons and their respective recombinases have been widely used in various bacteria for the stable and permanent expression of genes of interest (Lorenzo et al., 1998).

[0006] Among the diverse family of transposable elements, the Tn7 transposon stands out for its specific mechanism of integration into the chromosome. Tn7 is a 14,000 base pair DNA bacterial transposon discovered in Escherichia coli (Parks & Peters, 2007). Unlike other types of transposons, Tn7 transposition occurs in a specific, neutral region known as attTn7, from the English "attachment Tn7," in a specific orientation and at high frequency (Matsumoto et al., 2022). This recognition site by the Tn7 enzyme complex is located 25 base pairs of DNA downstream of the gene. glmS encodes glutamine-fructose-6-phosphate transaminase, an essential enzyme in prokaryotes and eukaryotes that plays an important role in the hexosamine biosynthetic pathway and is evolutionarily present in some bacterial phyla such as Proteobacteria, Firmicutes, and Bacteroidota (Wiles et al., 2018).

[0007] To generate new lineages carrying the introduction of exogenous genes into the genome, the mini-Tn7 transposon version was developed (Lorenzo et al., 1998). In this system, the genes of interest are inserted between two inverted 5'-3' and 3'-5' ends, called the left and right ends of Tn7, respectively. In synergy with the expression of genes encoding the transposases TnsA, TnsB, TnsC, and TnsD, they mobilize the genes of interest to the target recognition site (attTn7) through a "cut and paste" mechanism (May & Craig, 1996). TnsA performs DNA cleavage reactions at the 5' ends of Tn7, while TnsB processes DNA cleavage and joining reactions at the 3' ends of Tn7. Thus, the double-strand breaks underlying the excision of the Tn7 ends result from a collaboration between two active sites, one in TnsA and the other in TnsB (Sarnovsky, May & Craig, 1996).TnsC is an AAA+ ATPase protein that communicates with both the transposase complex (TnsAB) and the targeting module, TnsD. The latter protein is a specific DNA-binding protein that recognizes a sequence in the glmS gene (Walker et al., 2023). TnsD-mediated transposition is directed at high frequency to the attTn7 site in the transcriptional terminator of the glmS gene and has no detectable negative effect on the host (Parks & Peters, 2007). Finally, the 3' ends of the transposon are then joined to the target DNA (attTn7) by TnsB, producing a momentary conformation in which the transposon is covalently linked to the attTn7 recognition site at the 3' ends and is flanked by small “gaps” (DNA bases). (missing) at the 5' ends. Repair of these gaps by the host's own repair machinery produces a duplication of the target site by five base pairs, an inherent feature of transposon insertion (Peters & Craig, 2001).

[0008] The result of the entire process involving the mobilization of exogenous genes to the attTn7 recognition site located downstream of the glmS gene is the stable chromosomal insertion of the genes of interest flanked by the inverted ends of Tn7, free of genetic markers and transposases that are eliminated in subsequent cell divisions. The method explains how to perform insertions of large numbers of genes in Bradyrhizobium strains ensuring zero toxicity, without the use of nuclease-guided double-strand DNA cleavage methods, using only the synergistic action of the TnsABCD transposase complex for the specific insertion of genetic material between the donor DNA containing Tn7 transposon ends and the attTn7 target DNA site in the host cell.

[0009] US patent application 20240057611 A1 details a method for identifying and applying a microbial material that can enhance plant growth while mitigating nitrous oxide emissions. The material comprises a bacterial strain of Bradyrhizobium ottawaense, which stands out for its high nitrogen fixation capacity combined with the ability to reduce N2O emissions. The inventors discovered that the SG09, SF21, and SH12 strains, all belonging to the group, possess both capabilities, making them ideal for sustainable agriculture, but it does not address gene editing of these bacteria. Additionally, due to the high specificity of this species to its plant host, as is the case with rhizobia in general, it has little likelihood of widespread adoption in various other agricultural crops, even legumes. The description of the state of the patent is detailed below. The technique described in this patent also details methods for cultivating these bacteria and using them in plant cultivation to promote growth and reduce environmental impact. However, no solution is proposed for innovative precision breeding techniques.

[0010] The scientific article by Wasai-Hara et al. (2023) describes that the activity of the N2O reductase enzyme was approximately five times higher in 13 strains of B. ottawaense than the activity of this enzyme observed in the Bradyrhizobium diazoefficiens IISDA110 strain under anaerobic conditions. The authors explain that, unlike B. diazoefficiens, B. ottawaense strains have two transcription start sites for nitrous oxide reductase, i.e., promoting the reduction of N2O, which may contribute to the high expression of the nosZ gene. However, once again, nothing is taught about a modification / correction so that Bradyrhizobium strains can have the correct transcription of the nos operon that possesses an N2O reductase gene (nosZ), which reduces N2O to N2.

[0011] Sánchez et al. (2017) describe, through a scientific article, the possibility of mediation by the NasT gene in the expression mechanism of the nosRZDFYLX operon, responsible for reductions in N2O emissions, and suggest mechanisms of nitrogenous base deletion to increase the expression of nitrous oxide reductase in bacteria of the genus Bradyrhizobium diazoefficiens. Therefore, this work aimed at deleting the H1 clamp in the 5' leader region of the nosR gene mRNA, which plays a role in the termination of transcription of nos genes, and manipulating the two-component NasST system, which regulates the expression of the nosZ gene. Deletion of the H1 clamp can lead to a significant increase in the expression of nos genes, including nosZ. The NasS sensor protein inhibits the activity of the NasT transcription antiterminator in the presence of nitrate. Therefore, under aerobic conditions or in the absence of nitrate, a nasS mutant shows increased expression of nosZ due to the release of NasT from inhibition.The NasT protein binds. to the leader RNA nosR, facilitating the transcription of the reading of the nos operon and, therefore, increasing the expression of nosZ. In short, the scientific article by Sánchez et al. (2017) explains mechanisms of regulation of the nosZ gene in Bradyrhizobium diazoefficiens, but does not mention any genetic repair technique oriented towards the functioning of the nos-RZDFYLX operon.

[0012] US patent 20230257317 A1 describes methods and systems for generating and utilizing a genetically modified bacterium comprising a modification in a gene that regulates nitrogen fixation or assimilation, resulting in one or more of the following: constitutive expression of a nifA gene under nitrogen-limiting and non-nitrogen-limiting conditions, nifA activity under non-nitrogen-limiting conditions, decreased GlnD uridylyl-transferase activity, decreased GlnE adenylyl scavenging activity, and increased ammonium excretion.The patent describes a genetically modified version of Klebsiella variicola and Kosakonia sachhari, to delete the coding sequence of the nifl, ginE and ntrC genes, therefore not covering bacteria of the genera Bradyrhizobium, Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, and other nitrogen-fixing bacteria.

[0013] US patent 20210267214 A1 details materials and methods for reducing harmful atmospheric gases, such as greenhouse gases. In specific embodiments, the reduction of harmful atmospheric gases is achieved through the utilization and storage of enhanced vegetative carbon, as well as increased carbon sequestration in the soil. In some embodiments, the invention in question can be used to reduce the carbon footprint of an operator involved in, for example, agriculture, livestock production, waste management, or other industries. In certain embodiments, the invention The patent in question provides customizable products based on microorganisms, as well as methods for using these microorganism-based products for greenhouse gas reduction and / or enhanced carbon sequestration. The patent deals with a composition of microorganisms and involves the use of genetically modified microorganisms. For example, enhanced vegetative carbon utilization can be in the form of increased foliage in plants, increased stem and / or trunk diameter, enhanced root tissue growth, and / or increased plant number. In certain embodiments, the reduction of harmful atmospheric gases is achieved through enhanced agricultural fertilization practices and improved agricultural soil management.Improved agricultural fertilization practices can take the form of, for example, reducing nitrogen-rich fertilizers, as well as replacing some or all fertilizers, pesticides, and / or other soil amendments with a composition comprising one or more environmentally friendly soil microorganisms. The document suggests the use of several microorganisms; however, none of the genera Bradyrhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Nitrosomonas, Azospirillum, Nitrospirillum, and other Gram-negative nitrogen-fixing bacteria were mentioned.

[0014] US patent 20210163374 A1 discloses a bacterial composition, comprising at least one genetically modified bacterial strain that fixes atmospheric nitrogen in an agricultural system, wherein the bacterial strain in question comprises a modification in one or more genes selected from the group consisting of bcsll, bcslll, yjbE, fhaB, pehA, glgA, otsB, treZ, and cysZ. It further provides a bacterial composition and a method for increasing the colonization of a plant growth-promoting bacterial strain, wherein the growth-promoting bacterial strain has been remodeled to to increase the colonization of said plant. In a further aspect, the cited invention provides methods for increasing the nitrogen or nitrogen fixation available to a plant. The invention does not deal with the gene cluster in, nor does it specify modifications to, the genes involved in the denitrification process, which are the main objectives of the present invention. Objectives of the Invention

[0015] A primary objective of the present invention is to provide a modified microorganism, more specifically a modified nitrogen-fixing bacterium, in which the modification corrects the expression of the nos operon to allow the encoding of the nitrous oxide reductase enzyme in a bacterium that originally lacks the ability to reduce nitrous oxide (N2O) to dinitrogen (N2).

