Exploring Diazotrophic Diversity: Unveiling Nif Core Distribution and Evolutionary Patterns in Nitrogen-Fixing Organisms

doi:10.21203/rs.3.rs-4909524/v1

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Exploring Diazotrophic Diversity: Unveiling Nif Core Distribution and Evolutionary Patterns in Nitrogen-Fixing Organisms

https://doi.org/10.21203/rs.3.rs-4909524/v1

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Background

Diazotrophs carry out biological nitrogen fixation (BNF) using the nitrogenase enzyme complex (NEC), which relies on nitrogenase encoded by nif genes. Horizontal gene transfer (HGT) and gene duplications have created significant diversity among these genes, making it challenging to identify potential diazotrophs. Previous studies have established a minimal set of Nif proteins, known as the Nif core, which includes NifH, NifD, NifK, NifE, NifN, and NifB. This study aimed to identify potential diazotroph groups based on the Nif core and to analyze the inheritance patterns of accessory Nif proteins related to Mo-nitrogenase, along with their impact on N2 fixation maintenance.

Results

In a systematic study, 118 diazotrophs were identified, resulting in a database of 2,156 Nif protein sequences obtained with RAFTS³G. Using this Nif database and a data mining strategy, we extended our analysis to 711 species and found that 544 contain the Nif core. A partial Nif core set was observed in eight species in this study. Finally, we cataloged 662 species with Nif core, of which 52 were novel. Our analysis generated 10,076 Nif proteins from these species and revealed some Nif core duplications. Additionally, we determined the optimal cluster value (k = 10) for analyzing diazotrophic diversity. Combining synteny and phylogenetic analyses revealed distinct syntenies in the nif gene composition across ten groups.

Conclusions

This study advances our understanding of the distribution of nif genes, aiding in the prediction and classification of N₂-fixing organisms. Furthermore, we present a comprehensive overview of the diversity, distribution, and evolutionary relationships among diazotrophic organisms associated with the Nif core. The analysis revealed the phylogenetic and functional organization of different groups, identifying synteny patterns and new nif gene arrangements across various bacterial and archaeal species.

Diazotrophic diversity

Nitrogenase

RAFTS³G

SWeeP

nif core genes

Biological nitrogen fixation (BNF) is a crucial phase in the nitrogen cycle. This process is carried out predominantly by diazotrophic microorganisms, which possess the unique ability to fix nitrogen. Central to BNF are the nitrogenase enzymes, which catalyze the reduction of N₂ to NH₃ [1]. Nitrogenases, such as vanadium-dependent nitrogenase (V-nitrogenase) and iron-only nitrogenase (Fe-nitrogenase) play significant roles in BNF, particularly under conditions where molybdenum is limited [2]. The most widespread and well-studied of these enzymes is the Mo-nitrogenase, which is encoded by the nif genes. Diazotrophs use N₂ as a unique nitrogen source, even under different environmental conditions. However, significant diversity of the nif genes is required for BFN feasibility, which makes the structures encoded by the nif elements a target of investigation. In addition, some diazotrophs establish interactions with host organisms and have several evolutionary advantages[3]. Thus, the BNF mechanism has undergone several changes that have not been elucidated, resulting from successive horizontal and vertical transfers over billions of years [4–6].

Identifying and selecting new N₂-fixing organisms has been the objective of several works [7, 8]. Despite this, the in silico classification methods are significantly distinct. Some methodologies are limited to a single gene, usually nifH which encodes the nitrogenase NifH reductase [9]. This approach is also widely used as a phylogenetic molecular marker and a marker of new diazotrophic organisms [10–13]. However, it is reported that copies of nifH are present in non-fixing microorganisms, leading to bias in predicting new diazotrophs [14, 15]. Therefore, some studies suggest that the structural components of the Mo-nitrogenase framework (NifH, NifD and NifK) are needed for new nitrogen-fixing organism prediction [4]. Subsequently, it would need to include the elements that incorporate the biosynthesis of prosthetic groups, resulting in a minimal set composed of NifH, NifD, NifK, NifE, and NifN [16, 17].

In 2012, Dos Santos et al. proposed the core (the minimum set) of Mo-nitrogenase proteins by NifH, NifD, NifK, NifE, NifN and NifB, which are present in diazotrophic genomes, and these proteins are essential for BNF maintenance [7, 18]. Together, the functional and phylogenetic similarities of NifE with NifD and NifK with NifN suggest that the origin of nifEN is the duplication of an ancestor of the nifDK genes [19, 20]. However, the structural genes encoding nitrogenase (nifHDK) and Mo-Fe cofactor assembly proteins (nifENB) have similar phylogenetic distributions, indicating that all six genes co-evolved and remain together [21]. The relevance of the selection of Mo-nitrogenase core structures as the products of additional genes involved in the biosynthesis of prosthetic groups, we can obtain greater perceptions from them with the application of comparative methods [19]. Furthermore, the impacts involved in the transfer of Mo-nitrogenase and its implications in the construction of the nif gene cluster in Archaea and Bacteria genomes remain undefined. In addition, the classification of Mo-nitrogenases that conciliate the genes' dynamism and the complexity of the microorganisms involved is a great challenge.

