Exploring the Diversity of the Genus Nitrospirillum: Description of Six Novel Nitrogen-Fixing Bacteria Species

Exploring the Diversity of the Genus Nitrospirillum: Description of Six Novel Nitrogen-Fixing Bacteria Species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring the Diversity of the Genus Nitrospirillum: Description of Six Novel Nitrogen-Fixing Bacteria Species Jerri Edson Zilli, Stefan Shwab, Natália dos Santos Ferreira, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5600752/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this study, we characterized 86 plant growth-promoting bacterial strains belonging to the genus Nitrospirillum , isolated from diverse host plants and geographic regions. To investigate their evolutionary relationships, we employed phylogenetic analyses based on 16S rRNA and recA genes, as well as phylogenomic approaches including average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH). The classification of type strains was further supported by in silico analyses of chemotaxonomic markers, particularly genes involved in fatty acid biosynthesis and elongation, phospholipid and quinone production, and nitrogen fixation ( nifHDK operon). Phenotypic and chemotaxonomic characterization was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid methyl ester (FAME) profiling, and physiological assays. These included evaluations of nitrogen fixation capacity, antibiotic resistance, carbon source utilization, and enzymatic activity. This integrative approach provided detailed insight into the characteristics and diversity of the studied strains. Phylogenetic and genomic analyses revealed six novel taxa within the genus Nitrospirillum , in addition to the previously described species N. amazonense , N. iridis , and N. viridazoti . The distinctiveness of these new lineages was supported by both genomic metrics and phenotypic traits. All novel strains also exhibited ethylene production, confirming their nitrogen-fixing ability under in vitro conditions. Based on these findings, we propose the formal description of six novel species: Nitrospirillum bahiense sp. nov. (= BR 11865ᵀ, =UCCCB 233ᵀ), Nitrospirillum guanabarense sp. nov. (= BR 11163ᵀ, =UCCCB 228ᵀ), Nitrospirillum guaranorum sp. nov. (= BR 11164ᵀ, =UCCCB 229ᵀ), Nitrospirillum karajorum sp. nov. (= BR 11752ᵀ, =UCCCB 231ᵀ), Nitrospirillum guiasense sp. nov. (= BR 11828ᵀ, =UCCCB 232ᵀ), and Nitrospirillum pindoramense sp. nov. (= BR 11622ᵀ, =UCCCB 230ᵀ). Biological nitrogen fixation genomic analysis PGPR taxonomy bacteria diversity grasses MALDI-TOF MS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Plant growth-promoting bacteria (PGPB) play a pivotal role in modern agriculture by enhancing plant development through mechanisms such as biological nitrogen fixation and the production of growth-stimulating compounds. Their strategic application in agricultural systems increases the efficiency of agronomic inputs, diminishes reliance on chemical fertilizers, and markedly improves soil quality, thereby advancing sustainability in both agricultural and environmental practices [ 1 – 3 ]. Moreover, the supply of bio-based products intended for agricultural use is on the rise, with PGPB emerging as particularly prominent components. In numerous countries—especially across South America—PGPB have been commercially employed in agriculture for over a decade, with the inoculated area estimated to approach 10 million hectares [ 4 ]. Representative examples include bacteria from the genus Azospirillum , which is widely applied in maize cultivation [ 4 ], and Nitrospirillum , which is marketed as an inoculant for sugarcane [ 5 ]. The genus Nitrospirillum emerged from a taxonomic reclassification of the species Azospirillum amazonense , which was initially isolated from a variety of ecosystems following extensive research in the 1980s [ 6 , 7 ]. A modification in the standard semi-solid medium for isolating Azospirillum strains—achieved by incorporating 0.5 g L⁻¹ each of mannitol and sucrose into the original malate-based NFb medium [ 8 ]—permitted the isolation of a group of strains from Urochloa brizantha (formerly Brachiaria brizantha ) in the Amazon region [ 9 ]. Moreover, the optimization of a nitrogen-free medium by supplementing it with sucrose and adjusting the pH to a lower range (5.0–6.0), referred to as LGI or modified FAM, further facilitated the isolation of A. amazonense from diverse ecosystems [ 6 ]. Successful isolation of Nitrospirillum has since been achieved in other countries, with strains obtained from various regions and across distinct seasonal conditions [ 10 , 11 ]. The genus Nitrospirillum belongs to the phylum Proteobacteria, class Alphaproteobacteria, order Rhodospirillales, and family Rhodospirillaceae. The taxonomic classification has been established based on the phylogeny of specific genes, such as 16S rRNA, and whole-genome data from bacterial genomes [7, 12). Phenotypic characterization has involved additional analyses, including chemotaxonomic markers such as fatty acid profiles, quinone composition, and polar lipid patterns, as well as assessments of nitrogen fixation capacity, antibiotic tolerance, C-source utilization, and enzyme activities (7, 12). Ubiquinone Q-10 appears to be the major respiratory quinone, while C 18:1 ω 7c is the predominant fatty acid. Additionally, all species described exhibit biological nitrogen fixation, and in general, the strains of this genus do not have the ability to denitrify (12). In addition to Nitrospirillum amazonense , the species Nitrospirillum iridis was identified through isolation in Korea, and more recently, Nitrospirillum viridazoti has been documented [ 12 ]. N. viridazoti is distinguished by its ability to fix nitrogen and stimulate plant growth [ 5 , 13 ]. Furthermore, this species exhibits a distinctive partial denitrification pathway, wherein the enzyme nitrous oxide reductase exclusively catalyzes the reduction of nitrous oxide (N₂O) to dinitrogen (N₂) [ 12 ]. While N. iridis was isolated from Iris ensata , both N. amazonense and N. viridazoti were predominantly associated with grasses, including Andropogon gayanus , Axonopus sp., Bactris gasipaes , Brachiaria spp., Digitaria decumbens , Hyparrhenia rufa , Oryza sativa , Zea mays , Panicum pilosum , Paspalum virgatum , Pennisetum purpureum , Saccharum spp., and Sorghum vulgare . Notably, these bacteria have also been identified in non-grass hosts such as Ananas comosus , Cocos nucifera , Musa spp., among others [ 8 , 12 , 14 , 15 ]. Currently, an extensive collection of strains is under investigation, where genomic analyses are integrated with morphological and physiological comparisons to elucidate the taxonomy of this genus. In this study, we endeavored to enhance our understanding of bacterial diversity within the genus Nitrospirillum by characterizing 86 strains obtained from a variety of hosts and geographic regions. These strains have been deposited in the culture collection of the Johanna Döbereiner Biological Resource Center at Embrapa Agrobiologia over the past 35–40 years, representing a valuable repository for elucidating the evolutionary intricacies of Nitrospirillum . Employing a taxonomic framework grounded in both phylogenetic and phylogenomic analyses, we have proposed six novel species within the genus Nitrospirillum . This taxonomic contribution not only broadens our systematic understanding of bacterial classification but also underscores the critical importance of preserving and investigating microbial repositories to fully elucidate prokaryotic diversity. Materials and Methods Bacterial strains In the present study, we examined 86 bacterial strains obtained from the grass Brachiaria brizantha (three strains), B. humidicola (six strains), Sorghum vulgare (six strains), Zea mays (23 strains), and Oryza sativa (47 strains). The collection locations were in the Brazilian states of Bahia (northeast Atlantic Forest; 2 strains), Goiás (central-west Cerrado; 22 strains), and Rio de Janeiro (southeast Atlantic Forest; 62 strains) (Table S1). When deposited in the culture collection, the strains were characterized as gram-negative, non-sporulating cells with the capacity to fix nitrogen in a semi-solid medium. All these strains are stored at the Johanna Döbereiner Biological Resources Center (CRB-JD, WFCC 364, Seropedica, Rio de Janeiro, Brazil) (https://www.embrapa.br/agrobiologia/crb-jd) and are preserved long-term through lyophilization and storage at -80 °C in a culture medium containing 20% glycerol. The strains BR 11163 T , BR 11164 T , BR 11622 T , BR 11752 T , BR 11828 T , and BR 11865 T were also deposited at the University of Coimbra Bacteria Culture Collection (UCBCC, WFCC 1179 – Coimbra, Portugal) (https://ucccb.uc.pt/) and were assigned the accession numbers UCCCB 233 T , UCCCB 228 T , UCCCB 229 T , UCCCB 231 T , UCCCB 232 T , and UCCCB 230 T . Phylogenetic analyses Genomic DNA was extracted from the 86 strains using a Bacterial Genomic DNA Isolation Kit (Wizard; Promega, Madison, WI, USA) following the manufacturer's instructions. Segments of the 16S rRNA and recA genes were amplified via PCR using the primers 27F/1492R (16S rRNA) and 63F and 504R ( recA ) [16]. Subsequently, Sanger dideoxy DNA sequencing was performed. Forward and reverse contigs were used for gene sequence assembly using the Bionumerics package (v. 7). The 16S rRNA and recA sequences of the type strains were retrieved from the NCBI nucleotide database (www.ncbi.nlm.nih.gov). Sequences were compared with other sequences in the NCBI database using BLAST (https://blast.ncbi.nlm.nih.gov/) followed by analysis using MEGA 11 [17]. The evolutionary model was determined for alignment and tree topology using the JC+C model [18] for 16S rRNA gene sequences, assuming independent sites with identical mutation rates. For recA gene sequences, T92+G [19] was the best-fit model, which addresses transition-transversion and G+C-content biases. The SeaView tool was employed, incorporating maximum likelihood (ML) estimation of the PhyML software and ClustalX-generated alignment. Branch support was assessed using a nonparametric bootstrap test with 1000 replicates and an approximate likelihood ratio test (ALRT). The 16S rRNA sequences were also concatenated with the recA sequences for a more robust view of the phylogeny. Concatenation was performed with MEGA 11 using the JC+C model and ML estimation. Positive and negative selection assessment To evaluate the degree of neutrality between the sequences, identify those that do not fit the neutral theory model, and determine the balance between mutations and genetic drift, we used the neutrality test method proposed by Tajima [20]. Genomic analyses The genome sequences of strains BR 11865 T and BR 11622 T were sequenced at the DOE-Joint Genome Institute (JGI) as part of the Genomic Encyclopedia of Type Strains, Phase IV (KMG-V) to study the core and pan-genomes of soil and plant-associated prokaryotes (https://gold.jgi.doe.gov/studies?id=Gs0129091) using an Illumina NovaSeq 6000 platform. All genomes were first deposited in the RefSeq database and subsequently downloaded, along with the genomes of strains BR 11140T, BR 11142T (Y-1), and DSM 22198T, which were available in the RefSeq database (accessed in August 2023). The whole-genome sequences of strains BR 11752 T , BR 11163 T , BR 11164 T, and BR 11828 T were obtained using nanopore technology. High-molecular-weight DNA was extracted using a DNA Genomic Isolation Kit (Wizard; Promega), and libraries were constructed following the native barcoding genomic DNA protocol recommended by the manufacturer, after which sequencing was performed. The libraries were loaded onto a MinION flow cell model FLO-MIN 106 (version R10.3) and sequencing was monitored using the MinKNOW program (Oxford Nanopore Technologies). Base calling was performed using the Guppy program (Oxford Nanopore Technologies), data demultiplexing was performed using the Demultiplex program (version 1.2.1, available at https://github.com/jfjlaros/demultiplex), and sequencing data were evaluated using NanoPlot (version 1.24.0) [21]. Genomic sequences were assembled using the Flye program [22]. The genome sequences were deposited in the GenBank RefSeq database for automatic annotation. All genomes were processed in the BV-BRC platform [23], where genomic annotation was performed using the RAST toolkit (RASTtk). Phylogenomic analysis was conducted via the BV-BRC server's bacterial Phylogenetic Tree Service, constructing custom phylogenetic trees from user-selected genomes. The Codon Tree method employed single-copy BV-BRC PGFams and analyzed aligned coding DNA using RAxML [24]. The Comparative Systems Service, incorporating tools from the legacy PATRIC system, utilized a Protein Family Sorter [23, 25] to examine functional gene distribution across genomes (the “pan-genome”). Core genomes contain functional genes shared by all genomes, while accessory genomes include those shared by subsets. Pathway maps were generated via KEGG pathways [26] with default parameters. The average nucleotide identity values based on BLAST alignments (ANIb) from all pairwise genome comparisons were calculated using Pyani version 0.2.7, which is a Python module for calculating genomic metrics (https://github.com/widdowquinn/pyani) [27] Digital DNA–DNA hybridization (dDDH) was performed using the Genome Distance Calculator for Genome (GGDC; http://ggdc.dsmz.de/ ggdc. php), considering the recommended formula 2. Two genome sequences were considered to belong to the same species if the ANI and dDDH values were above 95–96% and 70%, respectively [28–30]. Genome-based analysis of chemotaxonomic markers Protein sequences of the respective genes, decaprenyl diphosphate synthase ( Dps ), 3-ketoacyl-CoA thiolase 2 (fadN-fadA-fadE operon), acyl-ACP phosphate acyltransferase ( PlsX ), and acyl-CoA thioesterase ( YciA ) were used to construct the phylogenetic topologies [12, 31-35]. The LG model (Le and Gascuel), an improved amino acid replacement matrix, was used for protein phylogenetics. Phylogenetic tree reconstruction was performed using the RaxML algorithm. Genome-based analysis of antimicrobial resistance The BV-BRC platform was used to analyze potential bacterial resistance to antibiotics [23]. The Genome Annotation Service in BV-BRC employs a k-mer-based approach to detect antimicrobial resistance (AMR) genes. This method leverages BV-BRC’s curated database of representative AMR gene sequence variants in BV-BRC. Each identified AMR gene was functionally annotated with its broad antibiotic resistance mechanism, the relevant drug class, and, in some cases, the specific antibiotic to which it confers resistance. nifHDK analysis Nitrogenase sequences, which comprise multiple subunits ( NifH, NifD , and NifK encoded by the nifHDK genes) — organized as an operon in these bacterial genomes —, were extracted from the set of genomes