[0016] A second objective of the present invention is to provide primer pairs, nucleotide sequences, and plasmids useful for modifying various nitrogen-fixing bacteria, including bacteria of the genera Azorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Methylobacterium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, and other Gram-negative nitrogen-fixing bacteria, for correction of the operon nos and consequent encoding of the nitrous oxide reductase enzyme.

[0017] A third objective of the present invention is to provide a method for preparing a microorganism of the genus Bradyrhizobium, or other nitrogen-fixing bacteria derived from the genera Azorhizobium, Azospirillum, Azotobacter, Ensifer, Gluconoacetobacter, Mesorhizobium, Methylobacterium, Nitrosomonas, Nitrospirillum, Pseudomonas, Rhizobium, Sinorhizobium, or other Gram-negative nitrogen-fixing bacteria, modified to correct the operons and consequently encode the nitrous oxide reductase enzyme. Brief Description of the Invention

[0018] One of the objectives of the present invention is to transform strains of nitrogen-fixing bacteria that do not encode the nitrous oxide reductase enzyme into strains that express said enzyme, through the correction or insertion of the functional nosRZDFYLX operon into their genome.

[0019] Another objective of the present invention is further achieved by detailing a nucleotide sequence which is responsible for repairing and reactivating the gene encoding the nitrous oxide reductase enzyme in bacteria of the genus Bradyrhizobium, or other nitrogen-fixing Gram-negative bacteria, for example, bacteria of the genera Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum or Nitrospirillum.

[0020] One of the objectives of the present invention is further achieved by a plasmid, which is responsible for providing the repair DNA sequences for the reactivation of the gene encoding the nitrous oxide reductase enzyme in bacteria of the genus Bradyrhizobium, or other nitrogen-fixing Gram-negative bacteria, for example, bacteria of the genera Azorhizobium, Azospirillum, Azotobacter, Ensifer, Gluconoacetobacter, Mesorhizobium, Methylobacterium, Nitrosomonas, Nitrospirillum, Pseudomonas, Rhizobium or Sinorhizobium.

[0021] One of the objectives of the present invention is achieved through the validation of a modified microorganism, preferably a microorganism of the genus Bradyrhizobium, which has had the repair and reactivation of the gene encoding the nitrous oxide reductase enzyme successfully performed. Description of the Drawings

[0022] Figure 1A depicts the nitrogen cycle involving nitrogen-fixing bacteria without the ability to synthesize nitrous oxide. reductase, demonstrating that at the end of the process N2O, which is an important greenhouse gas (GHG), ends up being released into the environment. Figure 1B shows the end of the nitrogen cycle involving nitrogen-fixing bacteria capable of synthesizing the enzyme nitrous oxide reductase, releasing only N2, an inert gas, into the environment at the end of the cycle.

[0023] Figure 2 illustrates the pSEVA231 plasmid containing 3,123 base pairs of DNA in its total sequence. This plasmid is mainly used for experiments isolating a gene or genes of interest because it has a relatively small size and characteristics necessary for easy propagation in E. coli DH10B bacteria. The genes of interest or exogenous DNA fragments to be isolated are inserted into the sequence called the multiple cloning site, which contains the recognition sites of several restriction enzymes commonly used for plasmid linearization. Additionally, the plasmid has the following characteristics: pBBR1 refers to the origin of replication of the broad-spectrum plasmid; neo expresses the neomycin phosphotransferase enzyme, conferring resistance to the antibiotic kanamycin; rep is the replicating protein of pBBR1; and an origin of transfer (oriT).

[0024] Figure 3 illustrates the recombinant plasmid pSEVA231 ■.■.nosRZDFLYX[B. ottawaense OO9T] consisting of a total of 12,537 base pairs of DNA in its total sequence. The plasmid harbors the genes isolated from the nosRZDFLYX operon of B. ottawaense OO9T between the restriction enzyme sequences Xba\ and Spel. The main function of the plasmid is to act as a donor of the operon genes, allowing for easier insertion into any other type of vector with more specific functionalities.

[0025] Figure 4 illustrates the mini-Tn7Gm plasmid, consisting of 4569 base pairs of DNA in its total sequence. The plasmid's main characteristic is the presence of two inverted sequences of the Tn7 transposon, called the "left end of the Tn7 transposon" and the "right end of the Tn7 transposon". In the middle of the inverted sequences is a multiple cloning site that allows the insertion of genes or DNA fragments of interest. In addition, adjacent to the site is a selection marker that confers resistance to the antibiotic gentamicin, encoded by the aacC1 gene, which is simultaneously flanked by two Flp recombinase recognition sequences, allowing its excision after the successful insertion of the Tn7 transposon and the genes of interest into the genome of the target bacterium.Additionally, the plasmid has the following characteristics: ColE1, refers to the origin of replication of the enterobacteria-specific plasmid; bla, located outside the inverted ends of the Tn7 transposon and expressing the Beta-lactamase enzyme, conferring resistance to the antibiotic ampicillin / carbenicillin; and, a transfer origin (oriT).

[0026] Figure 5 illustrates the recombinant plasmid mini-Tn7Gm ■.■.nosRZDFLYX[B. ottawaense OO9T] consisting of 13826 base pairs of DNA in its total sequence. The plasmid harbors the genes of the nosRZDFLYX operon of B. ottawaense OO9T, taken from the recombinant plasmid pSEVA231 .nosRZDFLYX[B. ottawaense OO9T] and cloned between the Spel restriction enzyme sequence. This plasmid acts as the main donor of the nos operon genes to be inserted into the attTn7 recognition site of the Bradyrhizobium bacterial genome, since any DNA sequence inserted between the inverted sequences of the Tn7 transposon is mobilized to the attTn7 of the target bacterium.

[0027] Figure 6 illustrates the pTNS3 plasmid, consisting of 9983 base pairs of DNA in its total sequence. The plasmid possesses the tnsABCD genes necessary to perform the transposition events of the Tn7 transposon in bacterial genomes with the attTn7 recognition site located downstream of the glmS gene and in the simultaneous presence of the mini-Tn7Gm vector. Additionally, the plasmid also presents the following characteristics: oriT refers to the origin of transfer; R6K refers to the origin of replication of the plasmid, functional only in the presence and action of the replicating protein Pir; bla expresses the Beta-lactamase enzyme, conferring resistance to the antibiotic ampicillin / carbenicillin; and plac / Pc are constitutive promoters.

[0028] Figure 7 illustrates the pFLP3 plasmid with a total DNA sequence length of 10925 base pairs. The plasmid possesses the flp gene, which encodes the Flp recombinase necessary for the excision of the aacC1 selection marker after successful transposition of the Tn7 transposon and the genes of interest in the genome of the target bacteria. Additionally, the plasmid also presents the following characteristics: oriT refers to the origin of transfer; ori1600 refers to the origin of replication of the broad-spectrum plasmid; bla expresses the Beta-lactamase enzyme conferring resistance to the antibiotic ampicillin / carbenicillin; tet expresses the efflux protein conferring resistance to the antibiotic tetracycline; cl is a lambda phage repressor protein; and sacB expresses the levanasucrase enzyme conferring sensitivity in the presence of sucrose.

[0029] Figure 8 illustrates the synergistic action of transposases TnsA, TnsB, TnsC, and TnsD for the integration of the RZDFLYX operon in conjunction with the selection marker aacC1 and the inverted sequences of the Tn7 transposon, at a specific location known as attTn7 present in the B. japonicum SEM IA genome. 5079. In this process, when both mini-Tn7Gm::nos-RZDFLYX[B. ottawaense OO9T] and pTNS3 plasmids are mobilized into B. japonicum SEMIA 5079 cells by electroporation or conjugation, the TnsA and TnsB proteins are produced, and then recognize the inverted sequences and break the DNA of these sequences. After this, the TnsD protein binds to the region of the glmS gene near the attTn7 site. Then, the TnsC protein interacts with the TnsD proteins, and the TnsAB complex mobilizes the transposon in the attTn7 region. After recognition and breakage of the attTn7 site by the TnsB protein, the same protein completes the transposition by binding the fragment to the 3' region of the attTn7 region. As a result, the new B. japonicum strain SEMIA 5079::Tn7Gm nosZ + It possesses the chromosomal integration of the nosRZDFLYX operon genes in addition to the inverted sequences of the Tn7 transposon and the aacC1 selection marker.