The diversity of diazotrophic organisms spans a wide range of bacterial and archaeal species, each exhibiting unique adaptations and regulatory mechanisms for nitrogen fixation [22]. This diversity is reflected in the genetic organization, regulatory elements, and accessory proteins associated with the nif genes, leading to variations in nitrogenase activity and efficiency [23–25]. Understanding the distribution and evolutionary relationships of these nif-containing microorganisms is essential for obtaining insights into their ecological roles, evolutionary history, and potential applications in agriculture and biotechnology [26].

In this context, extracting and analyzing whole genomes from various species and mapping genes or enzymatic complexes are great challenges. It is crucial to integrate techniques to reduce informational complexity, without the loss of biological meaning in a large volume of biological data [27]. Artificial intelligence (AI) has become a substantial strategy for computational and statistical methods for manipulating and extracting knowledge from BNF. Among many examples, AI has been applied to analyze a large volume of diazotrophic species information, reduce the complexity of available biological data, establish groups of organisms that describe similar patterns, and compare metrics in various data of nif genes [28], contributing significantly to overcoming the frontier of BFN knowledge.

This study aims to provide a comprehensive map of Archaea and Bacteria diazotrophic organisms with a complete Nif core, highlighting the diversity and distribution of nitrogen-fixing microorganisms. Through a systematic review of the literature and the use of advanced bioinformatics tools, we identified and classified diazotrophic organisms based on their nif gene content. Our analysis extends to bacterial proteomes, revealing the presence and absence of complete Nif cores, and highlighting potential new nitrogen-fixing species. We also explore the evolutionary relationships and functional adaptations of these organisms, providing new insights into the complexity and variability of nitrogen fixation. The findings fills significant gaps in the literature regarding the phylogeny of nif genes.

Map of diazotrophs with a complete Nif core

We obtained 118 references of organisms with the preliminary systematic review from the academic literature, of which 81 and 37 represented bacterial and archaeal genomes, respectively. RAFTS³G was used to identify 22 classes of Nif proteins from these 118 genomes: NifH, NifD, NifK, NifE, NifN, NifB, NifX, NifU, NifS, NifW, NifT, NifZ, NifM, NifJ, NifF, NifP, NifI1, NifI2, NifA, NifL, NifO, and NifX-associated. As a result, we generated 336 groups (a total of 2,156 Nif sequences) that constituted the Nif protein database. Data mining using the SWeeP method and Nif clusters obtained with RAFTS³G was developed to predict N₂-fixing species from bacterial ‘proteomes’ (the ‘proteome’ as defined in [29]) from the National Center for Biotechnology Information (NCBI) GenBank database. As a result, we enriched our potential diazotrophic list with 711 prokaryotic proteomes. Among them, 544 were identified a complete Nif core (defined as the minimum set composed of NifH, NifD, NifK, NifE, NifN and NifB) and 167 had partial or no similarity with some Nif proteins from the Nif core set (Additional File 2 - Table S1). Among the proteomes without the Nif core set, 87 (approximately 52%), such as Asaia bogorensis NBRC 16594, Rhizobium sp Y9, Synechococcus sp PCC 6312, and Agrobacterium tumefaciens, were unable to identify any Nif sequence similarity. In proteomes with some Nif core sets, 12 proteomes (approximately 7%) were identified as Nif proteins with significant similarities with NifD (e.g. Moorella thermoacetica) and with NifN (e.g. Aminobacter sp. MSH1) and 33 proteomes with NifH (approximately 20%), such as Clostridium botulinum CDC1436, Fibrobacter succinogenes subsp. succinogenes S85, Fusobacterium varium, Desulfitobacterium dehalogenans ATCC 51507, Megasphaera elsdenii 1414 among others. In proteomes with partial Nif core sets assembled, we highlight Thermacetogenium phaeum DSM 12270 (containing NifH, NifD, NifK, NifE, and NifB) and Clostridium drakei, Roseiflexus castenholzii DSM 13941, Roseiflexus sp RS1, Moorella thermoacetica, Caldicellulosiruptor kronotskyensis 2002, Candidatus Desulforudis audaxviator MP104C and Syntrophothermus lipocalidus DSM 12680 (containing NifH, NifD, NifK, and NifB) as described in Table 1.

Finally, merging the 544 species with a complete Nif core set and 118 identified from the literature, we obtained 662 archaeal and bacterial proteomes containing the Nif core set. Of these, we identified 52 species without literature linking the species (or genome reference) to BFN (Table 2). Additional File 2 - Chart S1 contains the list of diazotrophic organisms identified in this study, the genome accession number from GenBank and the bibliographic references for each organism identified as diazotrophic.