examined in this study using the BV-BRC/PATRIC database. The nucleotide sequences corresponding to nifH, nifD, and nifK were directly concatenated in order to use in alignments. Alignment was performed using JalView [36] and Clustal X [37]. The evolutionary model proposed for the tree topology [38] was the generalized time-reversible model of Tavaré from MEGA 11. A tree was generated from PhyML using the ML method. Mass spectrometry and fatty acid methyl ester (FAME) analysis The type strain candidates BR 11163 T , BR 11164 T , BR 11622 T , BR 11752 T , BR 11828 T , BR 11865 T , along with strains BR 11140 T and BR 11142 T were cultivated on DYGS and LGI solid media in Petri dishes at 30 °C for 24 and 48 h. An isolated colony from each strain was directly applied to a stainless steel MALDI plate to create a thin layer. Subsequently, it was promptly covered with 1 μL of a saturated α-cyano-4-hydroxy-cinnamic acid matrix (CHCA, 5 mg mL -1 in a solution of acetonitrile/water 50:50 with 0.1% trifluoroacetate) and air-dried before analysis. For external calibration, Escherichia coli DH5α was used as the standard strain, and each sample was spotted in quadruplicate to ensure reproducibility. Mass spectra were acquired using an Axima Performance iD Plus (Shimadzu Corp., integrated with the SARAMIS database) with a 60 Hz pulsed nitrogen laser. The analysis was conducted in the positive linear SARAMIS extraction mode, with a laser repetition rate of 50 Hz, five shots accumulated per profile, and a mass-to-charge ratio (m/z) of 2,000 to 20,000. Protein mass fingerprints for each colony were generated by accumulating 500 laser pulse cycles and were processed using the Shimadzu Biotech Launchpad software before export for analysis using the SARAMIS package. SuperSpectra were constructed by scrutinizing the mass spectra under multiple conditions for the same strains. This involved identifying consistent mass signals frequently appearing across the mass spectra and assigning weights to commonly agreed-upon peaks. Single-linkage cluster analysis was performed using SARAMIS parameters to produce a dendrogram of the Nitrospirillum strains (0.08% tolerance, range from 3,000 to 20,000 m/z). For FAME analyses, the strains BR 11163 T , BR 11164 T , BR 11622 T , BR 11752 T , BR 11828 T , BR 11865 T , along with strains BR 11140 T were grown on R2A medium [39] at 30 °C for 48 h. Fatty acid methyl esters were obtained from the fresh wet biomass according to the standard MIDI protocol and were separated, identified and quantified by GC (Agilent Technologies 7890B GC) with the Sherlock Microbial Identification System version 6.5 (MIDI) using the RTSBA6 database [40]. Morphology of colonies, cells, and physiological assays The strains BR 11163 T , BR 11164 T , BR 11622 T , BR 11752 T , BR 11828 T , and BR 11865 T , along with BR 11140 T and BR 11142T, were assessed through morphological and physiological assays. Their growth capabilities at different pH levels were evaluated by cultivating them on solid medium (LGI or modified FAM) at levels ranging from 5.0–8.0, followed by incubation for 5 days at 30 °C. To observe colony morphology, the bacteria were grown on homemade PDA medium and PDA medium supplemented with 10% crystal sugar and incubated for 7 days at 30 °C [8]. To assess cell morphology and pellicle formation in the medium, which are indicators of biological nitrogen fixation (BNF) activity, the bacteria were grown in 5 mL of nitrogen-free LGI semisolid medium at pH 7 in 10 mL glass flasks [8]. For micrography, bacterial samples were prepared following standard protocols [41] and subsequently visualized using Hitachi HT7800 transmission electron microscope set to an operating voltage of 100 kV. To confirm the BNF capability, bacteria grown in a semi-solid medium were evaluated for their ability to reduce acetylene. After 5 d of growth, the flasks were sealed with a chlorobutyl septum. One milliliter of gas was extracted from the headspace and replaced with acetylene. After 1 h of incubation, 1 mL of the headspace gas was directly injected into a gas chromatograph (Shimadzu 2010, CG-BID 2010 plus) coupled to a barrier discharge ionization detector (BID). The column (RTQBOND 0.53 mm ID; 20um; Cat 19742) was pre-conditioned for 0.5 min, the run temperature was 80 ºC for 6 min, with a detector temperature of 280 ºC and a helium flow rate of 50 mL/min. Ethylene production was estimated using a standard ethylene dose-response curve diluted with N 2 gas. Antibiotic sensitivity/tolerance tests were conducted using the Sensi-disc system (Cecon-Brazil) on a solid Dyg’s medium. The discs containing amoxicillin (10 µg), ampicillin (10 µg), rifampicin (15 µg), chloramphenicol (30 µg), azithromycin (15 µg), kanamycin (30 µg), gentamicin (10 µg), neomycin (30 µg), cephalexin (30 µg), penicillin (10 µg), tetracycline (30 µg), vancomycin (30 µg), streptomycin (10 µg), and erythromycin (25 µg) was used for tests. The discs were disposed on the culture medium after spreading 100 µL of the pre-inoculum obtained from bacterial growth in liquid DYGS medium, and plates were incubated at 28 °C for 4 days. Enzyme activities and substrate assimilation were evaluated by inoculating API 20NE strips (bioMérieux) and using the Biolog GNEIII kit (MicroPlate), following the manufacturer's instructions. Bacteria were cultured on DYGS solid medium [42] resuspended in a saline solution (0.85% NaCl) until the turbidity reached 4.0 on the McFarland scale, inoculated on the Biolog plates and then incubated for 36 h at 30 °C. Phenotypic data obtained using the Biolog kit were organized in a binary matrix, where 1 was considered a positive reaction and 0 was a negative reaction. Subsequently, the samples were grouped using the complete linkage and Pearson's distance measurement methods using the Heatmapper web service (http://www.heatmapper.ca/). Catalase activity was assessed by flooding a colony with 3% (v/v) H 2 O 2 and observing the presence of bubbles. The capacity to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, and zinc phosphate, production of indoles and siderophores, and cellulase activity were determined following methods described elsewhere [43]. Results and discussion 16S rRNA and recA phylogeny of the genus Nitrospirillum Initially, we investigated statistical methods capable of reinforcing the formation of different species from neutrality tests that evaluate genomic mutation rates. Our statistical analyses, aimed at interpreting signs of selection in our sequences using the Tajima method, revealed positive selection (>0) for both the 16S rRNA and recA genes. Positive selection as indicated by Tajima's D suggests an excess of intermediate-frequency alleles, which may result from population bottlenecks, structure, and/or balancing selection. Such evidence can be interpreted as indicative of a reduction in population size, balancing selection and sudden population contraction (additional files). These statistical values offer valuable biological insights, considering that the sequences do not fit the neutral theory model. Moreover, hypotheses can be formulated regarding the potential for excessive non-synonymous divergence among sequences. This aids in comprehending the evolutionary patterns of consistency across our samples, highlighting species differences and limited polymorphisms, and elucidating the topological patterns in our phylogenies [4,45]. The 16S rRNA gene phylogeny indicated that the 86 strains in this study were distributed among three main clades, with approximately 98.8% similarity within the genus Nitrospirillum (Fig. S1; Table S7). One of these clades was specific to the strain DSM 22198 T of N. iridis , and the second clade comprised 31 strains, including the strain BR 11142 T of N. amazonense . The third clade included the remaining new strains and BR 11140 T , the type strain of N. viridazoti (Fig. S1). The new strains were distributed across six clusters, each containing 3–46 strains, and exhibiting a similarity of more than 90.5% within each cluster. All these groups maintained distance from the three known Nitrospirillum species, except for strains BR 11169, BR 11162, and BR 11163 T , which positioned themselves close to strain BR 11142 T (Fig. S1). This clustering indicated the presence of new taxa; however, owing to the high conservation of the 16S rRNA sequences widely reported in the literature, it was not suitable for species-level inferences, including the genus Nitrospirillum [46]. In addition to the 16S rRNA gene phylogeny, we analyzed the sequences of the recA gene. Overall, this analysis provided greater resolution than the 16S rRNA analysis, with a more pronounced separation among the six clusters while keeping the same strains grouped (Fig. S2). All six clusters mentioned above were distant from the type strains BR 11140 T , BR 11142 T , and DSM 22198 T (Fig. S2). Notably, the group of strains BR 11169, BR 11162, and BR 11163 T , which were very close to BR 11142 T of N. amazonense in the 16S rRNA gene analysis, formed a distinct cluster in the recA analysis (Fig. S2). To obtain a better view of the strain phylogeny, taking into account that the topologies of the 16S rRNA and recA trees were similar, we concatenated those sequences (Fig. 1). The tree shows, as already observed in individual analyses, the new strains formed six distinct groups. Two clusters (highlighted in orange and brown) comprised three and four strains, respectively, isolated from Rio de Janeiro State. These clusters were positioned distantly from any type strain of the described species (Fig. 1). The third cluster (highlighted in purple) grouped 10 strains isolated from the States of Bahia, Rio de Janeiro, and Goiás. This cluster showed approximately 99% similarity with N. amazonense BR 11142 T . Similarly, the remaining three groups formed clades separated from the type strains of known species (highlighted in green, blue, and red), with a similarity below 99% among the clusters (Fig. 1). The strains of these three clusters were isolated from the states of Rio de Janeiro and Goiás and showed a broad distribution, especially for the cluster highlighted in red, which contained 46 strains. Although phylogenetic analyses of 16S rRNA and recA alone may not be sufficient for prokaryotic species circumscription, they can provide consistent information that aligns with genome-based analyses, serving as an initial source of information about a set of strains. Therefore, to confirm that the six distinct phylogenetic groups observed in the 16S rRNA and recA analyses represent new taxa, we proceeded with subsequent genomic analyses. Taxonomic distribution of sequenced genomes The genomes of six candidate strains were examined: BR 11163 T , BR 11164 T , BR 11622 T , BR 11752 T , BR 11828 T , and BR 11865 T . The general characteristics of these genome assemblies are listed in Table 1. All sequencing coverages (≥90X), completeness (>99.46%) and contamination (<1.31) were above the proposed minimal standard for taxonomic purposes [47, 48]. In general, the genome length of the new strains ranges between 6.3 and 7.1 Mb, with the GC content varying between 67.0% and 68.0%. These values are similar to those of the genomes of strains BR 11142 T and BR 11140 T that represent the formally described species N. amazonense , N. iridis , and N. viridazoti (Table 1). The topological arrangement of our phylogenomic tree showed evolutionary convergence among our new strains and the type strain of previously described species N. viridazoti and N. amazonense, whereas strain DSM 22198 T of N. iridis appeared to be evolutionarily divergent from our samples (Fig. 2). We discuss the phylograms for the two cases. In case 1, the BR 11865 T genome shared an evolutionary origin with BR 11142 T of N. amazonense species. In contrast, the BR 11164 T and BR 11163 T genomes showed an evolutionary connection with common ancestors of BR 11140 T of the N. viridazoti species. The genomes of the other strains (BR 11622 T , BR 11752 T , and BR 11828 T ) formed two distinct phylogenomic clades, indicating evolutionary divergence without genetic sharing with the discussed species. These findings align with the established concepts of species formation, emphasizing anagenesis in case 1 and cladogenesis in case 2. Previous studies have demonstrated how these concepts help determine species formation and understand evolutionary convergence and divergence [45]. To catalog the functional variability across our bacterial populations, whole functional genome analyses were performed to measure the representation of the entire collection of functional genomic sequences in our genome set (Fig. 3). The whole functional genome comprised 4453 protein families representing the pangenome, of which 2178 belong to the core. Unique protein families are shown next to each strain. The whole functional genome was composed of functional genes related to the metabolism of sulfur, nitrogen, and amino acids, whereas DNA and RNA processing functions were shared among all analyzed genomes. Finally, cell signaling and regulation as well as stress responses, defense, and virulence belonged to the most specific functional sequences of each genome. The ANIb percentages for the strain DSM 22198 T of N. iridis and strains BR 11163 T , BR 11164 T , BR 11622 T , BR 11752 T , BR 11828 T , and BR 11865 T were consistently less than 85% (Table 2). Similarly, a comparison between strain BR 11140 T and the new strains yielded values less than 94.1%. In another set of comparisons, the same strains against BR 11142 T of N. amazonense resulted in values consistently lower than 93.5%, except for strain BR 11185 T , which was 94.0% (Table 2). Comparisons among our new strains indicated that BR 11163 T , BR 11164 T , BR 11828 T , and BR 11865 T consistently exhibited ANI values less than 92.5% compared to all other strains (Table 2). In contrast, strains BR 11752 T and BR 11622 T showed values less than 93% in most comparisons and less than 95.5% in the comparison between themselves (Table 2). The dDDH values between the new strains and the type strains of N. amazonense , N. iridis , and N. viridazoti were less than 60.1% (Table 2). This maximum value was also observed among the new strains, except for the comparison between strains BR 11752 T and BR 11622 T , for which the value was 66.0% (Table 2). ANI and dDDH have emerged as valuable tools for delineating bacterial species, offering more precise and reproducible alternatives to traditional methods based on single genes. ANI calculates the average nucleotide identity between two genomic sequences, with values above a certain threshold (typically 95–96%) commonly considered indicative of species boundaries [27, 28]. Similarly, dDDH estimates the DNA–DNA hybridization based on genomic comparisons and provides a numerical measure of genomic relatedness between bacterial strains. Generally, dDDH values greater than 