[0030] Figure 9 illustrates the process of obtaining the new B. japonicum SEMIA 5079: :Tn7 nosZ strain. + Free from selection markers. In this process, the B. japonicum SEMIA 5079::Tn7Gm nosZ strains + Cells that tested positive for the nitrous oxide reduction phenotype are treated to incorporate the pFLP3 plasmid. This plasmid is inserted into nosZ cells by electroporation. + ...and subsequently, through the action of the Flp recombinase enzyme, the selection marker is excised from the genome, resulting in the B. japonicum lineage SEMIA 5079::Tn7 nosZ + The plasmid is eliminated during cell division by incubating the cells in mPSY or YMA medium without the addition of NaCl and supplemented with 100 g / L -1 sucrose.

[0031] Figure 10 illustrates the reduction of N2O comparing wild-type Bradyrhizobium japonicum strains (SEMIA 5079) (A), Bradyrhizobium ottawaense (B) and Bradyrhizobium japonicum mutant with corrected nosRZDFLYX operon (C). Detailed Description of the Invention

[0032] For the purposes of defining the present invention, the term "approximately" should be understood as an acceptable standard deviation, for example, a standard deviation of 5 to 10% from the descriptive value.

[0033] For the purposes of defining the present invention, the term "overnight" should be understood as any acceptable time interval between one working day and the next. Preferably, this term can be interpreted as a period of approximately 12 to 18 hours.

[0034] For the purposes of defining the present invention, the term "ambient temperature" means any acceptable working temperature, as readily understood by a person skilled in the art, for example, a temperature range of about 18°C ​​to 35°C.

[0035] The method described in this invention explains how to synthetically repair strains of Bradyrhizobium, or other Gram-negative nitrogen-fixing bacteria, ensuring zero toxicity and high specificity.

[0036] Biological nitrogen fixation (BNF) using diazotrophic bacteria is a safe technology for supplying nitrogen to plants. The vast majority of grains produced in Brazil, especially soybeans and beans, are inoculated, either in the seed or in the planting furrow, with nitrogen-fixing bacteria. Other plants receive inoculants from various genera, such as Azorhizobium, Azotobacter, Bradyrhizobium, Ensifer, Kosakonia, Mesorhizobium, Nitrobacter, Nitrosomonas, Rhizobium, or other Gram-negative bacteria capable of fixing atmospheric nitrogen very efficiently. This symbiosis completely eliminates or reduces the need for synthetic nitrogen supply to crops, guaranteeing cost reduction for the producer as well as substantially reducing GHG emissions from synthetic nitrogen fertilization. The environmental gain is significant because it replaces synthetic nitrogen fertilizer. Through biological nitrogen fixation (BNF), CO2 emissions are substantially reduced due to the absence of the synthetic nitrogen fertilizer production process and the potential N2O emissions resulting from the incomplete reduction of nitrogen in the form of nitrate in the soil. It is estimated that 50% of synthetic nitrogen applied to crops is lost through volatilization and / or leaching processes (Cantarella & Marcelino, 2008). This low efficiency in fertilizer use is eliminated when BNF technology is adopted. Some bacteria involved in BNF are also capable of completing the denitrification process, thus releasing N2, an inert gas, instead of N2O.However, as an example, of the commercial strains of the genera Bradyrhizobium and Rhizobium currently recommended by the Ministry of Agriculture, Livestock and Supply (MAPA) as inoculants in soybean and bean crops in Brazil, none express the genes of the nos operon, which prevents the encoding of the nitrous oxide reductase enzyme (Table 1). Therefore, these commercial strains, although contributing to BNF in an agronomically efficient way, are not able to reduce nitrous oxide emissions and in some cases may increase emissions of this gas. N2O is a gas approximately 300 times more potent in heat retention than CO2 and has a long permanence in the ecosystem (116 ± 9 years, Prather et al., 2015). Thus, there is a need to synthetically repair the nos operon in the genomic DNA of strains of these bacteria, whose related genes are responsible for the last stage of denitrification.In this way, the aim is to improve an efficient BNF system with a superior environmental benefit for the planet.

[0037] To mitigate the environmental damage caused by N2O emissions, it is necessary to develop technology that allows for the avoidance of N2O emissions without affecting food production. Among the plausible solutions is the use of diazotrophic bacteria capable of promoting the reduction... Complete nitrogen cycle management is highly desirable due to the possibility of releasing the inert gas N2 (Figure 1 B). In Brazil, as of August 2024, the Ministry of Agriculture, Livestock and Supply (MAPA) had granted 632 registrations for nitrogen-fixing inoculant products intended for soybean cultivation. However, despite the significant number of registrations and consequently products, all registrations are based on only four strains of bacteria of the genus Bradyrhizobium, recommended for soybean cultivation by MAPA, through Annex II of Normative Instruction IN 13 of 03 / 24 / 2011. The four strains are Bradyrhizobium diazoefficiens (SEMIA 5080), Bradyrhizobium elkanii (SEMIA 5019), Bradyrhizobium elkanii (SEMIA 587) and Bradyrhizobium japonicum (SEMIA 5079).Although all these strains possess in their genome the genes that encode the enzymes responsible for BNF, none of them is capable of expressing the nitrous oxide reductase protein, which is encoded by the nosZ gene (Zilli et al., 2021). In this context, the present invention describes the entire logical, methodological, and laboratory process to make the nosRZDFYLX operon functional and, therefore, lead to obtaining a mutant strain capable of transforming nitrous oxide gas into molecular nitrogen, reducing the harmful effects of greenhouse gases on the environment. Table 1: Genes involved in the denitrification process in the genome of bacteria used as inoculants in soybean cultivation. Genes related to denitrification were identified using Integrated Microbial Genomes (IMG) ( / / img.jgi.doe.gov / ) and the RAST / SEED-based prokaryotic genome annotation service ( / / rast.nmpdr.org / rast.cgi). The four main genes required for denitrification are in bold. Table adapted from Zilli et al. (2021).

[0038] In this method, we seek to introduce, through the transposition process, all gene clusters involved in the denitrification mechanism into Gram-negative nitrogen-fixing bacteria species that do not express the nitrous oxide reductase enzyme, including the four commercial Bradyrhizobium strains recommended for soybean cultivation and the three Rhizobium strains recommended for bean cultivation in Brazil. Brazil will be one of the first countries in the world to develop an environmentally sound technology that allows for continued food production with high productivity while reducing the greenhouse gas emission footprint, mainly by decreasing N2O emissions, which has a high potential to increase global warming.

[0039] Considering, for example, the soybean cultivation area in Brazil, around 44.6 million hectares in the 2023 / 24 season, the adoption of this technology would make it possible to reduce N2O emissions into the atmosphere by approximately 2.4 Tg of N per year, that is, 14.1% of total emissions or 21% of anthropogenic emissions (own data).

[0040] Thus, the present invention relates to Gram-negative nitrogen-fixing bacteria modified to express the enzyme nitrous oxide reductase, as well as to the genetic repair process. The method uses the technique of chromosomal insertion of exogenous genes into Gram-negative bacteria mediated by the mini-Tn7 transposon system. In this process, two vectors are inserted simultaneously by electroporation or conjugation into the target bacterium. The first vector, called mini-Tn7Gm. nosRZDFYLX[Bradyrhizobium ottawaense OO9T], includes the entire operon with the nosRZDFYLX genes originating from Bradyrhizobium ottawaense OO9T flanked by two inverted ends, a left end containing 166 base pairs of DNA and a right end containing 199 base pairs of DNA. The aim is to restore the reduction function of nitrous oxide (N2O) to molecular nitrogen (N2), and to the primer pairs, nucleotide sequences, and plasmids used in the aforementioned process for the repair of the microorganism.

[0041] The second vector, designated pTNS3, contains the tnsABCD genes which, when expressed, transpose the nosRZDFYLX operon into the attTn7 region of the target bacterium's genome. Subsequently, new strains harboring the nos operon in their genome are selected using the antibiotic gentamicin and confirmed by PCR. After confirmation and growth in culture media supplemented with gentamicin, the mutant bacteria are treated de novo to insert the FLP3 plasmid, which expresses the Flp recombinase and effects the exclusion of the gentamicin selection marker. This last plasmid is excluded from the bacterial populations using culture media supplemented with excess sucrose (100 g / L) (Choi et al., 2005; Choi & Schweizer, 2006).