RAFTS³G was utilized in 662 proteomes (a total of 3,143,063 protein sequences) and we generated 1,021,121 clusters: 327,558 sequences clustered with at least two sequences and 693,563 of the protein sequences were single. With the grouped sequences, we identified the major clusters containing Nif proteins (Additional File 2, Table S2), totaling a new database with 10,076 Nif sequences. The total number of Nif core sequences exceeds the number of proteomes analyzed: NifH (1,001), NifD (680), NifK (681), NifE (686), NifN (667), and NifB (669) (Additional File 2, Table S3). Additionally, we identified Skermanella pratensis and Skermanella sp. TT6 as the complete Nif core set in addition to the Nif protein accessories: NifA, NifW, NifP, NifS, NifU, NifX, NifXa, NifT and NifJ (Additional File 2, Chart S2) and for Asaia bogorensis NBRC 16594 [MTCC 4041] had no sequences of Nif core sequences observed in silico.

The k indicated to Nif core suggests that 10 diazotrophic groups

We determined the best number of clusters (k) for the Nif core proteome dataset using various statistical methods. From the 662 proteomes with a complete Nif core, we constructed SWeeP vectors for each species (Additional File 1, Figure S3). The k values were identified by comparing Hierarchical Clustering on Principal Components (HCPC) results with the Gap statistic, Elbow method, and Silhouette coefficient results. The Elbow method suggested optimal k values of 5, 10, 13, and 16; the Silhouette coefficients indicated 10, 13, and 18; and the Gap statistics suggested k = 8 (Additional File 1, Figure S4). In HCPC, the best k values were greater than 8 clusters (Additional File 1, Figure S5). We examined the k values 8, 10, and 13 and compared each cluster's consistency with the Nif core ordered dissimilarity heatmap (Figure 1 and Additional File 1, Figure S4). For k = 13, cyanobacteria species were subdivided into two groups (groups 6 and 7) and in heatmap were unique (Figure 1 A, in blue). For k = 8, all Paenibacillus species (Figure 1A, in orange) were grouped with Azotobacter spp. (group 3), but were proximity with Geobacter group in Heatmap. At k = 10, all cyanobacteria species were grouped into group 6, and Paenibacillus spp. were grouped in group 4 with Geobacter spp. (Figure 1, in red). Thus, k=10 was adopted for a more cohesive grouping. Group-phyla correlations are presented in Table 3.

The PCA-HCPC yields ten groups and four with great variance

PCA results of the Nif core proteins mapping the 10 groups were visualized in 2D and 3D (Additional File 1, Figure S7 A and B). PC1 and PC2 in PCA 2D explained 7.43% and 6.01% of the variance, respectively, indicating insufficient explained variance (PC < 10%) [30]. Therefore, a 3D visualization map was used (Figure 2). Groups with the highest auto values were identified in G1, G3, G6, and G10 (Figure 2 groups delimited in colorscolor).

The groups presented distinct phylogenetic and functional characteristics with significant synteny patterns

The 10 groups were arranged from the innermost to the outermost layer, consistent with the phylogenetic order (Figure 3), as follows: G3, G4, G6, G5, G2, G1, G7, G8, G9, and G10. These groups presented distinct phylogenetic and functional characteristics, with significant synteny patterns observed in several groups (Table 4 and Additional File 1, Figures S1-S16).

Group 3 (G3) constituted the majority of obligate anaerobic species of Archaea (Methanosarcina spp. division), Firmicutes (Clostridium spp. division), Fusobacteria, Spirochaetes, Verricumicrobia (Coraliomargarita akajimensis), Chloroflexi, Chlorobi, Bacteroidetes and Deltaproteobacteria (Desulfovibrio spp. division). We determined that this species had the most compact pattern of the general constitution of the nif genes (nifHI₁I₂DKENB) (Additional File 1 - Figure S8). Accessory structures such as NifX and NifV occur near the nif core (e.g., Ilyobacter polytropus DSM2926). C. akajimensis, the unique aerobic microorganism of G3, contained the positive regulator nifA, constituting the nifHDKENBVA cluster. The nifA sequences were observed simultaneously with the regulators anaerobic nifI₁I₂ in some species of Bacteroidetes, Spirochaetes, Verrucomicrobia and δ-proteobacteria.