70% indicate species boundary [29]. Both ANI and dDDH have been widely utilized in bacterial taxonomy, contributing to a more accurate and standardized approach for defining bacterial species. Therefore, our results, considering the phylogeny of 16S rRNA, recA , phylogenomic, MALDI-TOF profile, ANI values, and dDDH, confirmed that six new species were identified within the genus Nitrospirillum . The strains BR 11865 T , BR 11163 T , BR 11164 T , BR 11828 T , BR 11752 T , and BR 11622 T were designated as type strains of the new species that we named N. bahiense , N. guanabarense , N. guaranorum , N. karajorum , N. guiasense, and N. pindoramense, respectively. Chemotaxonomic markers Chemotaxonomic characterization has largely been replaced by genomic analysis for species delineation, although it remains valuable for genus characterization. Previous studies on Nitrospirillum species, including N. amazonense and N. iridis , show a predominance of the fatty acids C18:1 ω7c, C18:1 2-OH, and C16:0 3-OH. Polar lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyldimethylethanolamine, with ubiquinone Q10 being the major quinone [7, 46]. Here, we tested an alternative to traditional laboratory methods such as in silico chemotaxonomy. This approach involves the analysis of gene sequences involved in the synthesis and metabolism of fatty acids, polar lipids, and ubiquinone [32-35]. The analyzed DNA sequences focused on genes related to ubiquinone side chain synthesis (decaprenyl diphosphate synthase), fatty acid synthesis (acyl-CoA thioesterase), fatty acid metabolism and elongation (the fadN-fadA-fadE operon), and phospholipid synthesis (acyl-ACP phosphate acyltransferase) (Table S3). Phylogenetic trees were constructed using the protein sequences of the respective genes (Fig. S4) showed, in general, topologies in accordance with phylogenetic-based 16S rRNA and recA and the phylogram based on the whole genome (Fig. 1, Fig. 2, Fig. S1, Fig S2). As reported previously [12]. the analysis of chemotaxonomic marker genes showed that the type strains of N. amazonense and N. viridazoti generally diverged from each other and were positioned apart of strain DSM 22198 T of N. iridis and far from strain BR 11142 T of A. brasilense , which was used as an out-group (Fig. S4). These new strains consistently formed unique clades, particularly those related to ubiquinone production, fatty acid metabolism, and phospholipid synthesis (Fig. S4 A, B, D). For the four genes analyzed, the new strains formed three groups with greater or lesser similarity: i) the strains BR 11828 T , BR 11622 T , and BR 11752 T ; ii) BR 11163 T and BR 11164 T ; and iii) BR 11865 T , which tended to be grouped with strains BR 11140 T and BR 11142 T , except for the genes related to phospholipid synthesis ( YciA ), where BR 11140 T of N. viridazoti stands out (Fig. S4). Therefore, the analysis of chemotaxonomic marker genes was robust for confirming new Nitrospirillum species and corroborated the results of other phylogenetic and phylogenomic analyses. nifHDK phylogenetic analysis Considering the results obtained from the pan-core accessory genome steps, we conducted further analyses to clarify these results. Specifically, we explored the distribution of genes associated with the oxygen-sensitive nitrogenase complex, focusing on molybdenum nitrogenase. Molybdenum nitrogenase, encoded by nifH , nifD , and nifK genes is the most prevalent protein and is shared across all genomes. Phylogenetic analyses point to a functional relationship between Nitrospirillum genomes, considering their nitrogen fixation activities. The phylogeny of the nifHDK sequences correlated with the results obtained from the phylogenomic analysis, indicating the formation of the same phylogenetic clades. The only exception was the strain BR 11164 T , whose nifHDK genes were closer to DSM 22198 T , which was isolated in South Korea (Fig. 4). This was surprising, as the strain BR 11164 T was isolated in the same geographic area as the other new strains. Mass spectrometry and fatty acid analysis Direct colony application demonstrated high spectral quality and reproducibility. Furthermore, we observed high consistency between replicates for each strain under identical conditions. Although variations in the composition and characteristics of the culture medium and the bacterial growth time led to subtly different spectral profiles for the same strains, these distinctions did not significantly affect their overall identification. Consequently, there was no need for additional steps such as culture broth handling or protein extraction methods, as mentioned in other studies [49]. MALDI-TOF MS analysis generated distinct mass spectra of whole-cell proteins for all the studied strains, revealing 39–41 well-defined peaks of high quality (Fig. 5; Fig. S3). These peaks vary from 3,097.9 to 11,352.3 in mass. Using these protein mass profiles, a cladogram was constructed, which revealed that all strains shared similarity among no more than 85% (case of the strains BR 11662 T , BR 11828 T , and BR 11752 T ) or less than 75% in the case of other strains, including BR 11142 T of N. amazonense and BR 11140 T of N. viridazoti (Fig. 5). The observed similarity level confirms that the strains represent new taxa, [50] and the result corroborated the positioning of the strains in different groups in the phylogenetic 16S rRNA and recA tree and the phylogenomic tree (Fig. 1 and 2). The fatty acid profiles of our strains showed notable similarity of new strains with the strain BR 11140ᵀ, which was analyzed simultaneously. Summed Feature 8, which includes C 18:1 ω 7c and/or C 18:1 ω 6c, represented the dominant fraction in the profiles of all strains (ranging from 40.2% to 44.6%) (Table S99), like what has been previously reported for DSM 22198ᵀ ( N. iridis ) and BR 11142ᵀ ( N. amazonense ) [7]. Furthermore, C 18:1 2OH was consistently present in all strains, as were C 16:1 ω 5c (5.0%–6.9%) and C 16:0 3OH (5.2%–10.3%). Notably, strain BR 11865ᵀ exhibited the most divergent profile, with a substantially higher percentage of C 16:1 ω 5c (11.28%) and C 16:0 3OH (10.31%), and a lower level of C 16:0 (3.14%) compared to the other strains (Table S1). Altogether, the chemotaxonomic results clearly indicate that the proposed type strains for the new species exhibit a fatty acid composition consistent with that observed for members of the genus Nitrospirillum . Morphology of colonies, cells, and physiological assays All six candidate type strains of the new species grew at the tested pH levels in solid LGI medium, although more abundant growth was observed between pH 5 and 8. We did not thoroughly test different temperatures for the growth of these strains, but previous studies have shown that strains affiliated with the genus Nitrospirillum have an optimal growth temperature between 30 and 32 °C, and they can grow often at higher temperatures [15]. Analysis of the colony morphology on homemade potato agar medium [8] revealed that the strains formed white, circular, large, and flat colonies with raised margins. Most of the strains exhibited agglutinated, wrinkled colonies, a characteristic similar to that observed in N. amazonense (Fig. S5). However, this characteristic was not observed in the strains BR 11622 T , BR 11752 T , and BR 1182 T , which displayed slightly wet and elevated colonies. When the strains were grown on potato agar medium supplemented with 10% sucrose, strains BR 11164 T and BR 11752 T appeared to be less affected by the high sugar concentrations (Fig. S5). In contrast, strains BR 11163 T , BR 11622 T , BR 11828 T , and BR 11865 T exhibited moderate growth but formed smaller colonies. Strain BR 11140 T showed poor growth, with small colonies, whereas strain BR 11142 T produced only a few small colonies in the presence of 10% sugar, as previously observed [12]. Comparative analysis of cell morphology revealed that most of the new strains were curved or highly curved, spiral, or vibroid, and accumulated significant amounts of polyhydroxybutyrate (PHB) when grown in nitrogen-free LGI semi-solid medium (Fig. S6). BR 11865 T had longer spiral cells, whereas BR 11163 T had larger spiral cells. Transmission electron micrography also revealed a large accumulation of PHB in the cells of strains BR 11163ᵀ, BR 11164ᵀ, BR 11622ᵀ, BR 11752ᵀ, BR 11828ᵀ, and BR 11865ᵀ, as was also observed for strains BR 11142ᵀ and BR 11140ᵀ (Fig. S8). All analyzed strains exhibited polar monotrichous flagella and cell sizes ranging from 1.3 to 1.8 μm, which were generally slightly smaller than the sizes observed for strains BR 11142ᵀ and BR 11140ᵀ (Fig. S8). Overall, the new strains formed a characteristic veil-like pellicle on the subsurface of the semisolid LGI medium. Strains BR 11163 T , BR 11622 T , and BR 11142 T slightly acidified the medium, whereas strain BR 11865 T caused strong acidification unlike the other strains, which maintained a neutral pH (Fig. S7). The pellicle thickness varied among the strains, with strain BR 11164 T and BR 11865 T producing thicker pellicles than BR 11140 T . In contrast, the other strains, including strain BR 11142 T , showed thinner veil-like pellicles on the subsurface of the medium (Fig. S7). The formation of a pellicle on the subsurface of the semi-solid medium is reportedly an indicator of the capacity for BNF through nitrogenase activity [51]. To confirm this activity in the new Nitrospirillum strains, an assay was conducted to evaluate the ability to reduce acetylene, given that nitrogenase can reduce acetylene to ethylene [51]. It was observed that all six new candidate type strains, in addition to the strains BR 11140 T , BR 11142 T , and DSM 22198 T , produced between 496.2 and 648.6 nmol of C 2 H 4 mL -1 h -1 (Table S4), confirming the diazotrophic capacity of all strains. The amount of ethylene produced was similar to previously reported values for this type of assay with diazotrophic associative bacteria [52]. All tested strains tolerated chloramphenicol, azithromycin, tetracycline, and vancomycin and were sensitive to penicillin. The strain BR11865 T was sensitive to kanamycin, gentamicin, neomycin, and streptomycin, whereas strains BR 11140 T and BR 11142 T were not. The strains BR 11163 T and BR 11164 T were the most tolerant and differed from each other because BR 11163 T was tolerant to azithromycin. The strains BR 11828 T , BR 11622 T , and BR 11752 T were the most sensitive strains, with only the strain BR 11622 T and BR 11752 T being sensitive to amoxicillin, and only the latter being sensitive to ampicillin (Table S5). Bacterial antibiotic resistance investigated through the k-mer-based approach showed that only the gene sequences corresponding to the antimicrobial resistance (AMR)mechanism for efflux pumps varied depending on the genome analyzed. Other genes were present in all the strains, indicating antibiotic resistance targets (Table S6). The presence of AMR-related genes (even full-length genes) in each genome does not directly imply an antibiotic-resistant phenotype. Transmembrane proteins expel a wide range of toxic substances, including antibiotics, from the cell, thereby reducing the intracellular concentration of the drug and consequently its effectiveness [53]. In bacteria of the genus Nitrospirillum , which are important in terrestrial environments, particularly in nitrogen fixation, antibiotic resistance mediated by efflux pumps can be a critical adaptation that allows them to survive in soils contaminated with antimicrobial compounds [54]. Regarding source usage/tolerance, all strains showed a positive reaction in 25 sources tested by the API and Biolog kits, indicating that they could assimilate the carbon source or other nutrients, or were not inhibited by them. In contrast, none of the strains reacted in 16 tests. Thus, variability in responses was observed for more than 50 tests using the Biolog kit. Strain BR 11162 T showed the highest number of positive reactions (69 in total), followed by strain BR 11164 T . The other strains showed between 38 and 61 positive reactions. All strains were able to grow at pH 5–8, tolerate 1% sodium lactate, and only strains BR 11865 T and BR 11164 T did not grow in the presence of 1% NaCl; none of them showed a positive reaction at concentrations of 4 and 8% NaCl (Fig. S5). The heatmap generated using the Biolog test reaction data indicated that the tested strains had different phenotypic fingerprints (Fig. S8). Strains BR 11865 T , BR 11164 T , and BR 11622 T formed a cluster further away from BR 11142 T and BR 11140 T . In contrast, strains BR 11828 T , BR 11163 T , and BR 11752 T also formed a cluster. However, with greater similarity to strains typical of N. amazonense and N. viridazoti (Fig. S8). This analysis showed a slight discrepancy between the 16S rRNA and recA analyses. Strain BR 11865 T and BR 11164 T showed greater similarity to BR 11142 T and BR 11140 T than to BR 11828 T and BR 11752 T (Fig. S8). Overall, the enzymatic activities evaluated by the API kit were very similar among all new strains, with the only difference being strains BR 11164 T and BR 11865 T , which were negative for arginine dihydrolase. In contrast, the other strains were positive (Table S5). All new strains were positive for urease, catalase, and oxidase, which are typical enzymes for bacterial phenotypic characterization studies, whereas only strain BR 11622 T tested positive for cellulases (Table S5). None of the proposed type strains solubilized calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, or zinc phosphate. However, all strains exhibited siderophore production capacity, a trait indicated by halos in the culture medium, including strains BR 11142 T and BR 11140 T of the N. amazonense , and N. viridazoti , respectivelly It was also observed that all strains showed indole acetic production, as observed for strains BR 11142 T and BR 11140 T . In this characteristic, strain BR 11164 T stood out, producing more than 17 µg/mL indole compounds in 72 h (Table S5). Notably, in the API test analysis, indole production (L-tryptophan test) was negative for all strains, indicating the need for further investigation. Conclusion In conclusion, this study contributes to our understanding of bacterial diversity within the genus Nitrospirillum . The taxonomic framework based on phylogenetic and phylogenomic analyses provides a comprehensive view of the genus Nitrospirillum evolution. This genus is distributed across diverse biomes, including the Amazon, Atlantic Forest, and Cerrado, and possesses the ability to colonize dozens of plant species, with a particular focus on Poaceae, which is the most studied genus to date. ANI and dDDH support the proposition of six new species within the genus Nitrospirillum : N. bahiense , N. guanabarense , N. guaranorum , N. karajorum , N. guiasense and N. pindoramense . Analyses of nifHDK genes, mass spectrometry profiles, FAME and physiological