Nitrogen Fixation by Rhizobacterial Nif Mechanism: An Advanced Genetic Perspective

Abstract

The global population’s rising nutritional needs pose a challenge, demanding a 70% boost in agricultural efficiency to feed 10 billion people by 2050. This task is complicated by limited arable land and the imperative to reduce agrochemical usage. To overcome this, harnessing rhizobacteria and comprehending nif gene mechanisms to enhance nitrogen fixation is crucial. Nif genes encode enzymes, converting atmospheric nitrogen into vital ammonia found in diverse prokaryotes. Nitrogen-fixing bacteria, categorized as autogenous, symbiotic, and combined, collaborate with plants or independently fix nitrogen. Nitrogenase enzymes, represented by Mo, V, and Fe forms, enable this conversion. Nif operons, like nifRLA, nifHDK, nifENB, nifJ, nifUSVM, and nifWF, are pivotal in nitrogen fixation, synthesizing components, and regulating enzymes. Biotech advancements, like 2A peptides and gene manipulation, show promise in boosting crop yields. Translating rhizobacterial nitrogen fixation to cereals could revolutionize agriculture and global food security.

1. Introduction

The world population and its nutritional demand are gradually increasing in order to meet the dietary requirements of an estimated world population approaching 10 billion individuals by 2050, agricultural efficiency needs to be enhanced by approximately 70% [1]. This demanding task must be accomplished without expanding the available arable land and while maintaining or reducing the use of harmful agrochemicals such as fertilizers and pesticides [2]. Utilizing rhizobacteria and understanding nif gene mechanism in enhancing their nitrogen-fixing mechanism to increase agricultural yield is important in solving this ever-growing problem.

Nitrogen-fixing (nif) gene encodes enzymes responsible for the conversion of atmospheric nitrogen. These genes can be found as a core gene in diazotrophs, in free-living anaerobic bacteria capable of nitrogen fixation, such as Klebsiella pneumoniae, Azotobacter vinelandii Rhodospirillum rubrum, and Rhodobacter capsulatus, These genes are arranged in an operon [3]. They can also be found on plasmids together with other genes [4].

Nif gene clusters, which contain nitrogenase and various related enzymes, can be found in a wide range of prokaryotes, encompassing both bacteria and archaea [5]. On a global scale, the composition of prokaryotic communities involved in nitrogen fixation is primarily influenced by climatic conditions, with the majority of these groups showing a positive association with consistently warm or seasonally humid climates. Among the various soil characteristics, pH and nitrogen content have frequently been observed to exhibit the strongest correlations with the diversity of nitrogen-fixing groups [6]. Rhizobacteria can be categorized into three groups: nitrogen-fixing bacteria that can produce their own nitrogen, nitrogen-fixing bacteria that form a symbiotic relationship with plants, and nitrogen-fixing bacteria that can utilize both atmospheric nitrogen and other sources of nitrogen. To put a base for understanding the molecular mechanism of nitrogen fixation, we have to understand the forms of bacterial nitrogen fixation. Bueno Batista and Dixon, [3, 7] characterized nitrogen-fixing bacteria into autogenous, symbiotic, and combined nitrogen-fixing bacteria.

2. Autogenous and symbiotic nitrogen-fixing bacteria

Autogenous nitrogen-fixing bacteria refer to bacteria that are independent and capable of fixing nitrogen. Azotobacter vinelandii, cyanobacteria (or blue-green algae) Anabaena, and Nostoc are bacteria that fall into this category and exhibit notable nitrogenase activity and expression. It can efficiently perform nitrogen fixation even in the presence of oxygen, making it a prominent bacterium for studying autogenous nitrogen-fixing bacteria [3]. Azotobacter vinelandii requires oxygen and carries out nitrogen fixation using the Mo nitrogenase that is sensitive to oxygen. However, in situations where there is a limited availability of molybdenum (Mo), Azotobacter vinelandii can also fix nitrogen through alternative forms of nitrogenase known as the Vnf and Anf systems. These systems are genetically distinct from each other and provide additional pathways for nitrogen fixation when Mo is scarce [8]. Symbiotic nitrogen-fixing bacteria invade and multiply the rhizosphere then induce the formation of root nodules; the plant cell and the bacteria engage in an intimate association where the bacteria convert nitrogen to ammonia within the nodules while the plants provide shelter for the bacteria. Rhizobia species are a very good example of symbiotic bacteria [9]. We have reached a point where our knowledge of biological nitrogen fixation has advanced significantly, allowing us to consider the possibility of utilizing synthetic biology techniques to engineer symbiotic relationships. However, it’s important to note that currently, symbiotic nitrogen fixation is predominantly observed in legumes within agricultural systems [10].