Group 4 (G4) was composed of obligate anaerobes of Euryarcheota (Methanobacterium spp. division), Firmicutes (bacilares), Nitrospirae, and some facultative anaerobes or microaerobic organisms, such as Deferribacteres, Aquificales, Actinobacteria, Chrysogenetes, Epsilon and Deltaproteobacteria (Geobacter division). We identified an increase in nif genes constitution compared to the G3 (Additional File 1 - Figure S9). Notably in Methanotermococcus sp. IH1 and Heliobacterium modesticaldum Ice1 (nifI₁I₂HDKENXB), Kyrpidia spormannii EA1 (nifBVSHDKENXXaHesA), Paenibacillus spp. (nifBHDKENXXahesAV), Dissulfurispira thermophila T55J and Thermodesulfovibrio yellowstonii DSM 11347 (nifHDKENXI₁I₂B), Hydrogenobacter thermophilus TK6 and Thermocrinis albus DSM 14484 (nifI₁I₂TBWZVSAHDKXaENX), Denitrovibrio acetiphilus DSM 12809 (nifAZHDKTENXXaVB), Calditerrivibrio nitroreducens DSM 19672 (nifVBAZENXXaHDKT), Desulfurispirillum indicum S5 (nifVBASUPHDKTZENX), Frankia alni EA1 (nifVHDKENXXaWZBhesAhesB), Desulfuromonas soudanensis WTL (nifHDKENXI1I2B), Anaeromyxobacter sp. K (nifBHDKENXA), Geobacter pickeringii G13 (nifVHDKENXB), Sulfurospirillum barnesii SES3 and Arcobacter nitrofigilis DSM 7299 (nifVBHDKTAENXXaFWZQ). As presented, we identified variable-sized Nif cores in this group including different loci patterns to nifI₁I₂ in obligately anaerobic/facultative and nifA in facultative/aerobic organisms. Additionally, we identified the protein associated with NifX (we called the gene nifXa) and hesA and hesB genes in facultative/aerobic species.

Group 6 (G6) was composed of all the Cyanobacteria species. We identified the composition of the nif core with a relative pattern in cyanobacteria: nifPVZTBSUHDKENXXaW (Additional File 1, Figure S10). Proteins, such as fdxB, hesA, hesB, and Mo-nitrogenase C-terminal (we called the gene monC) have been reported to play significant roles in BNF presented in these cores.

Group 5 (G5) was composed of Alpha (Magnetococcus marinus MC-1 and Martelella endophytica YC6887), Beta (Azoarcus spp. division) and Gamma (Azotobacter spp. division) proteobacteria (Additional File 1 - Figure S11). In M. marinus, the pattern is the most compact compared with the other organisms in the group (nifABQHDKTYENXXaVZ), with the integration of the isc cluster. We identified the NifL protein in only one Alphaproteobacteria - M. endophytica (nifUSVWZMBHDKTYENXXaQLA). The Betaproteobacteria in this group were unique in that they contained the nifL. Among Halorhodospira spp., the only nitrogen-fixing species of Gammaproteobacteria is notable for not containing it (e.g., H. halophila SL1, nifENXXaABQVWZMHDKY). Clusters of genes adjacent to the nif elements that integrate other systems, such as iscAUS and rnfABCDEH, related to Fe-S incorporation and electron transfer respectively, which may also play a role in BNF, have been reported.

Group 2 (G2) was represented by gamma proteobacteria (Dickeya division). In G2, the genomic synteny of the species Tolumonas auensis, Brenneria goodwinii, Pectobacterium atrosepticum JG10-08, Dickeya fangzhongdai ND14b, and Kosakonia oryzae Ola 51 is represented in Additional File 1 Figure S12. We identified a cluster similar to Klebsiella variicola strain 342 [31] (nifJHDKTYENXUSVWZMFLABQ). In addition, we also identified genes related to the BNF process, namely fdxN, fdxB, and iscA (iscN).

Group 1 (G1) was a mixture of Gammaproteobacteria (Klebsiella division). The genomic synteny of Klebsiella quasivariicola KPN1705 and Raoultella ornithinolytica S12 is represented in Additional File 1, Figure S13. Among these three species and all the other species cataloged in G1 (Additional File 2, Table S5), we identified the synteny pattern nifJHDKTYENXUSVWZMFLABQ.

Group 7 (G7) was composed of alpha (Bradyrhizobium division), beta (Burkholderia division) and Gamma (Halothiobacillus division) proteobacteria, Acidithiobacilia, Nitrospirae (Leptospirillum spp.) and Verrucomicrobia (Methylacidiphilum spp.) (Additional File 1 - Figure S14). The composition of the nif genes revealed a pattern of nifABZ(Z)THDKENXXaQUSVPW. In most cases, there was a duplication of the nifZ gene (e.g., Azoarcus sp. KH32, nifABZ1Z2STHDKENXXaQW). Gene clusters such as isc, composed of hscA, iscA, iscN, iscU, iscS; rnf, composed of rnfABCDEH; and fix, composed of fixABCX, have been reported.

Group 8 (G8) was composed of Alphaproteobacteria (Rhodobacter spp. division) (Additional File 1 - Figure S15). Generally, the nif element pattern is presented as nifABZTHDKENXXaQUSVW. isc (iscANUS) and fix (fixABCX) clusters have also been reported near the nif elements.