[0042] In the end, selectively marker-free and genetically stable strains are generated, functionally expressing the nosRZDFYLX operon integrated into the chromosome. Thus, this technique aims to incorporate genes related to the denitrification process in nitrogen-fixing Gram-negative bacteria. For example, the New edited lines derived from the commercial lines Bradyrhizobium elkanii (SEMIA 587), Bradyrhizobium elkanii (SEMIA 5019), Bradyrhizobium japonicum (SEMIA 5079), Bradyrhizobium diazoefficiens (SEMIA 5080), used in soybean cultivation, Rhizobium tropici (SEMIA 4077, SEMIA 4080 and SEMIA 4088) used in bean cultivation, and Sinorhizobium melioti (SEMIA 116, SEMIA 134 and SEMIA 135) used in alfalfa cultivation, will be available to the Ministry of Agriculture, Livestock and Supply (MAPA) as alternatives to the currently available commercial lines. They will have the same proven agronomic efficiency, but will be able to completely reduce nitrate to N2, thus avoiding the release of an important greenhouse gas. The same operon can be inserted in the same way into other nitrogen-fixing Gram-negative bacteria, with the same expected result. oligonucleotide sequences

[0043] To that end, in a first approach, a pair of primers was constructed that amplifies a region within the nosR gene by PCR, where the primer pair comprises a forward primer and a reverse primer.

[0044] The oligonucleotides were designed following these parameters: 1) DNA sequences complementary to the template DNA containing a length between 18 and 38 nitrogenous bases, and with a GC (Guanine-Cytosine) percentage between 50 and 65%; 2) the ends of the 5' region of each specific oligonucleotide not complementary to the template DNA containing a length between 10 and 20 nitrogenous bases, and with a pairing temperature (Ta) not less than 50°C; 3) the ends of the 5' region of each specific oligonucleotide containing DNA sequences complementary to the ends of the 3' region of linear double-stranded DNA fragments.

[0045] Three oligonucleotide pairs were designed to amplify the B. nosRZDFYLX operon in three fragments by PCR. Ottawaense 009T with a total size of 9253 DNA base pairs. In one embodiment, the first pair of forward and reverse primers are designated as Frag1_OO9T_Fw, comprising or consisting of SEQ ID NO: 1, and Frag1_OO9T_Rv, comprising or consisting of SEQ ID NO: 2, respectively. The sequence SEQ ID NO: 1, comprising or consisting of the sequence: 5'-GCTCGGTACCCGGGGATCCTCTAGA-TTCGTACCTGTGGCGGCTTC-3', was designed to amplify a region of 2994 DNA base pairs. SEQ ID NO: 1 was designed to contain 22 DNA bases at the 5' end complementary to the 3' ends of the linearized pSEVA231 vector (GenBank accession: JX560328) (bases underlined), and 24 DNA bases at the 3' end complementary to the B. ottawaense OO9T genome. The sequence SEQ ID NO: 2, which comprises or consists of the sequence: 5'-TTCGCGGGTGG-CGGGCTGCATGCTTCGGTCAGAAC-3', was designed to include 36 DNA bases complementary to the genome of B. ottawaense OO9T.

[0046] In one embodiment, a second primer pair comprises the forward primer, designated Frag2_OO9T_Fw, comprising or consisting of SEQ ID NO: 3, and the reverse primer, Frag2_OO9T_Rv, comprising or consisting of SEQ ID NO: 4, designed to amplify a 3254 base pair region of DNA. SEQ ID NO: 3 sequences of 38 DNA bases and SEQ ID NO: 4 sequences of 38 DNA bases are also included. 37 DNA bases were designed to be complementary to the B. ottawaense OO9T genome. SEQ ID NO: 3 comprises or consists of the nucleotide sequence: 5'-AAGGCATGCAGCCCGCCAC-CCGCGAATTCCTCAAGAAC-3', and SEQ ID NO: 4 comprises or consists of the nucleotide sequence: 5'-CTCTCGTTTCAGCCTTGGCA-TAGAAGGCCTGCGTCTCG-3'.

[0047] In one embodiment, a third pair of initiators includes the forward initiator Frag3_OO9T_Fw, which comprises or consists of SEQ ID NO: 5, and the reverse initiator, Frag3_OO9T_Rv, which comprises or The sequences SEQ ID NO: 6, designed to amplify a 3,005 base pair region of DNA, are: SEQ ID NO: 5, which includes 35 DNA bases complementary to the B. ottawaense OO9T genome; and SEQ ID NO: 6, designed to contain 23 DNA bases at the 5' end complementary to the 3' ends of the linearized pSEVA231 vector (GenBank accession: JX560328) (bases underlined). SEQ ID NO: 5 comprises or consists of the nucleotide sequence: 5'-CCTTCTATGCCAGGCTGAAACGAGAGCCGGTCGCA-3', and SEQ ID NO: 6 comprises or consists of the nucleotide sequence: 5'-GTTTTCCCAGTCACGACGCGGCCGCAACTAGTGCGCAGGTTAGG-CCGACAATTCG-3'. Construction and Validation of Recombinant Nucleotide and Plasmid Sequences

[0048] The confirmation of the integration of the mini-Tn7 transposon containing the nosRZDFYLX operon was performed in the genome of Bradyrhizobium japonicum (SEMIA 5079) as an example, since the technique detailed above is compatible with other nitrogen-fixing Gram-negative bacteria. For this validation purpose, 2 primer pairs were designed that amplify in the downstream region of the left end of mini-Tn7 and upstream region of the right end of mini-Tn7. In B. japonicum SEMIA 5079, the forward primer was named glmSDown5079_Fw, which comprises or consists of SEQ ID NO: 7, and the reverse primer Tn7right, which comprises or consists of SEQ ID NO: 8. Both primers together amplify a 391 base pair region of DNA. The 22-base-pair DNA sequence SEQ ID NO: 7 is complementary to the near-terminal region of the glmS gene in SEMIA 5079, while the 23-base-pair DNA sequence SEQ ID NO: 8 is complementary to the right-end region of mini-Tn7. SEQ ID NO: 7 comprises or consists of the sequence: 5'-CGAAAGA-CCGTCTCCAACATG-3' and SEQ ID NO: 8 comprises or consists of 5'-CACAGCATAACTGGACTG ATTTC-3' .

[0049] However, another oligonucleotide pair, ORFUp5079_Rv, comprising or consisting of SEQ ID NO: 9, and Tn7Left, comprising or consisting of SEQ ID NO: 10, together amplify 562 base pairs of DNA. The 22-base DNA sequence SEQ ID NO: 9 is complementary to the left-hand region of mini-Tn7, while the 22-base DNA sequence SEQ ID NO: 10 is complementary to the near-start region of a coding sequence referred to here as an ORF. SEQ ID NO: 9 comprises or consists of the nucleotide sequence: 5'-CGTCTCGAACACCTGCTTCAAG-3'. SEQ ID NO: 10, in turn, comprises or consists of the nucleotide sequence: 5'-ATTAGCTTACGACGCTACACCC-3'.

[0050] Additionally, aiming for another PCR confirmation option, a final primer pair was established for amplification of a region of the aacC1 gene that acts as a selection marker. This oligonucleotide pair comprises the sequences Gmllp, which comprises or consists of SEQ ID NO: 11, of 20 DNA bases, and GmDown, which comprises or consists of SEQ ID NO: 12, of 20 DNA bases. SEQ ID NO: 11 comprises or consists of the sequence: 5'-TGGAGCAGCAACGATGTTAC-3'. SEQ ID NO: 12, in turn, comprises or consists of the sequence: 5'-TGTTAGGTGGCGGTACTTGG-3'.

[0051] The nucleotide sequences resulting from the amplifications above already include the CG repair base pair due to the use of the primer pairs described here.

[0052] In a preferred embodiment, said nucleotide sequence comprises or consists of SEQ ID NO: 13.

[0053] Thus, in one embodiment, the first six nucleotide sequences (SEQ ID NO: 1 - SEQ ID NO: 6) are relevant for the repair and reactivation of the gene encoding the nitrous oxide reductase enzyme in nitrogen-fixing Gram-negative bacteria. It should be noted that only the six sequences SEQ ID NO: 1 - SEQ ID NO: 6 are mandatory for the construction of the recombinant plasmid pSEVA231 ■.■.nosRZDFLYX[B. ottawaense OO9T] and subsequently the mini-Tn7Gm:: nosRZDFLYX[B. ottawaense OO9T] which provides nitrogen-fixing Gram-negative bacteria with the ability to express the entire enzymatic complex necessary for the correct functioning of the nitrous oxide reductase enzyme for the reduction of nitrous oxide gas. The sequences designated SEQ ID NO: 7 - SEQ ID NO: 12 are used to validate the effectiveness of mini-Tn7 transposon transfer containing the nosRZDFLYX operon genes in bacteria of the species Bradyrhizobium japonicum SEMIA 5079.Other bacteria from different Bradyrhizobium species, as well as from other genera of nitrogen-fixing Gram-negative bacteria, may have sequences equivalent to SEQ ID NO: 7 and SEQ ID NO: 9.