3. Rhizobacterial nif mechanism

3.1 The nif operons and genes

The set of genes responsible for the conversion of atmospheric nitrogen is known as the nif genes. In Klebsiella pneumoniae, these twenty (20) genes are enclosed within seven nif operons [11]. They play a crucial role in nitrogen fixation and are primarily present in nitrogen-fixing bacteria [12]. Klebsiella pneumoniae exists as a free-living bacterium, within its chromosome, there is a specific region spanning 24 kilobases (24-Kb) that contains a total of 20 nif genes [13]. These nif genes code for the entire nitrogen-fixing mechanism and its regulation. The important parts of this mechanism are the nitrogen complex, RNA polymerase sigma 54 factor, and the Pyruvate Flavodoxin oxidoreductase.

3.2 Nitrogenase enzyme complex

Nitrogenase is an intricate enzyme found in bacteria, responsible for converting dinitrogen (N₂) into ammonia (NH₃) through an ATP-dependent process. The enzyme is typically composed of two proteins: the catalytic molybdenum-iron protein (MoFeP) and the iron protein (FeP), which acts as its specific reductase [14]. Due to its remarkable stability, the conversion of dinitrogen (N₂) into ammonia (NH₃) by the nitrogenase enzyme complex demands a considerable amount of energy input [15]. Various nitrogen-fixing bacteria harbor three types of nitrogenase: molybdenum (Mo) nitrogenase, vanadium (V) nitrogenase, and iron-only (Fe) nitrogenase. Among them, molybdenum nitrogenase is the most extensively studied and well-characterized type, commonly found in diazotrophs like legume-associated rhizobia [16].

The nitrogenase (nif) genes encompass a range of components, including the nitrogenase structural genes responsible for forming the nitrogenase enzyme [17]. Additionally, there are genes involved in activating the Fe protein, molybdenum nitrogenase biosynthesis, iron-molybdenum cofactor biosynthesis, electron donation to nitrogenase, and regulatory genes essential for the expression of nif genes [12]. The nif regulon consists of factors that can activate or inhibit the production of proteins required for nitrogen fixation based on the prevailing environmental conditions [3].

Nitrogenase exists in three distinct isoforms, each containing a different metal in the M-Cluster. In molybdenum nitrogenase, the molybdenum occupies the M-Cluster. Another isoform, known as vanadium nitrogenase, substitutes molybdenum with vanadium in the M-Cluster. The third variant, termed Iron Nitrogenase or Iron Nitrogenase, has iron in place of molybdenum in its M-Cluster [18]. The most studied molybdenum nitrogenase is presented in Figure 1 below.

Figure 1.

Molybdenum nitrogenase. Collectively built from our study using paint Microsoft software.

3.2.1 Molybdenum (Mo) nitrogenase

The predominant role of molybdenum nitrogenase lies in facilitating the major portion of biological nitrogen fixation, a prokaryotic metabolic process crucial for shaping the global biogeochemical cycles of nitrogen and carbon [19]. The molybdenum version of nitrogenase (Mo-N2ase) has undergone thorough characterization and exhibits the highest level of catalytic activity. The Mo-N2ase system comprises two-component metalloproteins that work together to catalyze the ATP-dependent transformation of N₂ into NH₃. These components are the molybdenum iron protein and the iron protein. The MoFe protein is a tetramer made up of α2β2 subunits and contains two metalloclusters, namely the P-cluster and the R-homocitrate FeMo-cofactor (FeMoCo) cluster [20].