Group 9 (G9) included the Alphaproteobacteria Gluconacetobacter diazotrophicus PA1 5, Hartmannibacter diazotrophicus, Methylobacterium nodulans ORS 2060, Mesorhizobium erdmanii NZP2014, Rhizobium leguminosarum GLR17, and Sinorhizobium meliloti. The nif genes were similar in constitution, represented by nifABZTHDKENXXaQUSV(P)W. Only the nif genes in Neorhizobium galegae, Sinorhizobium fredii, Ensifer sojae, and other species of Rhizobium spp. were impracticable because these organisms have BNF genes in plasmid organelles. A cluster of fix and nod genes was found adjacent to the nif cluster. Several hypothetical sequences identified by RAFTS³G showed homology with IS group insertases (e.g., iss3).

G10 constituted the largest number of Rhizobium species and the Sinorhizobium americanum (CCGM7/CFNEI 73) and Sinorhizobium fredii NXT3 (Additional File 1 - Figure S16). The nif sequences of this group were identified exclusively in plasmids and the nif genes are constituted by nifABZTUSWHDKENXXa. For Rhizobium species (i.e. Rhizobium sp. TAL182) there were three structural components of the NifH forming the nifHDK (listed as NifH2, NifD2 and NifK2) and another copy of nifH (NifH3) generally with nifQ borderer, constituting the nifQH3H2D2K2ABZTUSWHDKENXXa cluster. Additional proteins include Nod proteins, Fix proteins, insertase-related proteins (e.g. ISS) and cytochrome P (CYP450).

Duplication of the Nif core in some Cyanobacteria species and Azoarcus sp KH32C

The tree obtained from nitrogen-fixing cyanobacteria was divided into non-heterocyst-forming (NFH) and heterocyst-forming (HF) clades (Figure 4). In NFH, there are diazotrophs of the orders Oscillatoriales, Chroococcidiopsidales, Pleurocapsales and Chroocococcales. The HF cluster was composed of Nostocales, Stigonematales and Synechococcales.

We report the duplication of Nif core proteins in Aulosira laxa NIES-50 , Azoarcus sp KH32C, Calothrix brevissima NIES-22, Nostoc carneum NIES -2107, Tolypothrix tenuis PCC 7101 and Trichormus variabilis ATCC 29413 suggesting the existence of different loci with two distinct nif clusters related with the Nif core or “Nif core2” - Nif core2 was adopted to call the second Nif core set identified (Additional File 2, Chart S3). A comparison of the Nif core and Nif core2 distributions in the phylogenetic tree revealed different topologies of Aulosira laxa, Calothrix brevissima, Nostoc carneum, Tolypothrix tenuis and Trichormus variabilis. The tree topology for these species, with the Nif core2, suggests the BNF related to HF characteristics.

The divergence observed in the Nif core tree compared to the proteome tree highlights that species like Azoarcus olearius, Azoarcus communis, and Azoarcus sp. BH72 were distinct from Azoarcus sp. KH32C and Azoarcus sp. CIB (Figure 5). Similarly, Azospira oryzae PS and Azospira sp. I09 formed distinct groups from Azospira restricta DSM 18626. In conforming, the HCPC-PCA suggests that the species of Azoarcus and Azospira should be divided into distinct groups—specifically, Azoarcus sp. KH32C and Azoarcus sp. CIB were grouped into G8—Azoarcus sp. KH32C (with Nif core2) was grouped into G7 (Figure 4). A. olearius (DQS4 and BH72 strains) and A. comunis (TSNA42 and TSPY31 strains) were grouped into G5. In parallel, the Azospira restricta DSM 18626 was grouped in G5, while A. oryzae OS and Azospira sp. I09 were grouped in G8. Based on the nif gene cluster annotation, Azoarcus spp. and Azospira spp. grouped in G5, G7, and G8 exhibited similar intragroup nif gene compositions (Additional File 2, Chart S4).

This study provides a comprehensive map of diazotrophic organisms with a Nif core, revealing significant findings on the diversity and distribution of nitrogen-fixing microorganisms. We constructed 336 clusters related to the 22 different types of Nif proteins in these organisms, comprising a robust database of 10,076 Nif sequences. We expanded our analysis to the prokaryotic genomes, identifying 711 species, with 544 containing the complete Nif core set (NifHDKENB) and 167 with partial or no similarity with some Nif protein from the Nif core. Among the organisms lacking the Nif core, 52% did not contain any sequences from the Nif core set and 48% showed some similarity with the Nif proteins. In proteomes with a partially Nif core set assembled, we highlight 33 containing only NifH, seven organisms containing NifHDKB, and Thermacetogenium phaeum DSM 12270 containing NifHDKEB. Garcia et al. (2020) proposed the following sequential evolutionary events of nitrogenase complex formation: 1) NifHDKB, 2) NifHDKEB, 3) NifHDKENB, and 4) Vnf/AnfHDK [32]. These findings suggest a possible "missing link" in the evolution of nitrogen-fixing mechanisms and the distribution of nitrogen-fixing capabilities among bacterial genomes. Additionally, these findings support new evolutionary hypotheses regarding the loss or functional alteration of Mo-nitrogenase between fixing and non-fixing species. Finally, we analyzed 662 archaeal and bacterial proteomes containing the Nif core. Notably, we identified 52 potential diazotrophs. This includes Skermanella pratensis and Skermanella sp. TT6, two previous species related to the inability to fix nitrogen [33]. Additionally, with the proteomes, we obtained protein clusters containing Nif proteins. Here, we identified Nif core sequences that exceeded the number of proteomes (51,21% for NifH and an average of 2,72% for the NifD, NifK, NifE, NifN and NifB proteins), suggesting Nif duplications in some proteomes.