tests confirmed the distinctiveness of the proposed species and its ability to fix nitrogen in vitro . These findings expand our systematic understanding of bacterial classification and demonstrate the critical role of preserving and investigating microbial repositories to elucidate prokaryotic diversity. The biotechnological potential of this genetic resource is elucidated based on the results presented here. Description of Nitrospirillum bahiense sp. nov. Nitrospirillum bahiense (ba.hi.en’se. N.L. neut. adj. bahiense , in reference to the state of Bahia, Brazil, in honor of its cultural and historical importance). This species displays major characteristics of the genus. The cells are Gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. Optimum temperature for growth is approximately 30 °C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11865 T (UCCCB 233 T ) is resistant to amoxicillin, ampicillin, chloramphenicol, azithromycin, penicillin, tetracycline, and vancomycin and sensitive to rifampicin, kanamycin, gentamicin, neomycin, cephalexin, streptomycin, and erythromycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, sucrose, α -D-glucose, D-mannose, D-galactose, L-fucose, and others, but does not grow in D-raffinose, D-serine, and D-sorbitol. The enzymatic tests for the type strain showed positive reaction to glucokinase, urease, β-glucosidase, catalase, and oxidase, and negative reactions to nitrate reduction. The ability to produce ethylene, indoles, and siderophores was observed, but the capability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, and zinc phosphate was not observed. The cell size of strain BR 11865 T ranges from 1.7 to 1.8 μm. Strains of N. bahiense were isolated from the roots Brachiaria humidicola , B. brizantha, and Zea mays . The species were distinguishable by molecular and genomic approaches, and via MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11865 T was isolated from the roots of B. humidicola in Bahia, Brazil. The genome of the type strain has a size of 7.0 Mbp with a DNA G+C content of 67.5%. Description of Nitrospirillum guanabarense sp. nov. Nitrospirillum guanabarense (gua.na.bar.en’se. N.L. neut. adj. guanabarense , named after the indigenous word for the city of Rio de Janeiro, Brazil, honors the cultural and historical significance of the term). This species displays major characteristics of the genus. The cells are Gram-negative and non-sporulating. All strains grow well on LGI and DYGS media. Optimum temperature for growth is approximately 30 °C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11163 T (UCCCB 228 T ) is resistant to amoxicillin, ampicillin, rifampicin, chloramphenicol, cephalexin, penicillin, and erythromycin and is sensitive to azithromycin, kanamycin, gentamicin, neomycin, vancomycin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, sucrose, α -D-glucose, D-mannose, D-galactose, L-fucose, and D-fructose, and tolerate 1% NaCl. The enzymatic tests for the type strain showed a positive reaction to glucokinase, urease, β-glucosidase, catalase, and oxidase, and a negative reaction to nitrate reduction. The ability to produce ethylene, indoles, and siderophores was observed for the type strain but not to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, or zinc phosphate. The cell size of strain BR 111163 T ranges from 1.2 to 1.5 μm. Strains of N. guanabara were isolated from the roots of Sorghum vulgare . The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. Type strain BR 11163 T was isolated from the roots of Sorghum vulgare in Rio de Janeiro, Brazil. The genome of the type strain has a size of 7.1 Mbp with a DNA G+C content of 67.6%. Description of Nitrospirillum guaranorum sp. nov. Nitrospirillum guaranorum ( gua.ra.no ’rum. N.L. gen. pl. n. guaranorum , named in honor of the Guarani people, the principal indigenous population originating from the Southeast of Brazil). This species displays major characteristics of the genus. The cells are Gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. Optimum temperature for growth is approximately 30 °C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11164 T (UCCCB 229 T ) is resistant to amoxicillin, ampicillin, rifampicin, azithromycin, chloramphenicol, cephalexin, penicillin, and erythromycin and sensitive to kanamycin, gentamicin, neomycin, vancomycin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, sucrose, α -D-glucose, D-mannose, D-galactose, L-fucose, D-fructose, α -D-lactose, and D-turanose. The enzymatic tests for the type strain show a positive reaction to glucokinase, urease, β-glucosidase, catalase, and oxidase, and a negative reaction to nitrate reduction and cellulase. The ability to produce ethylene, indoles, and siderophores was observed, but not the capability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, and zinc phosphate. The cell size of strain BR 11164 T ranges from 1.5 to 1.7 μm. Strains of N. guaiasensis have been isolated from the roots Oryza sativa . The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11164 T was isolated from the roots of Oryza sativa in Rio de Janeiro, Brazil. The genome of the type strain has a size of 7.0 Mbp with a DNA G+C content of 68.2%. Description of Nitrospirillum karajorum sp. nov. Nitrospirillum karajorum (ka.ra.jo’rum. N.L. gen. pl. n. karajorum of the Karajá people, in reference to the indigenous Karajá people, longstanding inhabitants along the Araguaia River with territorial occupations across the states of Goiás, Tocantins, Mato Grosso, and Pará). This species displays major characteristics of the genus. The cells were Gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. Optimum temperature for growth is approximately 30 °C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11752 T (UCCCB 231 T ) was resistant at tested levels to penicillin, tetracycline, vancomycin, and erythromycin and sensitive to amoxicillin, ampicillin, rifampicin, chloramphenicol, azithromycin, kanamycin, gentamicin, neomycin, cephalexin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, α -D-glucose, D-mannose, D-galactose, L-fucose, D-fructose, acetic acid, and pectin, and tolerate 1% NaCl. The enzymatic tests for the type strain showed positive reaction to nitrate reduction, glucokinase, urease, β-glucosidase, arginase, citrase, β-galactosidase, and a negative reaction to cellulase. The ability to produce ethylene, indoles, and siderophores was observed for the strain BR 11752 T , but its ability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, and zinc phosphate was not observed. The cell size of strain BR 11752 T ranges from 1.4 to 1.6 μm. Strains of N. karajorum have been isolated from the roots Oryza sativa . The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11752 T was isolated from the roots of Oryza sativa in Rio de Janeiro, Brazil. The genome of the type strain has a size of 6.5 Mbp and DNA G+C content of 67.3l%. Description of Nitrospirillum guiasense sp. nov. Nitrospirillum guiasense (gu.ias.en’se. N.L. neut. adj. guiasense about the Guaiás indigenous tribe, which originated from the name Goiás state, Brazil, in honor of the cultural and historical significance of this term). This species displays major characteristics of the genus. The cells were Gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. Optimum temperature for growth is approximately 30 °C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11828 T (UCCCB 232 T ) was resistant to ampicillin, penicillin, and amoxicillin, and sensitive to tetracycline, vancomycin, erythromycin, rifampicin, chloramphenicol, azithromycin, kanamycin, gentamicin, neomycin, cephalexin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, α -D-glucose, D-mannose, D-galactose, L-fucose, D-fructose and D-glucuronic acid, and tolerate 1% NaCl. The enzymatic tests for the type strain showed positive reaction to nitrate reduction, glucocinase, urease, β-glucosidase, arginase, citrase, β-galactosidase, and a negative reaction to cellulase. The ability to produce ethylene, indoles, and siderophore was observed, but not the capability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, or zinc phosphate. The cell size of strain BR 11828 T ranges from 1.5 to 1.8 μm. Strains of N. guaiasensis have been isolated from the roots Oryza sativa . The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11828 T was isolated from the roots of Oryza sativa in Rio de Janeiro, Brazil. The genome of the type strain has a size of 6.3 Mbp with a DNA G+C content of 67.7%. Description of Nitrospirillum pindoramense sp. nov. Nitrospirillum pindoramense (pin.do.ra.men’se. N.L. neut. adj. pindoramense , derived from the term “Pindorama”, which translates to "Land of Palm Trees" in reference to how indigenous people referred to the lands of Brazil before the arrival of the Portuguese) This species displays major characteristics of the genus. The cells were gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. The optimum temperature for growth is approximately 30 °C, and the optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11622 T (UCCCB 230 T ) is resistant to penicillin, ampicillin, tetracycline, vancomycin, and erythromycin and sensitive to vancomycin, erythromycin, amoxicillin, rifampicin, chloramphenicol, azithromycin, kanamycin, gentamicin, neomycin, cephalexin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, α -D-glucose, D-mannose, D-galactose, L-fucose, D-fructose, and acetic acid, and tolerate 1% NaCl. The enzymatic tests for the type strain showed a positive reaction to nitrate reduction, glucocinase, urease, β-glucosidase, citrase, β-galactosidase, arginase, and a negative reaction to cellulase. The ability to produce ethylene and indoles was observed, but not the capability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, or zinc phosphate. Strains of N. pindoramensis have been isolated from the roots Oryza sativa , Zea mays, and Brachiaria brizantha . The cell size of strain BR 11622 T ranges from 1.4 to 1.6 μm. The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11622 T was isolated from the roots of Zea mays in Rio de Janeiro, Brazil. The genome of the type strain has a size of 6.6 Mbp with a DNA G+C content of 67.2%. Declarations Ethics approval and consent to participate The authors have declared that no ethical issues exist. Consent for publication Not applicable. Availability of data and materials Sequence data that support the findings of this study have been deposited in the National Center for Biotechnology - NCBI. Information and Data is provided within the supplementary information files. Competing Interests The authors declare that there are no competing of interest. Funding Embrapa (Brazilian Agricultural Research Corporation; INCT Plant Growth–Promoting Microorganisms for Agricultural Sustainability and Environmental Responsibility (CNPq 465133/2014-2, Fundação Araucária-STI-043/2019, CAPES); CNPq Productivity Grant for some of researchers and FAPERJ (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro), projects E-26/201.074/2022 and E-26/210.303/2021. Authors' contributions JEZ coordinated the project, analyzed, and wrote the paper; SS, AFO, and EB performed the genomic analyses and assisted in writing the manuscript; NSF collaborated on DNA sequencing; JLSA, BJRA, ALS, and VMR collaborated on the nitrogen fixation test; LFWR collaborated on genome sequencing; FSD and KMOL collaborated on DNA sequencing and MALDI-TOF MS analyses; CNR collaborated on the phenotypic analysis; LHBS collaborated on MALDI-TOF MS analyses; and JIB collaborated on the phenotypic analysis and writing review. Acknowledgements We thank CNPq (Brazilian National Council for Scientific and Technological Development) for productivity grants awarded to some authors, CAPES (Coordination of Superior Level Staff Improvement) for student grants, and FAPERJ for Grant Program Cientista do Nosso Estado. We also thank Dr. Aharon Oren and Dr. Bernhard Schink for reviewing and suggesting new species names, Dr. Alane Vermelho for helping with the transmission electronic images and Dr. Paula Morais for helping with FAME analyzes. Ethical Approval Not applicable. References Soumare A, Diedhiou AG, Thuita M, Hafidi M, Ouhdouch Y, Gopalakrishnan S, et al. Exploiting biological nitrogen fixation: A route towards a sustainable agriculture. Plants. 2020;9. https://doi.org/.3390/plants9081011 Mohanty P, Singh PK, Chakraborty D, Mishra S, Pattnaik R. Insight into the role of PGPR in sustainable agriculture and environment. Frontiers in Sustainable Food Systems. 2021;5. https://doi.org/10.3389/fsufs.2021.667150. Nam JH, Thibodeau A, Qian YL, Qian MC, Park SH. Multidisciplinary evaluation of plant growth promoting rhizobacteria on soil microbiome and strawberry quality. AMB Express. 2023;13. https://doi.org/10.1186/s13568-023-01524-z. Santos MS, Nogueira MA, Hungria M. Outstanding impact of Azospirillum brasilense strains ab-v5 and ab-v6 on the Brazilian agriculture: Lessons that farmers are receptive to adopt new microbial inoculants. Rev Bras Cienc Solo. 2021;45. https://doi.org/10.36783/18069657RBCS20200128. Reis VM, Rios FA, Braz GBP, et al. Agronomic performance of sugarcane inoculated with Nitrospirillum amazonense (BR 11145). Revista Caatinga. 2020;33. https://doi.org/10.1590/1983-21252020v33n406rc. Magalhães FM, Baldani JI, Souto SM, Kuykendall JR, Döbereiner J. A new acid-tolerant Azospirillum species. An Acad Bras Cienc. 1983;55:417–30. Lin SY, Hameed A, Shen FT, Liu YC, Hsu YH, Shahina M, Lai WA, Young CC. Description of Niveispirillum fermenti gen. nov., sp. nov., isolated from a fermentor in Taiwan, transfer of Azospirillum irakense (1989) as Niveispirillum irakense comb. nov., and reclassification of Azospirillum amazonense (1983) as Nitrospirillum amazonense gen. nov. A Van Leeuwenhoek. 2014; 105. https://doi.org/10.1007/s10482-014-0176-6. Baldani JI, Reis VM, Videira SS, Boddey LH, Baldani VLD. The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists. Plant Soil. 2014;384. https://doi.org/10.1007/s11104-014-2186-6. Magalhães FMM. Nitrogen fixing bacteria isolated from diverse soils and grass roots in Amazônia. In: Vose P., Ruschel A. P., editors. Associative N 2 -Fixation. New York: CRC Press; 1981. p. 39–48. Reis FB, Silva MF, Tekeira KRS, Urquiaga S, Reis VM. Identification of Azospirillum amazonense isolates associated to Brachiaria spp. at different stages and growth conditions, and bacterial plant hormone production. Rev Bras Cienc Solo. 2004;28. https://doi.org/10.1590/s0100-06832004000100011. Hartmann A, Baldani JI. The Genus Azospirillum. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes: Volume 5: Proteobacteria: Alpha and Beta Subclasses. New York, NY: Springer New York; 2006. p. 115–40. Baldani JI, dos Santos Ferreira N, Shwab S, Reis VM, de Barros Soares LH, Simões-Araujo JL, et al. Nitrospirillum viridazoti sp. nov., an Efficient nitrogen-fixing species isolated from grasses. Curr Microbiol. 