[0054] In a preferred embodiment, the nucleotide sequences can also be modified to include one or more regulatory elements of the gene of interest.

[0055] In a preferred embodiment, the construction of the first recombinant plasmid containing the amplified DNA sequences of B. ottawaense OO9T was performed by joining four linear double-stranded DNA fragments, producing the plasmid named pSEVA231 ■.■.nosRZDFLYX[B. ottawaense OO9T], which comprises or consists of SEQ ID NO: 14. To this end, amplifications of three DNA fragments from the B. ottawaense OO9T template DNA were first performed by PCR using the oligonucleotide pair SEQ ID NO: 1 - SEQ ID NO: 2 for the first fragment, SEQ ID NO:3 - SEQ ID NO: 4 for the second fragment, and SEQ ID NO: 5 - SEQ ID NO: 6 for the third fragment using the high-fidelity enzyme Q5® High-Fidelity DNA Polymerase (New England Biolabs Inc., NEB). A 25 pL reaction composed of: Q5 Reaction Buffer 1X, Q5 High GC Enhancer 1X, 200 pM dNTPs, 500 nM of the oligo pair SEQ ID NO: 1 - SEQ ID NO: 2, or SEQ ID NO: 3 - SEQ ID NO: 4, or SEQ ID NO: 5 - SEQ ID NO: 6, 0.1-1.0 pg of template DNA and nuclease-free water, was transferred to the Mastercycler® Nexus Thermal Cyclers thermocycler (Eppendorf AG, Hamburg) under the following parameters: Initial denaturation: 98°C, 30 sec; 35 cycles (Denaturation: 98°C, 15 sec; Pairing: 65°C, 20 sec; Extension: 72°C, 30 sec / kb); Final extension: 72 °C, 2 mins, and maintenance at 10 °C. PCR products were subjected to electrophoresis on 1% agarose gel and stained with SYBR Safe DNA Gel Stain (Thermo Scientific™, Waltham, Massachusetts, USA).When required, Gel Loading Dye, Purple (6X) (New England Biolabs Inc., NEB) was used to visualize the migration of DNA fragments during electrophoresis. The electrophoretic run was performed at 80 V for 50 to 80 minutes in 1X TAE buffer (Composition: 40 mM Tris-Base, 1 M glacial acetic acid, and 50 mM EDTA, pH 8.0). After identifying the band visualized on the agarose gel corresponding to the expected size of the amplified DNA fragments, it was necessary to extract them from the gel. For this, the Wizard® SV Gel and PCR Clean-Up System kit (Promega, Madison, Wisconsin, USA) was used, allowing the extraction of both isolated and purified DNA fragments from the agarose gel.

[0056] In a preferred method, the fourth linear DNA fragment was obtained by extracting and purifying the pSEVA231 plasmid using the commercial Wizard® Plus SV Minipreps DNA Purification System kit (Promega, Madison, Wisconsin, USA) following the manufacturer's instructions. The purified plasmid DNA It was then digested with restriction enzymes Xba and Hind (FastDigest - Thermo Scientific, Waltham, Massachusetts, USA) for 60 minutes at 37°C, following the manufacturer's recommendations. The digested product was subjected to electrophoresis on a 1% agarose gel stained with SYBR Safe DNA Gel Stain (Thermo Scientific™, Waltham, Massachusetts, USA). Finally, linearized plasmid DNA was extracted and purified from the agarose gel using the Wizard® SV Gel and PCR Clean-Up System kit (Promega, Madison, Wisconsin, USA).

[0057] In a preferred embodiment, the 4 isolated and purified linear double-stranded DNA fragments are used to construct the recombinant plasmid pSEVA231 .nosRZDFLYX[B. ottawaense OO9T]. For this, a 20 L reaction was prepared containing: 0.15 pmol of the first fragment, 0.15 pmol of the second fragment, 0.15 pmol of the third fragment, and 0.05 pmol of the previously digested pSEVA231 vector; 10 pL of Hot-Fusion enzyme solution (100 mM Tris pH 7.5, 10 mM MgCl2, 200 pM of each dNTP, 10 mM DTT, 5% PEG-8000, 75x10⁻¹² 4 U of T5 exonuclease (New England Biolabs Inc., NEB) and 5x1 O' 2 The reaction was then transferred to a thermocycler and incubated for 1 hour at 50°C, and slowly cooled at a rate of 0.1°C per second until it reached 20°C in a total of 5 minutes.

[0058] In another preferred embodiment, the previously described plasmid construct was first stored and propagated in E. coli DH10B cells (Durfee et al., 2008) using the heat shock transformation technique. A culture of E. coli DH10B cells was prepared to make them chemocompetent following the protocol described by Sharma, Gil, and Wengier (2017). Thus, 10 pL of the plasmid construct reaction was mixed with 100 pL of chemocompetent E. coli DH10B cells and incubated on ice for 30 After 90 minutes, the solution was transferred to a heating block and incubated at 42°C for 90 seconds, then quickly transferred to ice and kept there for another 2 minutes. The DNA and cell mixture was then inoculated into 900 pL of SOC culture medium and incubated on a rotary shaker for 1 hour at 37°C and 220 rpm. The culture was centrifuged and the cells were suspended in 100 pL of sterile saline solution (0.85% NaCl) and then spread onto Petri dishes containing solid LB culture medium containing 20 pg / pL of the antibiotic kanamycin and incubated for 16-18 hours at 37°C.

[0059] In a preferred embodiment, verification of the correct insertion of the two Bradyrhizobium DNA fragments into the pSEVA231 plasmid can be confirmed by colony PCR using the commercial kit GoTaq® Green Master Mix (Promega, Madison, Wisconsin, USA) in a 15 pL reaction containing: GoTaq® Green Master Mix 2X, 500 nM of the oligonucleotide pair SEQ ID NO: 3 - SEQ ID NO: 4 or 500 nM of the universal oligonucleotide pair M13, 5% DMSO, 1-500 ng of template DNA and nuclease-free water. The amplification parameters were as follows: Initial denaturation: 95°C, 2 min; 25 cycles (Denaturation: 95°C, 30 sec; Pairing: 55°C, 20 sec; Extension: 72°C, 1 min / kb); Final temperature: 72°C, 5 min, and maintenance at 10°C.

[0060] In a preferred embodiment, after obtaining the first recombinant plasmid pSEVA231 .nosRZDFLYX[B. ottawaense OO9T], the fragment containing the entire nosRDZFLYX operon, consisting of 9253 base pairs of DNA, was excised from this plasmid by digestion using Xba and Spel enzymes (FastDigest - Thermo Scientific, Waltham, Massachusetts, USA) for 60 minutes at 37°C, following the manufacturer's recommendations. The digested product was subjected to electrophoresis on a 1% agarose gel stained with SYBR Safe DNA Gel Stain (Thermo Scientific™, Waltham, USA). (Massachusetts, USA). The linearized plasmid DNA was then extracted and purified from the agarose gel using the Wizard® SV Gel and PCR Clean-Up System kit (Promega, Madison, Wisconsin, USA). Additionally, the mini-Tn7Gm plasmid was also digested using the Spel enzyme following the same procedure described previously.

[0061] In a preferred embodiment, linearized fragments of the nosRDZFLYX operon and the mini-Tn7Gm plasmid were used to construct the final recombinant plasmid named mini-Tn7Gm::nosRZDFLYX[B. ottawaense OO9T], comprising or consisting of SEQ ID NO: 15. For this, a 10 L reaction was prepared containing: 0.15 pmol of the linear nosRZDFLYX fragment and 0.05 pmol of the previously digested mini-Tn7Gm vector; 1X of the T4 DNA ligase enzyme reaction buffer; 800 U of T4 DNA ligase enzyme (New England Biolabs Inc., NEB) and nuclease-free water. The reaction was then transferred to a thermocycler and incubated for 1 hour at 16°C, followed by an additional incubation at 65°C for 20 minutes. After the incubations were complete, the newly constructed plasmid was stored and propagated in chemocompetent E. coli DH10B cells, and subsequently transferred to E. coli S17-1 strains (Simon; Priefer; Pühler, 1983) by heat shock as described above.The verification of the correct insertion of the nosRZDFLYX operon into this new mini-Tn7Gm plasmid was performed by PCR following the same procedure described previously in the construction of the pSEVA231 ::nos-RZDFLYX[B. ottawaense OO9T] plasmid.