Through phylogenetic analysis of Nif core, combined with the identification of ten distinct groups, provides significant insights into the evolutionary and biological characteristics of nitrogen-fixing bacteria. These groups exhibit varying gene patterns and associated regulatory elements, reflecting adaptations to different ecological niches and symbiotic relationships. The prokaryotic gene clusters understanding may offer a rich toolbox for synthetic biology, because it facilitates the transfer of complete functions across evolution [34]. Group 3 showcases a diverse array of obligate anaerobes with a compact nif gene structure, highlighting the evolutionary conservation of the nif core among these ancient lineages. The regulation is predominantly mediated by the paralogs nifI₁I₂, a mechanism switch-in and switch-off depending on levels of 2-oxoglutarate [35]. Additionally, the presence of accessory structures like NifX and NifV suggests functional adaptations to enhance nitrogen fixation efficiency [36, 37]. Group 4 includes obligate anaerobes, facultative aerobes, and aerobes, all of which are diazotrophs with increased complexity in their nif genes and distinct nif gene cluster patterns and regulations. The presence of the NifX-associated protein (NifXa), encoded by the nifXa gene, suggests evolutionary innovations within this group. This protein, similar to NifX, may offer advantages under specific environmental conditions, such as adapting to microaerobic or aerobic environments [37, 38]. Group 6 consolidates all cyanobacteria, emphasizing the conserved nature of the nif core within this phylum. The inclusion of genes like fdxB, hesA, hesB, and monC (Mo-nitrogenase C-terminal protein) underscores the importance of these elements to electron transferring and final assembly and stabilizing of Mo-nitrogenase in cyanobacteria [39]. Group 5 reveals the integration of nif elements with other systems, such as isc (iron-sulfur cluster) and rnf (ferredoxin-NAD + reductase) gene clusters, in free-living proteobacteria species. This integration may be involved in enhancing nitrogen fixation through improved iron-sulfur cluster assembly and electron transfer mechanisms, including the rnf cluster was related to specific roles of BNF in microaerobic and aerobic conditions [40–42]. Group 2 and Group 1 exhibit patterns similar to known nitrogen-fixing Klebsiella variicola strain 342 indicating conserved nitrogen-fixing mechanisms across these groups. The presence of genes like fdxN, fdxB, and iscA (iscN) in Group 2 may play crucial roles in redox processes and in the maintenance of BFN [43, 44]. Group 7 and Group 8 show distinct patterns of nif gene duplication and integration with isc and fix clusters, suggesting evolutionary pressures to optimize nitrogen fixation under varying environmental conditions, from arid soils to aquatic environments [45]. Group 9 highlights the complexity of nif gene organization in alphaproteobacteria, with BNF genes often located in plasmids (Rhizobiales). The presence of adjacent fix and nod gene clusters indicates co-evolution of nitrogen fixation and nodulation capabilities [46]. Group 10 consists of a great number of Rhizobium species with nif genes located exclusively on plasmids. These species often have duplicated nifHDH and nifH genes, as well as some insertion sequences (ISS group). This indicates significant horizontal gene transfer of nitrogen-fixing capabilities and highlights the complexity of nif genes dynamics in these organisms[47, 48].

The phylogenetic analysis of duplicated Nif cores reveals distinct clades, suggesting specialization and adaptation to different environmental contexts. The Nif core proteins duplicated in five cyanobacteria species and Azoarcus sp. KH32C points to the presence of duplication loci related to Mo-nitrogenase dependent fixation. Curiously, cyanobacteria species were divided into NHF (non-heterocyst-forming) and HF (heterocyst-forming) clusters, and the duplication of nif core genes suggests that nif genes co-evolved as NHF species evolved into HF organisms [49]. The Nif core tree's topology when compared to the proteome tree indicates that Azoarcus and Azospira species are organized into distinct evolutionary groups. Raittz and colleagues reported separate origins of nif genes in the Azoarcus species, showing that Azoarcus sp. CIB and Azoarcus sp. KH32 were separated from Azoarcus genera [50]. Additionally, we report another nif loci in Azoarcus sp. KH32 which is related to Herbaspirillum spp. clade.