3.2.2 Vanadium (V) nitrogenase

Vanadium nitrogenases are enzymes capable of reducing atmospheric nitrogen into a biologically useful form. The enzymes also possess the unique ability to convert carbon monoxide (CO) into hydrocarbons, similar to the industrial Fischer-Tropsch process that turns CO and hydrogen into liquid hydrocarbons, a process that can only be performed by vanadium nitrogenase enzyme [21]. Most importantly, they serve as a substitute for molybdenum nitrogenase in situations where molybdenum is unavailable. Although it represents a significant biological use of vanadium, living organisms seldom rely on vanadium nitrogenase [22].

3.2.3 The iron only nitrogenase

Fe-only nitrogenase functions without the need for any additional heterometals other than iron, yet its specific architecture and cofactor structure have remained undisclosed thus far [22]. Fe-only nitrogenases consist of two components, namely the Fe protein (AnfH) and the FeFe protein (AnfDGK). However, compared to the other two nitrogenases (Mo and V nitrogenases), there is relatively limited knowledge regarding the biosynthesis and catalytic properties of the Fe-only nitrogenase. Among the three types of nitrogenases, the Fe-only nitrogenase is considered the simplest, as it relies on fewer gene products for its function [23].

3.3 RNA polymerase and the sigma 54 factor

In prokaryotes, the regulation of transcription frequently entails direct interaction between regulatory proteins and RNA polymerase. In the case of Sigma 54 RNA polymerase holoenzyme, regulatory proteins associated with enhancer regions located distantly from the promoter interact with the polymerase through DNA looping. The s54-dependent nifA promoter in Klebsiella pneumoniae is activated when the growth conditions are limited [24].

3.4 Pyruvate flavodoxin oxidoreductase

Pyruvate flavodoxin oxidoreductase is encoded by nifJ gene from operon nifJ, which is an oxidoreductase enzyme responsible for transferring electrons from pyruvate to flavodoxin, thus reducing nitrogenase [25]. This enzyme facilitates the conversion of pyruvate and acetyl-CoA and is commonly known as pyruvate: ferredoxin oxidoreductase (PFOR). It utilizes three substrates: pyruvate, CoA, and oxidized ferredoxin, and converts them into three products: acetyl-CoA, CO2, and reduced ferredoxin [26].

4. The nif mechanism and regulation

4.1 NifRLA operon

This operon activates the nigfA gene using RNA polymerase sigma 54 protein and kick starts the transcription of structural proteins in NifHDK [27]. The NifA protein is classified within the group of enhancer-binding proteins responsible for activating gene expression alongside RNA polymerase, specifically the specialized sigma factor σ54 (RpoN). This interaction enables the polymerase core to recognize −24/−12 promoters [28]. But the mechanism depends on the availability or absence of oxygen and ammonia. These environmental factors determine the activation of all the nif genes [29]. From nifRLA operon, nifL gene is deactivated in the absence of oxygen. Its primary function is to inhibit the transcription of nifA gene. The flavoprotein contain FAD that serves as a redox sensing cofactor that transduces molecules responsible for conveying the oxygen status to the nifL protein [30]. The fumarate nitrate functions here as a reduction regulator. In the absence of oxygen, the nifL protein exists in its reduced form with FADH₂ as the cofactor, and in this state, it cannot inhibit the activity of the nifA protein [3]. However, in the presence of oxygen, the nifL protein becomes oxidized with FAD as the cofactor, leading it to inhibit the nifA protein and consequently shutting down all other operons [24]. When a significant concentration of ammonium ions is found in the environment, it suppresses the transcription of both nitrogenase and other nif genes. NH4+ serves as a co-repressor for glutamine synthetase by covalently modifying it through adenylylation. This modified enzyme then attaches to the nifR region of the nifRLA operon, leading to the inhibition of nifL and nifA gene transcription. Consequently, σ-RNA polymerase is unable to initiate the transcription of other genes [31]. Under conditions of limited nitrogen availability during growth, the inhibitory effect of nifL protein on the nifA protein is counteracted by the Glnk protein’s antagonistic action on the nifL proteins [32]. The repressor binding site encoded by nifR situated between the promoter of the nifRLA operon deactivates the entire operon when there is availability of ammonia in the environment [29, 30, 33].