2024;81:144. https://doi.org/10.1007/s00284-024-03665-1. Reis VM, Rios FA, Braz GBP, Constantin J, Hirata ES, Biffe DF. Agronomic performance of sugarcane inoculated with Nitrospirillum amazonense (BR 11145). Revista Caatinga. 2020;33. https://doi.org/10.1080/07352680091139213. Magalhães FMM, Döbereiner J. Ocorrência de Azospirillum amazonense em alguns ecossistemas da Amazônia. Rev Microbiol. 1984;15:246–52. Baldani JI, Baldani VLD. History on the biological nitrogen fixation research in graminaceous plants: Special emphasis on the Brazilian experience. Anais da Academia Brasileira de Ciencias. 2005;77. https://doi.org/10.1590/S0001-37652005000300014. Gaunt MW, Turner SL, Rigottier-Gois L, Lloyd-Macgilp SA, Young JPW. Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int J Syst Evol Microbiol. 2001;51. https://doi.org/10.1099/00207713-51-6-2037. Tamura Koichiro, Glen Stecher, Kumar Sudhir. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38, 3022-3027. https://doi.org/10.1093/molbev/msab120. Jukes TH, Cantor CR. Evolution of Protein Molecules. New York: Academic Press. 1969. pp. 21–132. Tamura K. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol Biol Evol. 1992;9. https://doi.org/10.1093/oxfordjournals.molbev.a040752. Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989;123. https://doi.org/10.1093/genetics/123.3.585. De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. NanoPack: Visualizing and processing long-read sequencing data. Bioinformatics. 2018;34. https://doi.org/10.1093/bioinformatics/bty149. Kolmogorov M, Bickhart DM, Behsaz B, Gurevich A, Rayko M, Shin SB, et al. metaFlye: scalable long-read metagenome assembly using repeat graphs. Nat Methods. 2020;17. https://doi.org/10.1038/s41592-020-00971-x. Olson RD, Assaf R, Brettin T, Conrad N, Cucinell C, Davis JJ, et al. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res. 2023;51 1 D. https://doi.org/10.1093/nar/gkac1003. Stamatakis A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30. https://doi.org/10.1093/bioinformatics/btu033. Overbeek R, Begley T, Butler RM, Choudhuri J V., Chuang HY, Cohoon M, et al. The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 2005;33. https://doi.org/10.1093/nar/gki866. Kanehisa M, Goto S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Research. 2000;28. https://doi.org/10.1093/nar/28.1.27. Pritchard L, Glover RH, Humphris S, Elphinstone JG, Toth IK. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. Anal Methods. 2016;8:12–24. doi:10.1039/c5ay02550h. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 2007;57. https://doi.org/10.1099/ijs.0.64483-0. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013;14. https://doi.org/10.1186/1471-2105-14-60. Hayashi Sant’Anna F, Bach E, Porto RZ, Guella F, Hayashi Sant’Anna E, Passaglia LMP. Genomic metrics made easy: what to do and where to go in the new era of bacterial taxonomy. Critical Reviews in Microbiology. 2019;45. https://doi.org/10.1080/1040841X.2019.1569587. Yamano R, Yu J, Jiang C, Harjuno Condro Haditomo A, Mino S, Sakai Y, Sawabe T. Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea gen. nov. PloS one. 2022; 17:e0271174. https://doi.org/10.1371/journal.pone.0271174. Volodina E, Steinbüchel A. (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB') from fatty acid degradation operon of Ralstonia eutropha H16. AMB Express. 2014;28: 4-69. https://doi:0.1186/s13568-014-0069-0. Alvarez-Santullano Villegas NP, Mardones MS, Durán RE, Donoso R, González A, Sanhueza C, Navia R, Acevedo F, Pérez-Pantoja D, et al. Genome-wide metabolic reconstruction of the synthesis of Polyhydroxyalkanoates from sugars and fatty acids by Burkholderia sensu sato species. Microorganisms. 202;9:1290. https://doi.org/10.3390/microorganisms9061290. Sastre DE, Pulschen AA, Basso LGM, Benites Pariente JS, Marques Netto CGC, Machinandiarena F, Albanesi D, Navarro MVAS, de Mendoza D, Gueiros-Filho FJ. The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis. J Biol Chem. 2020;295:2148-2159. https://doi:10.1074/jbc.RA119.011147. Suzuki K, Okada K, Kamiya Y, Zhu XF, Nakagawa T, Kawamukai M, Matsuda H (1997) Analysis of the decaprenyl diphosphate synthase ( dps ) gene in fission yeast suggests a role of ubiquinone as an antioxidant. J Biochem 121:496–505. https://doi.org/10.1093/oxfordjournals.jbchem.a021614. Waterhouse AM, Procter JB, Martin DMA, et al. Jalview Version 2-A multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25: https://doi.org/10.1093/bioinformatics/btp033. Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23: https://doi.org/10.1093/bioinformatics/btm404. Tavaré S. Some probabilistic and statistical problems in the analysis of DNA sequences. American Mathematical Society: Lect. Math. Life Sci. 1986; 17, 57–86. Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. In: MIDI Technical Note 101. Newark, Delaware, USA: MIDI Inc; 1990. Reasoner DJ, Geldreich EE. A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol. 1985;49: https://doi.org/10.1128/aem.49.1.1-7.1985. Lindhout T, Lau PCY, Brewer D, Lam JS. Truncation in the core oligosaccharide of lipopolysaccharide affects flagella-mediated motility in Pseudomonas aeruginosa PAO1 via modulation of cell surface attachment. Microbiology (Reading). 2009;155: https://doi.org/10.1099/mic.0.030510-0. Rodrigues Neto J, Malavolta Júnior VA, Victor O. Meio simples para o isolamento e cultivo de Xanthomonas campestris pv. citri Tipo B. Sum Phyt. 1986;12–16. Ribeiro NV da S, Vidal MS, Barrios SCL, et al. Genetic diversity and growth promoting characteristics of diazotrophic bacteria isolated from 20 genotypes of Brachiaria spp. Plant Soil. 2020;451: https://doi.org/10.1007/s11104-019-04263-y. Oliveira A, Teixeira P, Azevedo M, et al. Corynebacterium pseudotuberculosis may be under anagenesis and biovar Equi forms biovar Ovis: A phylogenic inference from sequence and structural analysis. BMC Microbiol. 2016;16: https://doi.org/10.1186/s12866-016-0717-4. Vaux F, Trewick SA, Morgan-Richards M. Lineages, splits and divergence challenge whether the terms anagenesis and cladogenesis are necessary. Biol. J. Linn. Soc . 2016;117 Chung EJ, Park TS, Kim KH, Jeon CO, Lee HI, Chang WS, Aslam Z, Chung YR. Nitrospirillum irinus sp. nov., a diazotrophic bacterium isolated from the rhizosphere soil of Iris and emended description of the genus Nitrospirillum . A van Leeuwenhoek. 2015;108(3):721–729. https://doi.org/10.1007/s10482-015-0528-x. Chun J, Oren A, Ventosa A, et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol. 2018;68: https://doi.org/10.1099/ijsem.0.002516. De Lajudie PM, Andrews M, Ardley J, et al. Minimal standards for the description of new genera and species of rhizobia and agrobacteria. Int J Syst Evol Microbiol. 2019;69: https://doi.org/10.1099/ijsem.0.003426. Wang J, Wang H, Cai K, et al. Evaluation of three sample preparation methods for the identification of clinical strains by using two MALDI-TOF MS systems. J Mass Spect. 2021;56: https://doi.org/10.1002/jms.4696. Gaia V, Casati S, Tonolla M. Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting. Syst Appl Microbiol. 2011;34: https://doi.org/10.1016/j.syapm.2010.11.007. Reis VM, Olivares FL, Döbereiner. Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat. World J Microbiol Biotechnol. 1994 ;10: https://doi.org/10.1007/BF00144460. Santi C, Bogusz D, Franche C. Biological nitrogen fixation in non-legume plants, An Botany. 2013;111: https://doi.org/10.1093/aob/mct048. Henderson PJF, Maher C, Elbourne, LDH, Eijkelkamp BA, Paulsen IT, Hassan KA. Physiological functions of bacterial “Multidrug” efflux pumps. Chem. Rev. 2021;121, 9, 5417–5478. https://pubs.acs.org/doi/10.1021/acs.chemrev.0c01226. Huang L, Wu C, Gao H, Xu C, Dai M, Huang L, Hao H, Wang X, Cheng G. Bacterial multidrug efflux pumps at the frontline of antimicrobial resistance: An overview. Antibiotics. 2022;13. https://doi.org/10.3390/antibiotics11040520. Mutanda I, Sun J, Jiang J, Zhu D. Bacterial membrane transporter systems for aromatic compounds: Regulation, engineering, and biotechnological applications. Biotechnol Adv. 2022;59. https://doi.org/10.1016/j.biotechadv.2022.107952. Tables Table 1 General features of the strains genomic sequence assemblies available in the NCBI genome database. Strains Genome Length (Mb) GC Content (%) No. of contigs/scaffolds Contig L50 Contig N50 (Mb) Genome coverage (x) NCBI RefSeq assembly NCBI Accession number BR 11140 T (Y-2) 7.5 67.5 73 13 0.23 201 GCF_007827975.1 GCA_007827975.1 BR 11142 T (Y-1) 6.9 67.5 26 6 0.39 251 GCF_029594735.1 GCA_029594735.1 BR 11163 T 7.1 67.5 5 2 2.00 200 GCF_034653355.1 GCA_034653355.1 BR 11164 T 7.0 68.0 4 2 1.9 133 GCF_034653275.1 GCA_034653275.1 BR 11622 T 6.6 67.0 48 8 0.30 228 GCF_007827955.1 GCA_007827955.1 BR 11752 T 6.5 67.3 4 2 1.8 112 GCF_036348415.1 GCA_036348415.1 BR 11828 T 6.3 67.7 5 2 1.8 99 GCF_034427435.1 GCA_034427435.1 BR 11865 T 7.0 67.5 56 10 0.25 213 GCF_007828025.1 GCA_007828025.1 DSM 22198 T 7.1 67.0 58 9 0.83 211 GCF 014205765.1 GCA_014205765.1 Table 2 Average nucleotide identity (ANIb) and digital DNA-DNA hybridization (dDDH) values between N. iridis DSM 22198 T , N. viridazoti BR 111140 T , N. amazonense BR 11142 T and the proposed new species. Strain/Species DSM 22198 T BR 11142 T BR 11140 T BR 11865 T BR 11163 T BR 11164 T BR 11828 T BR 11752 T BR 11622 T ANIb DSM 22198 T ( N. iridis ) 100.0 85.1 84.9 84.9 85.0 85.0 84.5 84.4 84.5 BR 11142 T ( N. amazonense ) 85.1 100.0 93.4 94.0 91.2 91.8 86.9 86.9 87.0 BR 11140 T ( N. viridazoti ) 84.9 93.4 100.0 94.1 91.5 91.3 86.8 86.9 86.9 BR 11865 T ( N. bahiense ) 84.9 94.0 94.1 100.0 91.7 91.6 86.9 87.0 87.0 BR 11163 T ( N. guanabarense ) 85.0 91.2 91.5 91.7 100.0 90.1 87.0 87.0 87.1 BR 11164 T ( N. guaranorum ) 85.0 91.8 91.3 91.6 90.1 100.0 87.2 86.8 86.9 BR 11828 T ( N. guiasense ) 84.5 86.9 86.8 86.9 87.0 87.2 100.0 92.5 92.7 BR 11752 T ( N. karajorum ) 84.4 86.9 86.9 87.0 87.7 86.8 92.5 100 .0 95.5 BR 11622 T ( N. pindoramense ) 84.5 87.0 86.9 87.0 87.1 86.9 92.7 95.5 100.0 dDDH DSM 22198 T ( N. iridis ) 100.0 29.1 28.6 28.8 29.0 28.9 28.1 27.9 28.0 BR 11142 T ( N. amazonense ) 29.1 100.0 52.6 60.1 44.8 44.2 32.2 32.3 32.1 BR 11140 T ( N. viridazoti ) 28.6 52.6 100.0 55.6 43.6 46.1 32.5 32.5 32.6 BR 11865 T ( N. bahiense ) 28.8 60.1 55.6 100.0 45.5 45.0 32.3 32.5 32.4 BR 11163 T ( N. guanabarense ) 29.0 44.8 43.6 45.5 100.0 40.2 32.4 32.5 32.5 BR 11164 T ( N. guaranorum ) 28.9 44.2 46.1 45.0 40.2 100.0 33.3 32.3 32.2 BR 11828 T ( N. guiasense ) 28.1 32.2 32.5 32.3 32.4 33.3 100.0 48.4 49.0 BR 11752 T ( N. karajorum ) 27.9 32.3 32.5 32.5 32.5 32.3 48.4 100.0 66.0 BR 11622 T ( N. pindoramense ) 28.0 32.1 32.6 32.4 32.5 32.2 49.0 66.0 100.0 Additional Declarations No competing interests reported. Supplementary Files SupplementarMaterialmaro2025.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5600752","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449742308,"identity":"bcc6764b-523f-49c2-a6c9-c8744ce2b9aa","order_by":0,"name":"Jerri Edson Zilli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACxgYgkcDAwMPHztj4gIHhAFiUmSgtbMyMzQZEaYEDNmYGNgmitDC39z778IChToaNmbmt4mPOnXz+2c0PmAv34HFYz3HjGQkMbCCHtd2cue2Z5Yw7xwyYZzzDo2VGGjPQLzxgLbd5tx02YLiRYMDMcwCPlvnPQFokwFqK/wK1yN9I/4Bfyww2kBYDsBZmRqAWgxs5BGzpATnMIAEcyJK9254ZGN45U3B4Bh4thu3HmBl/VNTZ87O3P/zwc9sdA7nb7RsfF+DT0gAiDZCF4LGDA8hjCkngUz8KRsEoGAUjEQAAI6tKHE5+sDkAAAAASUVORK5CYII=","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":true,"prefix":"","firstName":"Jerri","middleName":"Edson","lastName":"Zilli","suffix":""},{"id":449742309,"identity":"6535b1f0-a7ca-4453-b82a-726af239beaf","order_by":1,"name":"Stefan Shwab","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Shwab","suffix":""},{"id":449742310,"identity":"b3962d99-9244-4238-ba10-fb1f0abe06c1","order_by":2,"name":"Natália dos Santos Ferreira","email":"","orcid":"","institution":"Universidade Federal Rural do Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Natália","middleName":"dos Santos","lastName":"Ferreira","suffix":""},{"id":449742311,"identity":"8b1489c6-a28c-48b3-8b67-289649dc799b","order_by":3,"name":"Veronica Massena Reis","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Veronica","middleName":"Massena","lastName":"Reis","suffix":""},{"id":449742312,"identity":"36dd37a9-42ae-4725-a721-d74943aa58e2","order_by":4,"name":"Alberto Fernandes de Oliveira Junior","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"Fernandes de Oliveira","lastName":"Junior","suffix":""},{"id":449742313,"identity":"342ce0a1-b388-41db-a729-81598427102e","order_by":5,"name":"Jean Luiz Simões-Araujo","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"Luiz","lastName":"Simões-Araujo","suffix":""},{"id":449742314,"identity":"ccc7631f-a152-46eb-a2c0-c2b121f406a4","order_by":6,"name":"Luis Henrique de Barros Soares","email":"","orcid":"","institution":"Universidade Federal Rural do Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"Henrique de Barros","lastName":"Soares","suffix":""},{"id":449742315,"identity":"0a9b064a-c796-40a6-ae82-e1cdd8800f1a","order_by":7,"name":"Fernanda dos Santos Dourado","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Fernanda","middleName":"dos Santos","lastName":"Dourado","suffix":""},{"id":449742316,"identity":"085c3848-f2a1-4069-ba51-d7ffe610f781","order_by":8,"name":"Evelise Bach","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul (UFRGS), IB- UFRGS","correspondingAuthor":false,"prefix":"","firstName":"Evelise","middleName":"","lastName":"Bach","suffix":""},{"id":449742317,"identity":"6bf9f3ff-6cde-45e7-8ab7-0ed04cf5be9d","order_by":9,"name":"Luiz Fernando Wurdig Roesch","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Luiz","middleName":"Fernando Wurdig","lastName":"Roesch","suffix":""},{"id":449742318,"identity":"667498c5-bca2-4246-93de-96e8998e1387","order_by":10,"name":"Carolina Nachi Rossi","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"Nachi","lastName":"Rossi","suffix":""},{"id":449742319,"identity":"21f11a28-0810-48d0-85e1-504c75ab06ef","order_by":11,"name":"Karinne Motta de Oliveira Lima de Souza","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Karinne","middleName":"Motta de Oliveira Lima","lastName":"de Souza","suffix":""},{"id":449742320,"identity":"e95ff9a8-4c22-450c-a9aa-88fb348a8485","order_by":12,"name":"Bruno José Rodrigues Alves","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Bruno","middleName":"José Rodrigues","lastName":"Alves","suffix":""},{"id":449742321,"identity":"05fb8b8f-74b3-4b97-be9c-067e1d5a3503","order_by":13,"name":"Andréia Loviane Silva","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"Andréia","middleName":"Loviane","lastName":"Silva","suffix":""},{"id":449742322,"identity":"9eddd964-a5f0-4095-9b0b-2208ca603999","order_by":14,"name":"José Ivo Baldani","email":"","orcid":"","institution":"Empresa Brasileira de Pesquisa Agropecuária","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Ivo","lastName":"Baldani","suffix":""}],"badges":[],"createdAt":"2024-12-07 23:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5600752/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5600752/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81680631,"identity":"d67b31e1-da47-4136-bb87-f17a0a157c54","added_by":"auto","created_at":"2025-04-30 09:01:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":142647,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on 16S rRNA-\u003cem\u003erecA\u003c/em\u003e concatenated genes of \u003cem\u003eNitrospirillum\u003c/em\u003e strains proposed as new species and the type strains \u003cem\u003eN. iridis\u003c/em\u003e DSM 22198\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eN. viridazoti\u003c/em\u003e BR 11140\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eN. amazonense\u003c/em\u003e BR 11142\u003csup\u003eT\u003c/sup\u003e. Bootstrap values over 50%, 500 repetitions, are shown. \u003cem\u003eA. brasilense \u003c/em\u003eSp7\u003csup\u003eT\u003c/sup\u003e was used as the outgroup.