In conclusion, our study provides a comprehensive overview of the diversity, distribution, and evolutionary relationships of diazotrophic organisms with complete Nif cores. These findings underscore the evolutionary diversification of nitrogen-fixing bacteria, with distinct phylogenetic and functional characteristics shaped by ecological and symbiotic interactions. The identified groups provide a framework for further exploration of the molecular mechanisms underlying biological nitrogen fixation and its evolutionary significance across different bacterial lineages. Further research is needed to explore the functional roles and regulatory mechanisms of Nif proteins in various environmental settings, which could have important implications for agriculture, biotechnology, and ecological conservation.

Defying the proteome and Nif core sets

Proteome set: Proteomes obtained from complete prokaryotic genomes. Nif core set: the 6 sequences that integrate the Nif core (NifH, NifD, NifK, NifE, NifN and NifB). We included bifunctional proteins related to gene fusion, such as NifEN and NifNB proteins. In the case of repeated or duplicated protein sequences (e.g.: NifH), we disregard sequences unrelated to homology or synteny with the other Nif core sequences.

Vectorial representation of NCBI proteomes

We extracted 10,324 proteomes from the complete genomes of bacterial organisms downloaded from the NCBI release 227. For data manipulation, we used SWeeP (available at: https://sourceforge.net/projects/spacedwordsprojection/) with the following parameters: mask "11011" for spaced words and 1369 to the projection of coordinates according to the material described and published in De Pierri et al. (2020).

Diazotrophic species obtained from the literature

The collection of diazotrophs aims to acquire a list of validated organisms by identifying the core set of Mo-nitrogenases or performing biochemical assay (e.g.: acetylene reduction standard tests). We used the literature scans of Dos Santos and collaborators (2012) and the catalog described in Chapman & Hall (2005). From this literature, we extracted the genomic data available in NCBI. We obtained and vectorized the Proteomes of each genome, using the same settings described in the previous section for SWeeP.

Data mining strategy of diazotrophic candidates

The objective of this step was to create a data mining strategy to search for new diazotrophic organisms. We represented the proteomes of obtained diazotrophic species through the literature into SWeeP in compact vectors, which we used to make up the Class 1 set (the true class), in the training and classification of the Artificial Neural Network (ANN). We selected the MultiLayer-Perceptron neuronal network class (MLP), configured at 5:3:1 (values for the input layer, hidden layers, and the output layer, respectively). The Class 0 set was composed of 5,000 non-fixing species proteomes (non-independent type test, training performed to debug). The creation steps of the SWeeP ANN were illustrated in Additional File 1 - Figure S1. We developed ANN in the MATLAB® environment licensed for the 2019b version (https://www.mathworks.com/products/matlab.html).

RAFTS³G applied to identify homologous Nif proteins (I)

The RAFTS³G tool [23] (available in: https://sourceforge.net/projects/rafts-g/) was adopted for this work to obtain homologous Nif protein clusters, starting from the identification of 22 known Nif classes: NifH, NifD, NifK, NifE, NifN, NifB, NfX NifU, NifS, NifV, NifP, NifW, NifZ, NifM, NifT, NifF, NifJ, NifO, NifA NifL, NifI1, and NifI2. We generated homologous clusters from the diazotrophic Proteomes and we extracted those proteins containing each of the 22 Nif classes. The Nif proteins group information composed our first Nif database (I). The RAFTS³G parameter was 0.4 similarity between sequences with binary search.

Nif prediction in diazotrophic candidates

After obtaining the Nif (I) protein base we created, we used the RAFTS3 search tool (available at: https://sourceforge.net/projects/rafts3) to search for Nif sequences in the candidate organisms selected by the network. Here, the Nif base served as a query structure (subject) for the RAFTS3 tool [51]. The objective of this search for Nif proteins was to identify all the 6 Nif core structures of Mo-nitrogenase as a second criterion in the step of selecting new N2-fixing organisms.

A systematic review to validate diazotrophic species

After localising the Nif core sequences on the organisms’ proteomes, we performed a systematic literature scanning of validated diazotrophs in studies between 2012 and 2024. We used the three main research databases: Expasy (SIB institute) (https://www.expasy.org/), PubMed (NCBI) (https://pubmed.ncbi.nlm.nih.gov/), and Scholar (Google) (https://scholar.google.com.br/).

RAFTS³G to identify homologous Nif proteins (II)

With the information on new diazotrophic proteomes, we accomplished a clustering strategy of the sequences to identify Nif homologous Groups (II). The RAFTS³G [27] was configured in 0.5 of similarity between sequences with binary search.

PCA SWeeP of Nif core dataset

We used the rSWeeP version 3.20 [52] to integrate the vectors with R functions. We built the Nif core PCA dataset and visualized it in 2D utilising the Factoshiny package [53]. We visualized the PCA in 3D using the plotPCA script (Additional file 1 – Figure S2).