4.2 NifHDK operon

This operon contained structural genes designated as nifH, nifD, and nifK [27], The nitrogenase enzyme complex is formed through the expression of several genes found in the nif operon. The nifD and nifK genes code for dinitrogenase, while nifH encodes for dinitrogenase reductase. The process of N2 fixation is tightly controlled at the genetic level, involving transcriptional and translational modifications of the nifHDK gene products [34].

4.3 NifEN and nifBQ

The nifEN and nifBQ genes are responsible for encoding cofactor assembly proteins that act as molecular scaffolds for the formation of Fe-Mo clusters. While not all alternative nitrogenases were found to have their own cofactor synthesis genes (nifENB), the phylogenetic analysis focused solely on the Mo-Fe nitrogenase due to this limitation. But nifEN is tolerant to varied expression levels [35]. Additionally, organisms with fused nifEN and nifNB genes were identified based on their sequence length [17]. NifB and nifEN are believed to be the exclusive nif-specific components essential for assembling an active nitrogenase, and their roles cannot be replaced by other enzyme activities within the host cell. NifB acts as an enzyme dependent on S-adenosylmethionine, responsible for the rearrangement and catalytic transformation of two [4Fe-4S] clusters into a [8Fe-9S-C] cluster known as nifB-co. Subsequently, this nifB-co cluster is bound and transferred to nifEN [25]. Arguably, nifQ on its own, because of limited information, is declared unessential for the assembly of Mo-Fe protein in Klebsiella pneumoniae but Bennett [25, 36, 37] reported that nifQ together with nifY, nifO, nifZ can code for nitrogenase cofactor in Azotobacter, Rhodobacter, and Klebsiella oxytoca.

4.4 NifJ operon

The operon comprises solely the nifJ gene, which encodes the pyruvate-flavodoxin-oxidoreductase protein. This protein facilitates the conversion of pyruvate, flavodoxin, or ferrodoxin to acetyl CoA while transferring electrons to nitrogenase enzymes [37, 38].

4.5 NifUSVM operon

The genes nifU and nifS encode the components of nitrogenase responsible for assembling [2Fe-2S] and [4Fe-4S] clusters essential for growth in nitrogen-fixing conditions. NifUS plays a crucial role in providing [Fe-S] clusters, which serve as metabolic building blocks for the biosynthesis of FeMo-Co. Surprisingly, in nifUS mutants, the expression of nifB was reduced, but the assembly of nifB’s [Fe-S] clusters was compensated by other non-nif machinery responsible for [Fe-S] cluster assembly. This indicates that nifUS is not indispensable for synthesizing active nifB [39], as presented in Figure 2.

Figure 2.

Mechanism of nitrogen fixation and regulation. Built from our study.

NifV is responsible for producing homocitrate, which serves as the cofactor in the active site. Additionally, Fe-S clusters are highly susceptible to damage caused by reactive oxygen species, leading to the oxygen sensitivity observed in numerous enzymes containing Fe-S cofactors [1, 25]. In the presence of nifM, the monomeric subunit of the Fe protein comprised approximately 10% of the entire cell protein. On the other hand, when nifM was absent, the nifH protein constituted 4.7% of the whole-cell protein, but it displayed no observable activity in whole-cell extract assays. The role of nifM is to convert the nifH peptide into the functional Fe protein of nitrogenase. Without the presence of nifM, only an inactive form of the nifH polypeptide is produced [40].

4.6 NifWF operon

NifW was observed to interact with an apo-form of the MoFe protein lacking FeMo-Co, containing immature P-clusters, but its specific biochemical role remains unidentified [41]. The binding of the accessory protein NifW is associated with a reduction in the distance between the clusters, along with slight alterations in their coordination. These findings suggest that NifW plays a conformational role in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer together before their fusion, which could be vital in complex cellular environments [42]. The nifF gene facilitates the transfer of electrons from the nifJ protein to the Fe protein of nitrogenase.

4.7 NifT, nifY and nifZ

NifT and nifZ genes from different nif operons were termed unessential and/or undetermined by Poudel et al. [38]. This is because they cannot technically code for anything on their own, many scholars combine them under the function of an entire operon, not a specific gene. However, nifY code is a chaperone for the apo Mo-fe protein in the molybdenum nitrogenase complex [36, 37].