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5600752/v1/c3e40284c667fc5267603cb2.png"},{"id":81680632,"identity":"28deab89-69dc-4ff9-8e67-62338d37fb07","added_by":"auto","created_at":"2025-04-30 09:01:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41826,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenomic tree of \u003cem\u003eNitrospirillum\u003c/em\u003e species, including the strains proposed as new species and the type strains \u003cem\u003eN. iridis\u003c/em\u003e DSM 22198ᵀ, \u003cem\u003eN. viridazoti\u003c/em\u003e BR 11140ᵀ, and \u003cem\u003eN. amazonense\u003c/em\u003e BR 11142ᵀ. Bootstrap values over 50%, 500 repetitions, are shown. \u003cem\u003eA. brasilense \u003c/em\u003eSp7\u003csup\u003eT\u003c/sup\u003e was used as the outgroup.\u003c/p\u003e","description":"","filename":"Fig2revised.png","url":"https://assets-eu.researchsquare.com/files/rs-5600752/v1/03bb84b312702b994d58ee87.png"},{"id":81680634,"identity":"d7bf5873-d52c-4b5b-8e0f-125ce4d95aae","added_by":"auto","created_at":"2025-04-30 09:01:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":150072,"visible":true,"origin":"","legend":"\u003cp\u003eFlower-plot of protein families describing pan, core and the unique genome. BR 11142\u003csup\u003eT\u003c/sup\u003e, BR 11140\u003csup\u003eT\u003c/sup\u003e and DSM 22198\u003csup\u003eT\u003c/sup\u003e are type strains of the species \u003cem\u003eN. amazonense\u003c/em\u003e, \u003cem\u003eN. iridis \u003c/em\u003eand\u003cem\u003e N. viridazoti\u003c/em\u003e, respectively.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5600752/v1/1f61f68c47fef2907aa2b9b9.png"},{"id":81680633,"identity":"16d8f75e-e3d0-4c41-8ffa-f3ec3ab940fa","added_by":"auto","created_at":"2025-04-30 09:01:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39022,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional phylogenetic distribution of nitrogen fixation genes, \u003cem\u003enifHDK\u003c/em\u003egenes. Bootstrap values over 50%, 500 repetitions, are shown. \u003cem\u003eA. brasilense \u003c/em\u003eBR 11669 was used as the outgroup.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5600752/v1/5b168d8d448ef9b57264e80b.png"},{"id":81681421,"identity":"acb671bd-89c5-4bf3-88d2-e9748db7f265","added_by":"auto","created_at":"2025-04-30 09:09:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32177,"visible":true,"origin":"","legend":"\u003cp\u003eDendrogram of \u003cem\u003eNitrospirillum\u003c/em\u003espp. constructed from mass spectrum using the SARAMIS single-linkage cluster analysis. The samples were obtained from bacteria cultivation on LGI and Dygs culture media at 30°C during 24 and 48hour incubation and analyzed in triplicate. The scale bar indicates the similarity (%). Accumulated number of peaks shows as datacount is listed.\u003c/p\u003e","description":"","filename":"Fig05.png","url":"https://assets-eu.researchsquare.com/files/rs-5600752/v1/901c780d8e33c8005989130b.png"},{"id":85303981,"identity":"983b44f7-b458-49af-a62f-4d59262d1aae","added_by":"auto","created_at":"2025-06-24 12:32:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1637968,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5600752/v1/1b5c3eee-a204-4869-a628-ef8c56fa2665.pdf"},{"id":81680636,"identity":"bd5f57c1-a7f3-4cf9-90f4-7ead5fc96b46","added_by":"auto","created_at":"2025-04-30 09:01:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1674199,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarMaterialmaro2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5600752/v1/ec09d524a244905ae5a6f081.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the Diversity of the Genus Nitrospirillum: Description of Six Novel Nitrogen-Fixing Bacteria Species","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant growth-promoting bacteria (PGPB) play a pivotal role in modern agriculture by enhancing plant development through mechanisms such as biological nitrogen fixation and the production of growth-stimulating compounds. Their strategic application in agricultural systems increases the efficiency of agronomic inputs, diminishes reliance on chemical fertilizers, and markedly improves soil quality, thereby advancing sustainability in both agricultural and environmental practices [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, the supply of bio-based products intended for agricultural use is on the rise, with PGPB emerging as particularly prominent components. In numerous countries\u0026mdash;especially across South America\u0026mdash;PGPB have been commercially employed in agriculture for over a decade, with the inoculated area estimated to approach 10\u0026nbsp;million hectares [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Representative examples include bacteria from the genus \u003cem\u003eAzospirillum\u003c/em\u003e, which is widely applied in maize cultivation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and \u003cem\u003eNitrospirillum\u003c/em\u003e, which is marketed as an inoculant for sugarcane [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eNitrospirillum\u003c/em\u003e emerged from a taxonomic reclassification of the species \u003cem\u003eAzospirillum amazonense\u003c/em\u003e, which was initially isolated from a variety of ecosystems following extensive research in the 1980s [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A modification in the standard semi-solid medium for isolating \u003cem\u003eAzospirillum\u003c/em\u003e strains\u0026mdash;achieved by incorporating 0.5 g L⁻\u0026sup1; each of mannitol and sucrose into the original malate-based NFb medium [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u0026mdash;permitted the isolation of a group of strains from \u003cem\u003eUrochloa brizantha\u003c/em\u003e (formerly \u003cem\u003eBrachiaria brizantha\u003c/em\u003e) in the Amazon region [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, the optimization of a nitrogen-free medium by supplementing it with sucrose and adjusting the pH to a lower range (5.0\u0026ndash;6.0), referred to as LGI or modified FAM, further facilitated the isolation of \u003cem\u003eA. amazonense\u003c/em\u003e from diverse ecosystems [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Successful isolation of \u003cem\u003eNitrospirillum\u003c/em\u003e has since been achieved in other countries, with strains obtained from various regions and across distinct seasonal conditions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eNitrospirillum\u003c/em\u003e belongs to the phylum Proteobacteria, class Alphaproteobacteria, order Rhodospirillales, and family Rhodospirillaceae. The taxonomic classification has been established based on the phylogeny of specific genes, such as 16S rRNA, and whole-genome data from bacterial genomes [7, 12). Phenotypic characterization has involved additional analyses, including chemotaxonomic markers such as fatty acid profiles, quinone composition, and polar lipid patterns, as well as assessments of nitrogen fixation capacity, antibiotic tolerance, C-source utilization, and enzyme activities (7, 12). Ubiquinone Q-10 appears to be the major respiratory quinone, while C\u003csub\u003e18:1\u003c/sub\u003e\u003cem\u003eω\u003c/em\u003e7c is the predominant fatty acid. Additionally, all species described exhibit biological nitrogen fixation, and in general, the strains of this genus do not have the ability to denitrify (12).\u003c/p\u003e \u003cp\u003eIn addition to \u003cem\u003eNitrospirillum amazonense\u003c/em\u003e, the species \u003cem\u003eNitrospirillum iridis\u003c/em\u003e was identified through isolation in Korea, and more recently, \u003cem\u003eNitrospirillum viridazoti\u003c/em\u003e has been documented [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eN. viridazoti\u003c/em\u003e is distinguished by its ability to fix nitrogen and stimulate plant growth [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, this species exhibits a distinctive partial denitrification pathway, wherein the enzyme nitrous oxide reductase exclusively catalyzes the reduction of nitrous oxide (N₂O) to dinitrogen (N₂) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While \u003cem\u003eN. iridis\u003c/em\u003e was isolated from \u003cem\u003eIris ensata\u003c/em\u003e, both \u003cem\u003eN. amazonense\u003c/em\u003e and \u003cem\u003eN. viridazoti\u003c/em\u003e were predominantly associated with grasses, including \u003cem\u003eAndropogon gayanus\u003c/em\u003e, \u003cem\u003eAxonopus\u003c/em\u003e sp., \u003cem\u003eBactris gasipaes\u003c/em\u003e, \u003cem\u003eBrachiaria\u003c/em\u003e spp., \u003cem\u003eDigitaria decumbens\u003c/em\u003e, \u003cem\u003eHyparrhenia rufa\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003ePanicum pilosum\u003c/em\u003e, \u003cem\u003ePaspalum virgatum\u003c/em\u003e, \u003cem\u003ePennisetum purpureum\u003c/em\u003e, \u003cem\u003eSaccharum\u003c/em\u003e spp., and \u003cem\u003eSorghum vulgare\u003c/em\u003e. Notably, these bacteria have also been identified in non-grass hosts such as \u003cem\u003eAnanas comosus\u003c/em\u003e, \u003cem\u003eCocos nucifera\u003c/em\u003e, \u003cem\u003eMusa\u003c/em\u003e spp., among others [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, an extensive collection of strains is under investigation, where genomic analyses are integrated with morphological and physiological comparisons to elucidate the taxonomy of this genus. In this study, we endeavored to enhance our understanding of bacterial diversity within the genus \u003cem\u003eNitrospirillum\u003c/em\u003e by characterizing 86 strains obtained from a variety of hosts and geographic regions. These strains have been deposited in the culture collection of the Johanna D\u0026ouml;bereiner Biological Resource Center at Embrapa Agrobiologia over the past 35\u0026ndash;40 years, representing a valuable repository for elucidating the evolutionary intricacies of \u003cem\u003eNitrospirillum\u003c/em\u003e. Employing a taxonomic framework grounded in both phylogenetic and phylogenomic analyses, we have proposed six novel species within the genus \u003cem\u003eNitrospirillum\u003c/em\u003e. This taxonomic contribution not only broadens our systematic understanding of bacterial classification but also underscores the critical importance of preserving and investigating microbial repositories to fully elucidate prokaryotic diversity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eBacterial strains\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, we examined 86 bacterial strains obtained from the grass \u003cem\u003eBrachiaria\u003c/em\u003e \u003cem\u003ebrizantha\u003c/em\u003e (three strains), \u003cem\u003eB. humidicola\u003c/em\u003e (six strains), \u003cem\u003eSorghum vulgare\u003c/em\u003e (six strains), \u003cem\u003eZea mays\u003c/em\u003e (23 strains), and \u003cem\u003eOryza sativa\u003c/em\u003e (47 strains). The collection locations were in the Brazilian states of Bahia (northeast Atlantic Forest; 2 strains), Goi\u0026aacute;s (central-west Cerrado; 22 strains), and Rio de Janeiro (southeast Atlantic Forest; 62 strains) (Table S1). When deposited in the culture collection, the strains were characterized as gram-negative, non-sporulating cells with the capacity to fix nitrogen in a semi-solid medium.\u003c/p\u003e\n\u003cp\u003eAll these strains are stored at the Johanna D\u0026ouml;bereiner Biological Resources Center (CRB-JD, WFCC 364, Seropedica, Rio de Janeiro, Brazil) (https://www.embrapa.br/agrobiologia/crb-jd) and are preserved long-term through lyophilization and storage at -80 \u0026deg;C in a culture medium containing 20% glycerol.\u003c/p\u003e\n\u003cp\u003eThe strains BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, and BR 11865\u003csup\u003eT\u0026nbsp;\u003c/sup\u003ewere also deposited at the University of Coimbra Bacteria Culture Collection (UCBCC, WFCC 1179 \u0026ndash; Coimbra, Portugal) (https://ucccb.uc.pt/) and were assigned the accession numbers UCCCB 233\u003csup\u003eT\u003c/sup\u003e, UCCCB 228\u003csup\u003eT\u003c/sup\u003e, UCCCB 229\u003csup\u003eT\u003c/sup\u003e, UCCCB 231\u003csup\u003eT\u003c/sup\u003e, UCCCB 232\u003csup\u003eT\u003c/sup\u003e, and UCCCB 230\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhylogenetic analyses\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted from the 86 strains using a Bacterial Genomic DNA Isolation Kit (Wizard; Promega, Madison, WI, USA) following the manufacturer\u0026apos;s instructions. Segments of the 16S rRNA and \u003cem\u003erecA\u003c/em\u003e genes were amplified via PCR using the primers 27F/1492R (16S rRNA) and 63F and 504R (\u003cem\u003erecA\u003c/em\u003e) [16]. Subsequently, Sanger dideoxy DNA sequencing was performed. Forward and reverse contigs were used for gene sequence assembly using the Bionumerics package (v. 7). The 16S rRNA and \u003cem\u003erecA\u003c/em\u003e sequences of the type strains were retrieved from the NCBI nucleotide database (www.ncbi.nlm.nih.gov). Sequences were compared with other sequences in the NCBI database using BLAST (https://blast.ncbi.nlm.nih.gov/) followed by analysis using MEGA 11 [17]. The evolutionary model was determined for alignment and tree topology using the JC+C model [18] for 16S rRNA gene sequences, assuming independent sites with identical mutation rates. For \u003cem\u003erecA\u003c/em\u003e gene sequences, T92+G [19] was the best-fit model, which addresses transition-transversion and G+C-content biases. The SeaView tool was employed, incorporating maximum likelihood (ML) estimation of the PhyML software and ClustalX-generated alignment. Branch support was assessed using a nonparametric bootstrap test with 1000 replicates and an approximate likelihood ratio test (ALRT). The 16S rRNA sequences were also concatenated with the \u003cem\u003erecA\u003c/em\u003e sequences for a more robust view of the phylogeny. Concatenation was performed with MEGA 11 using the JC+C model and ML estimation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePositive and negative selection assessment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the degree of neutrality between the sequences, identify those that do not fit the neutral theory model, and determine the balance between mutations and genetic drift, we used the neutrality test method proposed by Tajima [20].