HCPC method applied in PCA of Nif core

We obtained estimates for the number of groups (k) using the HCPC method and compared the results with Gap statistics [54], the Elbow method [55], and the silhouette coefficient [56]. We adopted a maximum of 20 clusters generated by each method to balance granularity and interpretability. The consensusclusterplus function in R (v4.3) was utilized for HCPC analysis [57], while the fviz_nbclust function from the factoextra package [58] (https://rpkgs.datanovia.com/factoextra/index.html) facilitated Gap statistics, Elbow method, and silhouette coefficient evaluations. This approach ensures that the clustering analysis captures meaningful patterns without overfitting or oversimplifying the data structure.

Heatmaps created to compare groups

The heatmap technique was divided into two moments: 1) we created an ordered heat map from the ordered dissimilarity matrix, to verify the species grouped consistency, and to compare the k value more weighted in the division of groups. We used the coldiss function [59] in R. 2) we built a heatmap from the Euclidean distance matrix ordered for Nif core groups and Proteomes to compare if the Nif core information had some divergence about proteome information. In this case, we used standard scripts in MATLAB® and we constructed two heatmaps of the Euclidean distance between Nif core groups and the distance from the Proteomes versus Nif core distance of obtained groups.

SWeeP applied to build phylogenetic trees

We built the SWeeP [29] vectors for all diazotrophic proteomes in two main approaches: 1) with Proteome data and 2) with Nif core data. The parameters used for SWeeP were spaced words "11011" and the matrix (W) of 1369 coordinates. We calculated the Euclidean distance for the vectors in each approach. We used the NJ method for phylogenetic construction. We evaluated the branches' reliability using the bootstrap method, a function available at the Analyses of Phylogenetics and Evolution (APE) version 5.6-1 package [60] with a sampling of 1,000 replicates. The protocol for this step was illustrated in Additional File 1 - Figure S3. We visualized the Dendrograms in Dendroscope [61] release 3.8.1 (https://software-ab.informatik.unituebingen.de/download/dendroscope3/welcome.html). To compare the topology between the Nif core and the Proteome trees we used the tanglegram algorithm (available in the Dendroscope tool). (https://software-ab.informatik.uni-tuebingen.de/download/dendroscope/manual.pdf).

Genomic synteny with SyntTax web

We used the SyntTax web tool [62] to search for prokaryotic synteny (available at: https://archaea.i2bc.paris-saclay.fr/SyntTax/). The advantage is that SyntTax has its relational base of complete genomes available and updated with the NCBI base. The search was made from the sequences in proteins by blocks of synteny. The use of SyntTax is limited to the chromosomal data of the species, therefore ineffective if there is plasmidial gene synteny, for example. We searched for syntenic blocks in the species available using Nif core proteins (NifH, NifD, NifK, NifE, NifN, and NifB) extracted from Klebsiella variicola strain 342 [31] for bacteria and in Methanosarcina mazei strain Go1 [63] for archaea species. We adopted 10% as the minimum BLAST score (standard algorithm).

Acknowledgements

Federal University of Paraná (UFPR), National Council for Scientific and Technological Development (CNPq), CAPES (Coordination for the Improvement of Higher Education Personnel) & Araucária Foundation for supporting this work. The Department of Biochemistry, Biological Sciences Sector – Federal University of Paraná (UFPR) and Laboratory of Artificial Intelligence Applied to Bioinformatics, Professional and Technical Education Sector – SEPT (UFPR), Brazil for technical and infrastructure support.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All the data supporting our findings are contained within the manuscript. The supplementary data as Nif core and Proteome prokaryotic FASTA files, the SWeeP matrices, RAFTS³G generated clusters, and synteny relatories are also available at: https://drive.google.com/drive/folders/1VRsizmEEKhiNidgkB4C7mg-5HjQTV84H?usp=share_link

Competing interests

The author(s) declare no competing interests.

Funding

B.T.L.N. received a joint from CAPES (Coordination for the Improvement of Higher Education Personnel) & Araucária Foundation for PhD studies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the paper.

Authors' contributions

B.T.L.N. authored the main manuscript, conducted the data analyses, and prepared all figures and tables. R.B.R.C. wrote and reviewed the manuscript. R.T.R. and F.O.P. contributed with advanced expertise in the field. All authors reviewed this manuscript.

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Exploring Diazotrophic Diversity: Unveiling Nif Core Distribution and Evolutionary Patterns in Nitrogen-Fixing Organisms

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Abstract

Figures

Introduction

RESULTS

Discussion

Methods

Defying the proteome and Nif core sets

Vectorial representation of NCBI proteomes

Diazotrophic species obtained from the literature

Data mining strategy of diazotrophic candidates

RAFTS³G applied to identify homologous Nif proteins (I)

Nif prediction in diazotrophic candidates

A systematic review to validate diazotrophic species

RAFTS³G to identify homologous Nif proteins (II)

PCA SWeeP of Nif core dataset

HCPC method applied in PCA of Nif core

Heatmaps created to compare groups

SWeeP applied to build phylogenetic trees

Genomic synteny with SyntTax web

Declarations

References

Tables

Additional Declarations

Supplementary Files

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Version 1