[0062] In an even more preferred embodiment, the integration of the mini-Tn7 transposon into the B. japonicum SEMIA 5079 genome containing the nosRZDFLYX operon genes of B. ottawaense OO9T (mini-Tn7Gm: .nosRZDFLYX[B. ottawaense OO9T]) was performed by electroporation (Guerinot, Morisseau & Klapatch, 1990; Hattermann & Stacey, 1990), and optionally, when necessary, by tri-parental conjugation between an E. coli S17-1 donor cell (mini-Tn7Gm::nosRZDFLYX[B. ottawaense OO9T]), another E. coli S17-1 donor cell (pTNS3), and a recipient cell of the nitrogen-fixing bacterium of interest (Hmelo et al., 2015). The following describes the method of simultaneously inserting the mini-n7Gm::nosRZDFLYX[B. ottawaense OO9T] and pTNS3 plasmids by electroporation into B. japonicum SEMIA 5079 cells: B. japonicum SEMIA 5079 cells were cultured in modified mPSY medium (1.0 g ■ L). -1 of yeast extract, 3.0 g ■ L -1 of peptone, 100 mg ■ L -1 of MgSO4-7H2O and 100 mg ■ L -1of NaCl in 3 mM potassium phosphate buffer, pH 6.8) in a rotary shaker at 30°C and 180 rpm for up to 7 days until reaching an optical density of 0.4-0.6 (600 nm). Subsequently, the culture was divided into 50 ml conical tubes and cooled on ice and centrifuged at 4°C, then sequentially washed in cold sterile distilled water in 10% glycerol, and finally homogenized in 3 ml of 10% glycerol (~10 12 cells ■ L -1 ), stored in 40 pl aliquots at -80 °C.

[0063] For electroporation, an aliquot of the treated cells was thawed, mixed with 500 ng of mini-Tn7Gm mosRZDFLYX[B. ottawaense OO9T] plasmid DNA and 500 ng of pTNS3 plasmid DNA, and kept on ice for 1 minute before being transferred to 0.2 cm cuvettes; then, electrical pulses were applied using a Gene Pulser (Bio-Rad Laboratories, Richmond, California, USA) with a 25 pF capacitor and different field strengths of 10.5-12.5 kV / cm for durations of 5-8 milliseconds and 15-20 milliseconds. Immediately after the pulse, 1 mL of mPSY medium was added to 10 mL test tubes and the cells were incubated on a rotary shaker at 30°C, 180 rpm, for approximately 20 hours; After recovery, dilutions were spread on mPSY selective medium supplemented with gentamicin (20-40 pg / mL). -1 ), giving rise to colonies transformed into 7- 15 days. Colonies with visually larger diameters were selected to verify the insertion of the transposon containing the nosRZDFYLX operon genes by PCR using the sequences SEQ7-SEQ8 or SEQ9-SEQ10, or SEQ11-SEQ12. Isolated colonies positive for the presence of the Tn7 transposon and nosRZDFYLX operon were isolated and incubated in YMA medium (0.5 g ■ L⁻¹). 1 K2HPO4, 0.2 g ■ L 1 MgSO4·7H2O, 0.1 g ■ L 1 NaCl, 5 g ■ L 1 Mannitol, and 15 g ■ L“ 1Agar) or mPSY for subsequent propagation, storage, and phenotypic validation of nitrous oxide reduction. Finally, after phenotypic validation for nitrous oxide reduction, B. japonicum SEMIA 5079 cells (mini-Tn7Gm-.-.nosRZDFLYX[B. ottawaense OO9T]) were chemically treated as previously described to insert the pFLP3 plasmid by electroporation under the same conditions of electrical pulse strength and time and recovery time. The cells were then incubated in mPSY or YMA solid medium without NaCl addition and supplemented with 100 g ■ L⁻¹ 1 Sucrose was incubated for 7-15 days at 30°C. Colonies with visually larger diameters that grew after this incubation time were selected for verification of gentamicin selection marker exclusion by PCR. Cells that did not show amplification of the aacC1 gene fragment were isolated, propagated, and stored at -80°C.

[0064] In a preferred embodiment, for the transfer of the Tn7 transposon and the nosRZDFYLX operon by tri-parental conjugation, the process is detailed below: A colony of B. japonicum SEMIA 5079 recipient cells, pre-cultured at 30°C in plates with YMA medium (0.5 g µL⁻¹) 1 K2HPO4, 0.2 g ■ L 1 MgSO4-7H2O, 0.1 g ■ L 1 NaCl, 5 g ■ L 1 Mannitol, and 15 g ■ L“ 1 Agar) was transferred to a glass test tube containing 5 ml of antibiotic-free liquid YM medium, and subsequently incubated on a rotary shaker at 30°C at a speed of 150 rpm for 5 to 7 days. One day before starting the conjugation process, a A colony of E. coli S17-1 donor cells (mini-Tn7Gm::nos-RZDFLYX[B. ottawaense OO9T]), and another E. coli S17-1 donor cell (pTNS3), pre-cultured at 37°C in solid LB medium supplemented with 20 pg ■ mL⁻¹ gentamicin, was transferred to a glass test tube containing 5 mL of LB medium supplemented with 20 pg ■ mL⁻¹ gentamicin and incubated on a rotary shaker overnight at 37°C and 180 rpm. On the day of the hybridization process between the strains, 500 pL of the recipient cell culture was transferred to a 1.5 mL microtube and, in parallel, 1.5 mL of the donor strain culture was also transferred to a 1.5 mL microtube. Therefore, the donor and recipient cells were centrifuged at 7,000 rpm in the Eppendorf MiniSpin® microcentrifuge for 5 minutes at room temperature.After that, both recipient and donor cells were suspended separately in 50 µL of liquid YM medium without added antibiotic, and subsequently both cell volumes were gently mixed in a single 1.5 mL microtube. The mixture was then transferred without spreading to a Petri dish containing antibiotic-free YMA medium and incubated in an incubator for 48–72 hours at 30°C. After the incubation period, a small portion of the mixed cell mass was transferred to a new 1.5 mL microtube containing 500 µL of antibiotic-free liquid YM culture medium and then incubated for 3 hours at 30°C without agitation. Then, the culture containing the mixture of both cells was centrifuged at 7000 rpm in an Eppendorf MiniSpin® microcentrifuge for 5 minutes at room temperature, then the supernatant was discarded and the cells were carefully suspended in 100 µL of saline solution (NaCl 0.85%) sterile and then the entire volume was spread into Petri dishes containing mPSY medium supplemented with 15 g ■ L. -1 of agar and 20-40 pg ■ mL -1 of gentamicin and 30 pg ■ mL -1 of nalidixic acid for the selection of the ex-conjugant recipient strain B. japonicum SEMIA 5079 (mini- n7Gm..nosRZDFLYX[B. ottawaense 009T]) and exclusion of E. coli donor cells. The cells were incubated for 7-15 days at 30 °C, and visually uniform colonies were verified by PCR following the parameters described previously. After confirmation of the presence of the nos operon, the cells underwent the same phenotypic validation procedure, and subsequent excision of the gentamicin selection marker for cells that tested positive for the nitrous oxide reduction phenotype.

[0065] According to data retrieved from the National Center for Biotechnology Information (NCBI), the largest database of sequenced genomes on the planet, as of October 1, 2025, there were 2.32 million sequenced bacterial genomes deposited in the collection; however, only 237 genomes have the nosZ gene annotated. This indicates a very low prevalence of this gene (less than 0.01%) in the bacterial population. Considering that this is the only gene that codes for the nitrous oxide reductase enzyme, its reintroduction into agrobiological systems is of fundamental importance for mitigating greenhouse gas emissions and also has practical implications, such as the possibility of using it as a marker for traceability of plant products that have been inoculated with nitrogen-fixing microorganisms that have undergone gene editing to insert the operon into their genomes, as in the present invention.

[0066] With the aim of further increasing the effectiveness of traceability in plant product supply chains, a preferred approach involves inserting a "barcode" sequence, i.e., a unique fragment of approximately 25 nucleotides positioned downstream of the 3'UTR region of the nosX gene, the last gene in the operon. The use of DNA barcode sequences attached to the 3'UTR region of the nosRZDFYLX operon, specifically the nosX gene, facilitates more precise traceability of the product. The operon, since the sequence defined as a barcode has the following characteristics: 1) a length of 25 to 30 DNA bases, increasing the specificity of pairing with the template; 2) the defined sequence has no biological activity per se; and 3) the sequence defined as a barcode, in conjunction with another oligonucleotide sequence that pairs with the nosX gene template, has 100% amplification and pairing.