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenomic analyses\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequences of strains BR 11865\u003csup\u003eT\u003c/sup\u003e and BR 11622\u003csup\u003eT\u003c/sup\u003e were sequenced at the DOE-Joint Genome Institute (JGI) as part of the Genomic Encyclopedia of Type Strains, Phase IV (KMG-V) to study the core and pan-genomes of soil and plant-associated prokaryotes (https://gold.jgi.doe.gov/studies?id=Gs0129091) using an Illumina NovaSeq 6000 platform. All genomes were first deposited in the RefSeq database and subsequently downloaded, along with the genomes of strains BR 11140T, BR 11142T (Y-1), and DSM 22198T, which were available in the RefSeq database (accessed in August 2023).\u003c/p\u003e\n\u003cp\u003eThe whole-genome sequences of strains BR 11752\u003csup\u003eT\u003c/sup\u003e, BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT,\u003c/sup\u003e and BR 11828\u003csup\u003eT\u003c/sup\u003e were obtained using nanopore technology. High-molecular-weight DNA was extracted using a DNA Genomic Isolation Kit (Wizard; Promega), and libraries were constructed following the native barcoding genomic DNA protocol recommended by the manufacturer, after which sequencing was performed. The libraries were loaded onto a MinION flow cell model FLO-MIN 106 (version R10.3) and sequencing was monitored using the MinKNOW program (Oxford Nanopore Technologies). Base calling was performed using the Guppy program (Oxford Nanopore Technologies), data demultiplexing was performed using the Demultiplex program (version 1.2.1, available at https://github.com/jfjlaros/demultiplex), and sequencing data were evaluated using NanoPlot (version 1.24.0) [21]. Genomic sequences were assembled using the Flye program [22]. The genome sequences were deposited in the GenBank RefSeq database for automatic annotation.\u003c/p\u003e\n\u003cp\u003eAll genomes were processed in the BV-BRC platform [23], where genomic annotation was performed using the RAST toolkit (RASTtk). Phylogenomic analysis was conducted via the BV-BRC server\u0026apos;s bacterial Phylogenetic Tree Service, constructing custom phylogenetic trees from user-selected genomes. The Codon Tree method employed single-copy BV-BRC PGFams and analyzed aligned coding DNA using RAxML [24]. The Comparative Systems Service, incorporating tools from the legacy PATRIC system, utilized a Protein Family Sorter [23, 25] to examine functional gene distribution across genomes (the \u0026ldquo;pan-genome\u0026rdquo;). Core genomes contain functional genes shared by all genomes, while accessory genomes include those shared by subsets. Pathway maps were generated via KEGG pathways [26] with default parameters.\u003c/p\u003e\n\u003cp\u003eThe average nucleotide identity values based on BLAST alignments (ANIb) from all pairwise genome comparisons were calculated using Pyani version 0.2.7, which is a Python module for calculating genomic metrics (https://github.com/widdowquinn/pyani) [27] Digital DNA\u0026ndash;DNA hybridization (dDDH) was performed using the Genome Distance Calculator for Genome (GGDC; http://ggdc.dsmz.de/ ggdc. php), considering the recommended formula 2. Two genome sequences were considered to belong to the same species if the ANI and dDDH values were above 95\u0026ndash;96% and 70%, respectively [28\u0026ndash;30].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenome-based analysis of chemotaxonomic markers\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eProtein sequences of the respective genes, decaprenyl diphosphate synthase (\u003cem\u003eDps\u003c/em\u003e), 3-ketoacyl-CoA thiolase 2 (fadN-fadA-fadE operon), acyl-ACP phosphate acyltransferase (\u003cem\u003ePlsX\u003c/em\u003e), and acyl-CoA thioesterase (\u003cem\u003eYciA\u003c/em\u003e) were used to construct the phylogenetic topologies [12, 31-35]. The LG model (Le and Gascuel), an improved amino acid replacement matrix, was used for protein phylogenetics. Phylogenetic tree reconstruction was performed using the RaxML algorithm.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenome-based analysis of antimicrobial resistance\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe BV-BRC platform was used to analyze potential bacterial resistance to antibiotics [23]. The Genome Annotation Service in BV-BRC employs a k-mer-based approach to detect antimicrobial resistance (AMR) genes. This method leverages BV-BRC\u0026rsquo;s curated database of representative AMR gene sequence variants in BV-BRC. Each identified AMR gene was functionally annotated with its broad antibiotic resistance mechanism, the relevant drug class, and, in some cases, the specific antibiotic to which it confers resistance.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003enifHDK analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNitrogenase sequences, which comprise multiple subunits (\u003cem\u003eNifH, NifD\u003c/em\u003e, and \u003cem\u003eNifK\u003c/em\u003e encoded by the \u003cem\u003enifHDK\u003c/em\u003e genes) \u0026mdash; organized as an operon in these bacterial genomes \u0026mdash;, were extracted from the set of genomes examined in this study using the BV-BRC/PATRIC database. The nucleotide sequences corresponding to \u003cem\u003enifH, nifD, and nifK\u003c/em\u003e were directly concatenated in order to use in alignments. Alignment was performed using JalView [36] and Clustal X [37]. The evolutionary model proposed for the tree topology [38] was the generalized time-reversible model of Tavar\u0026eacute; from MEGA 11. A tree was generated from PhyML using the ML method.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMass spectrometry and fatty acid methyl ester (FAME)\u0026nbsp;\u003c/em\u003eanalysis\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe type strain candidates BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, BR 11865\u003csup\u003eT\u003c/sup\u003e, along with strains BR 11140\u003csup\u003eT\u003c/sup\u003e and BR 11142\u003csup\u003eT\u003c/sup\u003e were cultivated on DYGS and LGI solid media in Petri dishes at 30 \u0026deg;C for 24 and 48 h. An isolated colony from each strain was directly applied to a stainless steel MALDI plate to create a thin layer. Subsequently, it was promptly covered with 1 \u0026mu;L of a saturated \u0026alpha;-cyano-4-hydroxy-cinnamic acid matrix (CHCA, 5 mg mL\u003csup\u003e-1\u003c/sup\u003e in a solution of acetonitrile/water 50:50 with 0.1% trifluoroacetate) and air-dried before analysis.\u003c/p\u003e\n\u003cp\u003eFor external calibration, \u003cem\u003eEscherichia coli\u003c/em\u003e DH5\u0026alpha; was used as the standard strain, and each sample was spotted in quadruplicate to ensure reproducibility. Mass spectra were acquired using an Axima Performance \u003cem\u003eiD\u003c/em\u003e Plus (Shimadzu Corp., integrated with the SARAMIS database) with a 60\u0026thinsp;Hz pulsed nitrogen laser. The analysis was conducted in the positive linear SARAMIS extraction mode, with a laser repetition rate of 50 Hz, five shots accumulated per profile, and a mass-to-charge ratio (m/z) of 2,000 to 20,000.\u003c/p\u003e\n\u003cp\u003eProtein mass fingerprints for each colony were generated by accumulating 500 laser pulse cycles and were processed using the Shimadzu Biotech Launchpad software before export for analysis using the SARAMIS package. SuperSpectra were constructed by scrutinizing the mass spectra under multiple conditions for the same strains. This involved identifying consistent mass signals frequently appearing across the mass spectra and assigning weights to commonly agreed-upon peaks. Single-linkage cluster analysis was performed using SARAMIS parameters to produce a dendrogram of the \u003cem\u003eNitrospirillum\u003c/em\u003e strains (0.08% tolerance, range from 3,000 to 20,000 m/z).\u003c/p\u003e\n\u003cp\u003eFor FAME analyses, the strains BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, BR 11865\u003csup\u003eT\u003c/sup\u003e, along with strains BR 11140\u003csup\u003eT\u003c/sup\u003e were grown on R2A medium [39] at 30 \u0026deg;C for 48 h. Fatty acid methyl esters were obtained from the fresh wet biomass according to the standard MIDI protocol and were separated, identified and quantified by GC (Agilent Technologies 7890B GC) with the Sherlock Microbial Identification System version 6.5 (MIDI) using the RTSBA6 database [40].\u003cem\u003eMorphology of colonies, cells, and physiological assays\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe strains BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, and BR 11865\u003csup\u003eT\u003c/sup\u003e, along with BR 11140\u003csup\u003eT\u003c/sup\u003e and BR 11142T, were assessed through morphological and physiological assays. Their growth capabilities at different pH levels were evaluated by cultivating them on solid medium (LGI or modified FAM) at levels ranging from 5.0\u0026ndash;8.0, followed by incubation for 5 days at 30 \u0026deg;C. To observe colony morphology, the bacteria were grown on homemade PDA medium and PDA medium supplemented with 10% crystal sugar and incubated for 7 days at 30 \u0026deg;C [8]. To assess cell morphology and pellicle formation in the medium, which are indicators of biological nitrogen fixation (BNF) activity, the bacteria were grown in 5 mL of nitrogen-free LGI semisolid medium at pH 7 in 10 mL glass flasks [8]. For micrography, bacterial samples were prepared following standard protocols [41] and subsequently visualized using Hitachi HT7800 transmission electron microscope set to an operating voltage of 100 kV.\u003c/p\u003e\n\u003cp\u003eTo confirm the BNF capability, bacteria grown in a semi-solid medium were evaluated for their ability to reduce acetylene. After 5 d of growth, the flasks were sealed with a chlorobutyl septum. One milliliter of gas was extracted from the headspace and replaced with acetylene. After 1 h of incubation, 1 mL of the headspace gas was directly injected into a gas chromatograph (Shimadzu 2010, CG-BID 2010 plus) coupled to a barrier discharge ionization detector (BID). The column (RTQBOND 0.53 mm ID; 20um; Cat 19742) was pre-conditioned for 0.5 min, the run temperature was 80 \u0026ordm;C for 6 min, with a detector temperature of 280 \u0026ordm;C and a helium flow rate of 50 mL/min. Ethylene production was estimated using a standard ethylene dose-response curve diluted with N\u003csub\u003e2\u003c/sub\u003e gas.\u003c/p\u003e\n\u003cp\u003eAntibiotic sensitivity/tolerance tests were conducted using the Sensi-disc system (Cecon-Brazil) on a solid Dyg\u0026rsquo;s medium. The discs containing amoxicillin (10 \u0026micro;g), ampicillin (10 \u0026micro;g), rifampicin (15 \u0026micro;g), chloramphenicol (30 \u0026micro;g), azithromycin (15 \u0026micro;g), kanamycin (30 \u0026micro;g), gentamicin (10 \u0026micro;g), neomycin (30 \u0026micro;g), cephalexin (30 \u0026micro;g), penicillin (10 \u0026micro;g), tetracycline (30 \u0026micro;g), vancomycin (30 \u0026micro;g), streptomycin (10 \u0026micro;g), and erythromycin (25 \u0026micro;g) was used for tests. The discs were disposed on the culture medium after spreading 100 \u0026micro;L of the pre-inoculum obtained from bacterial growth in liquid DYGS medium, and plates were incubated at 28 \u0026deg;C for 4 days.\u003c/p\u003e\n\u003cp\u003eEnzyme activities and substrate assimilation were evaluated by inoculating API 20NE strips (bioM\u0026eacute;rieux) and using the Biolog GNEIII kit (MicroPlate), following the manufacturer\u0026apos;s instructions. Bacteria were cultured on DYGS solid medium [42] resuspended in a saline solution (0.85% NaCl) until the turbidity reached 4.0 on the McFarland scale, inoculated on the Biolog plates and then incubated for 36 h at 30 \u0026deg;C. Phenotypic data obtained using the Biolog kit were organized in a binary matrix, where 1 was considered a positive reaction and 0 was a negative reaction. Subsequently, the samples were grouped using the complete linkage and Pearson\u0026apos;s distance measurement methods using the Heatmapper web service (http://www.heatmapper.ca/). Catalase activity was assessed by flooding a colony with 3% (v/v) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and observing the presence of bubbles. The capacity to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, and zinc phosphate, production of indoles and siderophores, and cellulase activity were determined following methods described elsewhere [43].\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cem\u003e16S rRNA and\u0026nbsp;\u003c/em\u003e\u003cem\u003erecA\u0026nbsp;phylogeny of the genus\u0026nbsp;Nitrospirillum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eInitially, we investigated statistical methods capable of reinforcing the formation of different species from neutrality tests that evaluate genomic mutation rates. Our statistical analyses, aimed at interpreting signs of selection in our sequences using the Tajima method, revealed positive selection (\u0026gt;0) for both the 16S rRNA and \u003cem\u003erecA\u003c/em\u003e genes. Positive selection as indicated by Tajima\u0026apos;s D suggests an excess of intermediate-frequency alleles, which may result from population bottlenecks, structure, and/or balancing selection. Such evidence can be interpreted as indicative of a reduction in population size, balancing selection and sudden population contraction (additional files). These statistical values offer valuable biological insights, considering that the sequences do not fit the neutral theory model. Moreover, hypotheses can be formulated regarding the potential for excessive non-synonymous divergence among sequences. This aids in comprehending the evolutionary patterns of consistency across our samples, highlighting species differences and limited polymorphisms, and elucidating the topological patterns in our phylogenies [4,45].