5. Recent advances in the manipulation of nif genes to optimize crop yield

To satisfy the ever-increasing population, there is a need to optimize crop production and increase yield using biotechnology. Using soil bioavailable microorganism transformation into nitrogen-fixing bacteria or optimizing the average concentration of nitrogen in the soil by manipulating the nif regulon will go a long way in solving food shortage in the world. Scientists advance in achieving this through:

1. Utilizing 2A peptides to simultaneously express multiple proteins involved in the synthesis of the complex nitrogenase system. When nif proteins are connected using 2A peptides, successful co-expression occurs in Saccharomyces cerevisiae. The inclusion of a 2A tail in nifH, nifB, and nifD does not impact the activity of these nif proteins, except for nifK, where the presence of the 2A tail results in a complete loss of activity. Co-expression of nine nif proteins (nifH, nifB, nifD, nifK, nifE, nifN, nifX, hesA, and nifV) from Paenibacillus polymyxa (WLY78) is effectively achieved in yeast. Moreover, yeast nifH, co-expressed with nifH from P. polymyxa, and nifS and nifU from Klebsiella oxytoca demonstrate the activity of the Fe protein [12].

2. Manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially engineer synthetic symbiotic relationships with plants through engineering nif clusters and engineering nitrogenase clusters in native and heterologous hosts, inducible expression in response to fixed nitrogen and oxygen, engineering symbiotic relationships between legumes and rhizobacters, and engineering electron carriers [25].

3. Mimicking microbial fixing mechanism of rhizobacteria in cereal crops to create cereals that can fix nitrogen and burst crop yield [25, 43, 44].

6. Conclusion

In conclusion, addressing the growing challenge of meeting the nutritional demands of an approaching world population of 10 billion by 2050 requires a significant enhancement in agricultural efficiency. This challenge must be met while avoiding the expansion of arable land and minimizing the use of harmful agrochemicals. To overcome these hurdles, harnessing the potential of rhizobacteria and understanding the mechanisms of the nitrogen-fixing (Nif) genes become pivotal.

The nif genes, responsible for the conversion of atmospheric nitrogen into biologically useful forms, play a central role in nitrogen fixation. These genes are found in diverse prokaryotes, and their arrangement varies from core operons to plasmid-located clusters. Nitrogenase, the enzyme complex encoded by nif genes, is a sophisticated machinery consisting of various components, each with distinct roles in catalyzing the conversion of nitrogen. Molybdenum nitrogenase is the most widely studied and characterized variant, but vanadium and iron-only nitrogenases also contribute to this process.

The regulation of nif gene expression is intricately linked to environmental factors such as oxygen and nitrogen availability. Complex regulatory networks involving proteins like nifA, nifL, and glnk modulate gene expression based on these factors. Furthermore, the assembly of active nitrogenase involves several cofactor assembly proteins, such as nifEN and nifB, which are crucial for forming functional Fe-Mo clusters.

Recent advancements in genetic engineering have opened avenues to optimize crop yields through the manipulation of nif genes. Techniques like utilizing 2A peptides to co-express multiple nif proteins and engineering nitrogenase gene expression in heterologous hosts show promise in improving nitrogen fixation efficiency and oxygen tolerance. Additionally, the possibility of creating synthetic symbiotic relationships between plants and nitrogen-fixing bacteria offers exciting prospects for enhancing agricultural productivity.

In a world where food security is a pressing concern, understanding the intricate mechanisms of nif genes and leveraging the potential of nitrogen-fixing bacteria stand as promising strategies to address the challenge of feeding a growing global population while ensuring sustainability and environmental responsibility.

Acknowledgments

I would like to express my sincere gratitude to all the researchers, scholars, and experts whose valued contributions have significantly enriched the information presented in this document. Their diligent work and dedication in the fields of agriculture, genetics, microbiology, and biochemistry have added substance for understanding the complex mechanisms of nitrogen fixation and its potential applications in addressing global food security challenges. Their insights and discoveries have been instrumental in shaping the content of this discussion.

Conflict of interest

I declare that there is no conflict of interest related to the content presented in this document. The information and perspectives shared are solely intended to provide an objective overview of the subject matter.