[0067] The barcode sequence design is randomly generated, with a GC percentage between 50 and 60%, and the specificity of the matching is performed using BLASTN (Basic Local Alignment Search Tool for Nucleotides) against other DNA sequences in the largest NCBI database, with some requirements such as not having coverage greater than 70% with the sequences in the database, and not having matching mainly in the 3' region of the barcode. Additionally, for the purpose of analyzing the specificity of amplification by PCR with the direct oligonucleotide of the nosX gene and the reverse barcode, the PRIM ER-BLAST software is used, which indicates whether the PCR by both oligonucleotides has off-targets. The absence of off-target amplifications will be an important parameter for determining the unique and restricted amplification of the nosRZDFYLX operon of this invention, which can be further confirmed by genome sequencing technologies. Validation of Nitrous Oxide Reductase Expression

[0068] An in-vitro experimental assay was conducted to confirm the expression of the nitrous oxide reductase enzyme by the new SEMIA 5079 strain with the inserted nosRZDFLYX operon. For comparison, the wild-type Bradyrhizobium japonicum strain (SEMIA 5079) (A), Bradyrhizobium ottawaense (B), and mutant Bradyrhizobium japonicum strain (C) with the inserted nosRZDFLYX operon were used (Figure 10). All strains were incubated for 4 days in 100 ml of HM culture medium supplemented with 0.1% (w / v) arabinose and 0.025% extract. yeast cultures were mixed and kept under agitation at 120 RPM and 28°C. After incubation, 1 ml of the culture of each strain, at a concentration of 10 8The CFU / mL count was transferred to hermetically sealed glass vials containing 9 mL of HM culture medium in liquid form without arabinose and yeast extract, according to the methodology described by Wasai-Hara et al. (2023). The assay involved a total of six replicates for each group, totaling one sample with an experimental n of 18 units and three treatment groups, one of which was a control. At the beginning of the experiment, the vials were hermetically sealed with rubber septa and their headspace was suctioned with a 60 mL syringe. Nitrous oxide (N2O) gas was then injected into each vial using 20 mL syringes, resulting in an approximate concentration of 100 ppmv with a purity above 99.99% within the vials. Sample collection was performed using 3 mL gas-tight syringes, with 0.5 mL being directly injected into the gas chromatograph.The N2O concentration was measured in all sample units at time zero and time one (24 hours later), using an Agilent 6890N gas chromatograph equipped with a Shincarbon ST100 / 120 column and a pulsed discharge helium ionization detector (PDHID). The carrier gas used was high-purity helium (99.999%). The difference between the final concentration (t1) and the initial concentration (t0) relative to the initial concentration represents the relative reduction in N2O emissions over 24 hours.

[0069] The analysis of variance rejected the null hypothesis at the 0.001 significance level that the mean emission reductions between the groups are equal. Post-hoc analysis using Tukey's test indicated no statistically significant difference between groups B and C, and both differed statistically from group A, the control group, represented by the wild-type strain. The mean N2O reduction due to group B was 31.5% and 23.5% for group C, compared to a 10.9% reduction in group A. control group (A). The assumptions for the mean comparison tests were met, with Bartlett's test indicating homoscedasticity (w > 0.88) and normal distribution by the Shapiro-Wilk test (p-value > 0.32) in the observations. The boxplot with the visual summary of the analysis can be seen in Figure (10).

[0070] The results of the experimental assay allow us to conclude that genetic editing of the commercial strain of Bradyrhizobium japonicum (SEMIA 5079), with the insertion of the nosRZDFLYX operon, reduces nitrous oxide emissions by an average of 23.5% over a 24-hour period, per ml of Bradyrhizobium at a concentration of 10 8 CFU / mL.

[0071] While examples of preferred embodiments have been described, it should be understood that the scope of the present invention encompasses other possible variations, being limited only by the content of the claims alone, including possible equivalents.

[0072] The following are embodiments of the present invention:

[0073] In a first embodiment, a pair of primers is described that amplify a region comprising the functional genes in Bradyrhizobium RZDFYLX by PCR.

[0074] In a preferred embodiment, the forward primer is Frag1_OO9T_Fw, which comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 1, and the reverse primer is Frag1_OO9T_Rv, which comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 2. In an alternative embodiment, the forward primer is Frag2_OO9T_Fw, which comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 3, and the reverse primer is Frag2_OO9T_Rv, which comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 4. In an alternative embodiment, the forward primer is Frag3_OO9T_Fw, which comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 5, and the reverse primer is Frag3_OO9T_Rv, which comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 6.

[0075] In a second embodiment, a nucleotide sequence is described for the repair and / or activation of the operon encoding the nitrous oxide reductase enzyme in nitrogen-fixing Gram-negative bacteria, optionally wherein said bacteria are of the genera Bradyrhizobium, Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, Kosakonia. In a preferred embodiment, the nucleotide sequence comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 13.

[0076] In a third embodiment, a plasmid is described comprising the pSEVA231 plasmid (GenBank accession: JX560328), plus the nucleotide sequence as defined above. In a preferred embodiment, the plasmid is pSEVA231::nos-RZDFYLX as defined in Figure 3. In a more preferred embodiment, the plasmid comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 14.

[0077] In an alternative embodiment, the plasmid comprises the mini-Tn7Gm plasmid (GenBank accession: AY599232), plus the nucleotide sequence as defined above. In a preferred embodiment, the plasmid is mini-Tn7Gm::nosRZDFYLX as defined in Figure 5. In a more preferred embodiment, the plasmid comprises or consists of the nucleotide sequence as defined in SEQ ID NO: 15.

[0078] In a fourth embodiment, the present invention describes a modified microorganism comprising the nucleotide sequence or plasmid as defined above.

[0079] In a preferred embodiment, the microorganism is a nitrogen-fixing Gram-negative bacterium, optionally belonging to the genera Bradyrhizobium, Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, or Kosakonia. In a preferred embodiment, the microorganism correctly expresses the nos operon, encoding the nitrous oxide reductase enzyme and other auxiliary proteins for complete denitrification to occur.In an alternative embodiment, Bradyrhizobium strains are selected from the group consisting of Bradyrhizobium diazoefficiens CNPSo 10, Bradyrhizobium diazoefficiens CNPSo 104, Bradyrhizobium diazoefficiens CNPSo 105, Bradyrhizobium diazoefficiens CNPSo 106, Bradyrhizobium diazoefficiens CNPSo 107, Bradyrhizobium diazoefficiens CNPSo 108, Bradyrhizobium diazoefficiens CNPSo 109, Bradyrhizobium diazoefficiens CNPSo 110, Bradyrhizobium diazoefficiens S14C, Bradyrhizobium diazoefficiens USDA 122, Bradyrhizobium diazoefficiens USDA 122-Bd, Bradyrhizobium diazoefficiens CB1809, Bradyrhizobium japonicum OPT_986_513, Bradyrhizobium japonicum OPT_986_514 and Bradyrhizobium japonicum OPT-986-515.

[0080] In a fifth embodiment, the present invention provides a method for preparing a microorganism comprising the repaired nosR gene, wherein the method comprises contacting the microorganism with a plasmid as defined above, wherein the microorganism is a nitrogen-fixing Gram-negative bacterium, optionally wherein said bacterium is of the genus Bradyrhizobium, Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, Kosakonia.

[0081] In a sixth embodiment, the present invention provides a microorganism produced by the method described above. In a preferred embodiment, the microorganism is of the genus Bradyrhizobium, Bradyrhizobium, Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, Kosakonia and other Gram-negative nitrogen-fixing bacteria comprising the nos-functional operon. Bibliographic References: Aryal, B., Gurung, R., Camargo, AF, Fongaro, G., Treichel, H., Mainali, B., ... & Puadel, SR (2022). Nitrous oxide emission in the altered nitrogen cycle and implications for climate change. Environmental Pollution, 120272. Bernhard, A. (2010) The Nitrogen Cycle: Processes, Players and Human Impact. Knowledge of Nature Education 3(10):25 Canfield, D. E., Glazer, A. N., & Falkowski, P. G. (2010). The evolution and future of Earth’s nitrogen cycle, science, 330(6001), 192-196. Cantarella, H., & Marcelino, R. (2008). Fontes alternativas de nitrogênio para a cultura do milho. Informações Agronômicas, 122, 12-14. Choi, K.-H., Gaynor, J. B., White, K. G., Lopez, C., Bosio, C. M., Karkhoff-Schweizer, R. R., & Schweizer, H. P. (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, 2(6), 443-448. Choi, K.-H., & Schweizer, H. P. (2006). Mini-tn7 insertion in bacteria with single atttn7 sites: Example pseudomonas aeruginosa. Nature Protocols, 7(1), 153-161. Gruber, N., & Galloway, J. N. (2008). An Earth-system perspective of the global nitrogen cycle. Nature, 451 (7176), 293-296. Guerinot, M. L., Morisseau, B. A., & Klapatch, T. (1990). Electroporation of Bradyrhizobium japonicum. Molecular and General Genetics MGG, 221(2), 287-290. Hattermann, D. R., & Stacey, G. (1990). Efficient DNA transformation of Bradyrhizobium japonicum by electroporation. Applied and Environmental Microbiology, 56(4), 833-836. Hmelo, L. R., Borlee, B. R., Almblad, H., Love, M. E., Randall, T. E., Tseng, B. S., Lin, C., Irie, Y., Storek, K. M., Yang, J. J., Siehnel, R. J., Howell, P. L., Singh, P. K., Tolker-Nielsen, T., Parsek, M. R., Schweizer, H. P., & Harrison, J. J. (2015). Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nature protocols, lOf W), 1820-1841 . Itakura, M., Uchida, Y., Akiyama, H., Hoshino, Y. T., Shimomura, Y., Morimoto, S., ... & Minamisawa, K. (2013). Mitigation of nitrous oxide emissions from soils by Bradyrhizobium japonicum inoculation. Nature Climate Change, 3(3), 208-212. Kuypers, M. M., Marchant, H. K., & Kartal, B. (2018). The microbial nitrogen-cycling network. Nature Reviews Microbiology, 16(5), 263-276. Lindstrom, K., & Mousavi, S. A. (2020). Effectiveness of nitrogen fixation in rhizobia. Microbial biotechnology, 13(5), 1314-1335. Lorenzo, V., Herrero, M., Sánchez, J. M., & Timmis, K. N. (1998). Mini-transposons in microbial ecology and environmental biotechnology. FEMS Microbiology Ecology, 27(3), 211-224. Matsumoto, A., Schlüter, T., Melkonian, K., Takeda, A., Nak- agami, H., & Mine, A. (2022). A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants. Plant Communications, 3(1), 100227. May, E. W., & Craig, N. L. (1996). Switching from cut-and-paste to replicative tn7 transposition. Science, 272(5260), 401-404. McKenzie, G. J., & Craig, N. L. (2006). Fast, easy and efficient: Site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event. BMC Microbiology, 6(1), 39. Parks, A. R., & Peters, J. E. (2007). Transposon tn 7 is widespread in diverse bacteria and forms genomic islands. Journal of Bacteriology, 189(5), 2170-2173. Peters, J. E., & Craig, N. L. (2001). Tn7: Smarter than we thought. Nature Reviews Molecular Cell Biology, 2(11), 806-814. Prather, M. J., Hsu, J., DeLuca, N. M., Jackman, C. H., Oman, L. D., Douglass, A. R., ... & Funke, B. (2015). Measuring and modeling the lifetime of nitrous oxide including its variability. Journal of Geophysical Research: Atmospheres, 120(11), 5693-5705. Ravishankara, A. R., Daniel, J. S., & Portmann, R. W. (2009). Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century, science, 326(5949), 123-125. Sánchez, C., Mitsui, H., & Minamisawa, K. (2017). Regulation of nitrous oxide reductase genes by A / asT-mediated transcription antitermination in Bradyrhizobium diazoefficiens. Environmental Microbiology Reports, 9(4), 389-396. https: / / doi.Org / 10.1111 / 1758-2229.12543 Sarnovsky, R. J., May, E. W., & Craig, N. L. (1996). The Tn7 trans- posase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products. The EMBO Journal, 15(22), 6348-6361. Walker, M. W. G., Klompe, S. E., Zhang, D. J., & Sternberg, S. H. (2023). Novel molecular requirements for CRISPR RNA-guided transposition. Nucleic Acids Research, 57(9), 4519-4535. Wiles, T. J., Wall, E. S., Schlomann, B. H., Hay, E. A., Parthasa- rathy, R., & Guillemin, K. (2018). Modernized tools for streamlined genetic manipulation and comparative study of wild and diverse proteobac- terial lineages. mBio, 9(5), e01877-18. Wasai-Hara, S., Itakura, M., Fernandes Siqueira, A., Takemoto, D., Sugawara, M., Mitsui, H., ... & Minamisawa, K. (2023). Bradyrhizo- bium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression. Scientific Reports, 13(1), 18862. Zilli, J. É., Pacheco, R. S., Gianluppi, V., Smiderle, O. J., Urquiaga, S., & Hungria, M. (2021). Biological N2 fixation and yield performance of soybean inoculated with Bradyrhizobium. Nutrient Cycling in Agroecosystems, 779(3), 323-336. https: / / doi.org / 10.1007 / s10705-021-10128-7.

Claims

CLAIMS 1. A pair of primers, characterized by the fact that they amplify a region comprising the functional genes in Bradyrhizobium RZDFYLX by PCR.

2. Primer pair according to claim 1, characterized in that the forward primer is Frag1_OO9T_Fw, comprising or consisting of the nucleotide sequence as defined in SEQ ID NO: 1, and the reverse primer is Frag1_OO9T_Rv, comprising or consisting of the nucleotide sequence as defined in SEQ ID NO:

2.

3. Primer pair according to claim 1, characterized in that the forward primer is Frag2_OO9T_Fw, comprising or consisting of the nucleotide sequence as defined in SEQ ID NO: 3, and the reverse primer is Frag2_OO9T_Rv, comprising or consisting of the nucleotide sequence as defined in SEQ ID NO:

4.

4. Primer pair according to claim 1, characterized in that the forward primer is Frag3_OO9T_Fw, comprising or consisting of the nucleotide sequence as defined in SEQ ID NO: 5, and the reverse primer is Frag3_OO9T_Rv, comprising or consisting of the nucleotide sequence as defined in SEQ ID NO:

6.

5. Nucleotide sequence, characterized in that it is for the repair and / or activation of the operon encoding the nitrous oxide reductase enzyme in nitrogen-fixing Gram-negative bacteria, optionally wherein said bacteria are of the genera Bradyrhizobium, Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, Kosakonia.

6. Nucleotide sequence according to claim 5, characterized in that it comprises or consists of the nucleotide sequence as defined in SEQ ID NO:

13.

7. Plasmid, characterized in that it comprises the pSEVA231 plasmid (GenBank accession: JX560328), plus the nucleotide sequence as defined in either of claims 5 or 6.

8. Plasmid according to claim 6, characterized in that it is pSEVA231::nosRZDFYLX as defined in Figure 3.

9. Plasmid, according to claim 7 or 8, characterized in that it comprises or consists of the nucleotide sequence as defined in SEQ ID NO:

14.

10. Plasmid, characterized in that it comprises the mini-Tn7Gm plasmid (GenBank accession: AY599232), with the nucleotide sequence added, as defined in claim 5 or 6.

11. Plasmid, according to claim 10, characterized in that it is the mini-Tn7Gm::nosRZDFYLX as defined in Figure 5.

12. Plasmid, according to claim 11, characterized in that it comprises or consists of the nucleotide sequence as defined in SEQ ID NO:

15.

13. Modified microorganism, characterized in that it comprises the nucleotide sequence as defined in claim 5 or 6, or plasmid as defined in any of claims 10 to 12.

14. Modified microorganism, characterized in that it is a nitrogen-fixing Gram-negative bacterium, optionally wherein said bacterium is of the genus Bradyrhizobium, Rhizo- bium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, Kosakonia.

15. Modified microorganism, according to claim 13 or 14, characterized in that it correctly expresses the nos operon, encoding the nitrous oxide reductase enzyme and other auxiliary proteins for complete denitrification to occur.

16. Modified microorganism, according to claim 13 or 14, characterized in that the Bradyrhizobium strains are selected from the group consisting of Bradyrhizobium diazoefficiens CNPSo 10, Bradyrhizobium diazoefficiens CNPSo 104, Bradyrhizobium diazoefficiens CNPSo 105, Bradyrhizobium diazoefficiens CNPSo 106, Bradyrhizobium diazoefficiens CNPSo 107, Bradyrhizobium diazoefficiens CNPSo 108, Bradyrhizobium diazoefficiens CNPSo 109, Bradyrhizobium diazoefficiens CNPSo 110, Bradyrhizobium diazoefficiens S14C, Bradyrhizobium diazoefficiens USDA 122, Bradyrhizobium diazoefficiens USDA 122-Bd, Bradyrhizobium diazoefficiens CB1809, Bradyrhizobium japonicum OPT_986_513, Bradyrhizobium japonicum OPT_986_514 and Bradyrhizobium japonicum OPT-986-515.

17. Method for preparing a microorganism comprising the repaired nosR gene, characterized in that it comprises contacting the microorganism with a plasmid as defined in any one of claims 6 to 12, wherein the microorganism is a nitrogen-fixing Gram-negative bacterium, optionally wherein said bacterium is of the genus Bradyrhizobium, Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, Kosakonia.

18. Microorganism, characterized in that it is produced by the method as defined in any one of claims 16 to 19.

19. Microorganism, according to claim 18, characterized in that the microorganism is of the genus Bradyrhizobium, Bradyrhizobium, Rhizobium, Methylobacterium, Mesorhizobium, Sinorhizobium, Gluconoacetobacter, Azorhizobium, Ensifer, Azotobacter, Pseudomonas, Nitrosomonas, Azospirillum, Nitrospirillum, Kosakonia and other nitrogen-fixing Gram-negative bacteria comprising the nos-functional operon.

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