\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA gene phylogeny indicated that the 86 strains in this study were distributed among three main clades, with approximately 98.8% similarity within the genus \u003cem\u003eNitrospirillum\u003c/em\u003e (Fig. S1; Table S7). One of these clades was specific to the strain DSM 22198\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN. iridis\u003c/em\u003e, and the second clade comprised 31 strains, including the strain BR 11142\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN. amazonense\u003c/em\u003e. \u0026nbsp;The third clade included the remaining new strains and BR 11140\u003csup\u003eT\u003c/sup\u003e, the type strain of \u003cem\u003eN. viridazoti\u003c/em\u003e (Fig. S1). The new strains were distributed across six clusters, each containing 3\u0026ndash;46 strains, and exhibiting a similarity of more than 90.5% within each cluster. All these groups maintained distance from the three known \u003cem\u003eNitrospirillum\u003c/em\u003e species, except for strains BR 11169, BR 11162, and BR 11163\u003csup\u003eT\u003c/sup\u003e, which positioned themselves close to strain BR 11142\u003csup\u003eT\u003c/sup\u003e (Fig. S1). This clustering indicated the presence of new taxa; however, owing to the high conservation of the 16S rRNA sequences widely reported in the literature, it was not suitable for species-level inferences, including the genus \u003cem\u003eNitrospirillum\u0026nbsp;\u003c/em\u003e[46].\u003c/p\u003e\n\u003cp\u003eIn addition to the 16S rRNA gene phylogeny, we analyzed the sequences of the \u003cem\u003erecA\u003c/em\u003e gene. Overall, this analysis provided greater resolution than the 16S rRNA analysis, with a more pronounced separation among the six clusters while keeping the same strains grouped (Fig. S2). All six clusters mentioned above were distant from the type strains BR 11140\u003csup\u003eT\u003c/sup\u003e, BR 11142\u003csup\u003eT\u003c/sup\u003e, and DSM 22198\u003csup\u003eT\u003c/sup\u003e (Fig. S2). Notably, the group of strains BR 11169, BR 11162, and BR 11163\u003csup\u003eT\u003c/sup\u003e, which were very close to BR 11142\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN.\u0026nbsp;amazonense\u003c/em\u003e in the 16S rRNA gene analysis, formed a distinct cluster in the \u003cem\u003erecA\u003c/em\u003e analysis (Fig. S2).\u003c/p\u003e\n\u003cp\u003eTo obtain a better view of the strain phylogeny, taking into account that the topologies of the 16S rRNA and \u003cem\u003erecA\u003c/em\u003e trees were similar, we concatenated those sequences (Fig. 1). The tree shows, as already observed in individual analyses, the new strains formed six distinct groups. Two clusters (highlighted in orange and brown) comprised three and four strains, respectively, isolated from Rio de Janeiro State. These clusters were positioned distantly from any type strain of the described species (Fig. 1). The third cluster (highlighted in purple) grouped 10 strains isolated from the States of Bahia, Rio de Janeiro, and Goi\u0026aacute;s. This cluster showed approximately 99% similarity with \u003cem\u003eN. amazonense\u003c/em\u003e BR 11142\u003csup\u003eT\u003c/sup\u003e. Similarly, the remaining three groups formed clades separated from the type strains of known species (highlighted in green, blue, and red), with a similarity below 99% among the clusters (Fig. 1). The strains of these three clusters were isolated from the states of Rio de Janeiro and Goi\u0026aacute;s and showed a broad distribution, especially for the cluster highlighted in red, which contained 46 strains.\u003c/p\u003e\n\u003cp\u003eAlthough phylogenetic analyses of 16S rRNA and \u003cem\u003erecA\u003c/em\u003e alone may not be sufficient for prokaryotic species circumscription, they can provide consistent information that aligns with genome-based analyses, serving as an initial source of information about a set of strains. Therefore, to confirm that the six distinct phylogenetic groups observed in the 16S rRNA and \u003cem\u003erecA\u003c/em\u003e analyses represent new taxa, we proceeded with subsequent genomic analyses.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTaxonomic distribution of sequenced genomes\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe genomes of six candidate strains were examined: BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, and BR 11865\u003csup\u003eT\u003c/sup\u003e. The general characteristics of these genome assemblies are listed in Table 1. All sequencing coverages (\u0026ge;90X), completeness (\u0026gt;99.46%) and contamination (\u0026lt;1.31) were above the proposed minimal standard for taxonomic purposes [47, 48]. In general, the genome length of the new strains ranges between 6.3 and 7.1 Mb, with the GC content varying between 67.0% and 68.0%. These values are similar to those of the genomes of strains BR 11142\u003csup\u003eT\u003c/sup\u003e and BR 11140\u003csup\u003eT\u003c/sup\u003e that represent the formally described species \u003cem\u003eN. amazonense\u003c/em\u003e, \u003cem\u003eN. iridis\u003c/em\u003e, and \u003cem\u003eN. viridazoti\u003c/em\u003e (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe topological arrangement of our phylogenomic tree showed evolutionary convergence among our new strains and the type strain of previously described species \u003cem\u003eN. viridazoti\u003c/em\u003e and \u003cem\u003eN. amazonense,\u0026nbsp;\u003c/em\u003ewhereas strain DSM 22198\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN. iridis\u003c/em\u003e appeared to be evolutionarily divergent from our samples (Fig. 2). We discuss the phylograms for the two cases. In case 1, the BR 11865\u003csup\u003eT\u003c/sup\u003e genome shared an evolutionary origin with BR 11142\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN. amazonense\u003c/em\u003e species. In contrast, the BR 11164\u003csup\u003eT\u003c/sup\u003e and BR 11163\u003csup\u003eT\u003c/sup\u003e genomes showed an evolutionary connection with common ancestors of BR 11140\u003csup\u003eT\u003c/sup\u003e of the \u003cem\u003eN. viridazoti\u003c/em\u003e species. The genomes of the other strains (BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, and BR 11828\u003csup\u003eT\u003c/sup\u003e) formed two distinct phylogenomic clades, indicating evolutionary divergence without genetic sharing with the discussed species. These findings align with the established concepts of species formation, emphasizing anagenesis in case 1 and cladogenesis in case 2. Previous studies have demonstrated how these concepts help determine species formation and understand evolutionary convergence and divergence [45].\u003c/p\u003e\n\u003cp\u003eTo catalog the functional variability across our bacterial populations, whole functional genome analyses were performed to measure the representation of the entire collection of functional genomic sequences in our genome set (Fig. 3). The whole functional genome comprised 4453 protein families representing the pangenome, of which 2178 belong to the core. Unique protein families are shown next to each strain. The whole functional genome was composed of functional genes related to the metabolism of sulfur, nitrogen, and amino acids, whereas DNA and RNA processing functions were shared among all analyzed genomes. Finally, cell signaling and regulation as well as stress responses, defense, and virulence belonged to the most specific functional sequences of each genome.\u003c/p\u003e\n\u003cp\u003eThe ANIb percentages for the strain DSM 22198\u003csup\u003eT\u0026nbsp;\u003c/sup\u003eof \u003cem\u003eN. iridis\u003c/em\u003e and strains BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, and BR 11865\u003csup\u003eT\u003c/sup\u003e were consistently less than 85% (Table 2). Similarly, a comparison between strain BR 11140\u003csup\u003eT\u003c/sup\u003e and the new strains yielded values less than 94.1%. In another set of comparisons, the same strains against BR 11142\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN. amazonense\u003c/em\u003e resulted in values consistently lower than 93.5%, except for strain BR 11185\u003csup\u003eT\u003c/sup\u003e, which was 94.0% (Table 2).\u003c/p\u003e\n\u003cp\u003eComparisons among our new strains indicated that BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, and BR 11865\u003csup\u003eT\u003c/sup\u003e consistently exhibited ANI values less than 92.5% compared to all other strains (Table 2). In contrast, strains BR 11752\u003csup\u003eT\u003c/sup\u003e and BR 11622\u003csup\u003eT\u003c/sup\u003e showed values less than 93% in most comparisons and less than 95.5% in the comparison between themselves (Table 2).\u003c/p\u003e\n\u003cp\u003eThe dDDH values between the new strains and the type strains of \u003cem\u003eN. amazonense\u003c/em\u003e, \u003cem\u003eN. iridis\u003c/em\u003e, and \u003cem\u003eN. viridazoti\u003c/em\u003e were less than 60.1% (Table 2). This maximum value was also observed among the new strains, except for the comparison between strains BR 11752\u003csup\u003eT\u003c/sup\u003e and BR 11622\u003csup\u003eT\u003c/sup\u003e, for which the value was 66.0% (Table 2).\u003c/p\u003e\n\u003cp\u003eANI and dDDH have emerged as valuable tools for delineating bacterial species, offering more precise and reproducible alternatives to traditional methods based on single genes. ANI calculates the average nucleotide identity between two genomic sequences, with values above a certain threshold (typically 95\u0026ndash;96%) commonly considered indicative of species boundaries [27, 28]. Similarly, dDDH estimates the DNA\u0026ndash;DNA hybridization based on genomic comparisons and provides a numerical measure of genomic relatedness between bacterial strains. Generally, dDDH values greater than 70% indicate species boundary [29]. Both ANI and dDDH have been widely utilized in bacterial taxonomy, contributing to a more accurate and standardized approach for defining bacterial species. Therefore, our results, considering the phylogeny of 16S rRNA, \u003cem\u003erecA\u003c/em\u003e, phylogenomic, MALDI-TOF profile, ANI values, and dDDH, confirmed that six new species were identified within the genus\u003cem\u003e\u0026nbsp;Nitrospirillum\u003c/em\u003e. The strains BR 11865\u003csup\u003eT\u003c/sup\u003e, BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, and BR 11622\u003csup\u003eT\u003c/sup\u003e were designated as type strains of the new species that we named \u003cem\u003eN. bahiense\u003c/em\u003e, \u003cem\u003eN. guanabarense\u003c/em\u003e, \u003cem\u003eN. guaranorum\u003c/em\u003e, \u003cem\u003eN. karajorum\u003c/em\u003e, \u003cem\u003eN. guiasense,\u003c/em\u003e and \u003cem\u003eN. pindoramense,\u0026nbsp;\u003c/em\u003erespectively.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChemotaxonomic markers\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eChemotaxonomic characterization has largely been replaced by genomic analysis for species delineation, although it remains valuable for genus characterization. Previous studies on \u003cem\u003eNitrospirillum\u003c/em\u003e species, including \u003cem\u003eN. amazonense\u003c/em\u003e and \u003cem\u003eN. iridis\u003c/em\u003e, show a predominance of the fatty acids C18:1 \u0026omega;7c, C18:1 2-OH, and C16:0 3-OH. Polar lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyldimethylethanolamine, with ubiquinone Q10 being the major quinone [7, 46].\u003c/p\u003e\n\u003cp\u003eHere, we tested an alternative to traditional laboratory methods such as \u003cem\u003ein silico\u003c/em\u003e chemotaxonomy. This approach involves the analysis of gene sequences involved in the synthesis and metabolism of fatty acids, polar lipids, and ubiquinone [32-35]. The analyzed DNA sequences focused on genes related to ubiquinone side chain synthesis (decaprenyl diphosphate synthase), fatty acid synthesis (acyl-CoA thioesterase), fatty acid metabolism and elongation (the fadN-fadA-fadE operon), and phospholipid synthesis (acyl-ACP phosphate acyltransferase) (Table S3). Phylogenetic trees were constructed using the protein sequences of the respective genes (Fig. S4) showed, in general, topologies in accordance with phylogenetic-based 16S rRNA and \u003cem\u003erecA\u003c/em\u003e and the phylogram based on the whole genome (Fig. 1, Fig. 2, Fig. S1, Fig S2). As reported previously [12]. the analysis of chemotaxonomic marker genes showed that the type strains of \u003cem\u003eN. amazonense\u003c/em\u003e and \u003cem\u003eN. viridazoti\u003c/em\u003e generally diverged from each other and were positioned apart of strain DSM 22198\u003csup\u003eT\u003c/sup\u003e\u003cem\u003e\u0026nbsp;of N. iridis\u003c/em\u003e and far from strain BR 11142\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eA. brasilense\u003c/em\u003e, which was used as an out-group (Fig. S4). These new strains consistently formed unique clades, particularly those related to ubiquinone production, fatty acid metabolism, and phospholipid synthesis (Fig. S4 A, B, D). For the four genes analyzed, the new strains formed three groups with greater or lesser similarity: i) the strains BR 11828\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, and BR 11752\u003csup\u003eT\u003c/sup\u003e; ii) BR 11163\u003csup\u003eT\u003c/sup\u003e and BR 11164\u003csup\u003eT\u003c/sup\u003e; and iii) BR 11865\u003csup\u003eT\u003c/sup\u003e, which tended to be grouped with strains BR 11140\u003csup\u003eT\u003c/sup\u003e and BR 11142\u003csup\u003eT\u003c/sup\u003e, except for the genes related to phospholipid synthesis (\u003cem\u003eYciA\u003c/em\u003e), where BR 11140\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN. viridazoti\u003c/em\u003e stands out (Fig. S4). Therefore, the analysis of chemotaxonomic marker genes was robust for confirming new \u003cem\u003eNitrospirillum\u003c/em\u003e species and corroborated the results of other phylogenetic and phylogenomic analyses.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003enifHDK phylogenetic analysis\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eConsidering the results obtained from the pan-core accessory genome steps, we conducted further analyses to clarify these results. \u0026nbsp; Specifically, we explored the distribution of genes associated with the oxygen-sensitive nitrogenase complex, focusing on molybdenum nitrogenase. Molybdenum nitrogenase, encoded by \u003cem\u003enifH\u003c/em\u003e, \u003cem\u003enifD\u003c/em\u003e, and \u003cem\u003enifK\u003c/em\u003e genes is the most prevalent protein and is shared across all genomes. Phylogenetic analyses point to a functional relationship between \u003cem\u003eNitrospirillum\u003c/em\u003e genomes, considering their nitrogen fixation activities. The phylogeny of the \u003cem\u003enifHDK\u003c/em\u003e sequences correlated with the results obtained from the phylogenomic analysis, indicating the formation of the same phylogenetic clades. The only exception was the strain BR 11164\u003csup\u003eT\u003c/sup\u003e, whose \u003cem\u003enifHDK\u003c/em\u003e genes were closer to DSM 22198\u003csup\u003eT\u003c/sup\u003e, which was isolated in South Korea (Fig. 4). This was surprising, as the strain BR 11164\u003csup\u003eT\u003c/sup\u003e was isolated in the same geographic area as the other new strains.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMass spectrometry and fatty acid analysis\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDirect colony application demonstrated high spectral quality and reproducibility. Furthermore, we observed high consistency between replicates for each strain under identical conditions. Although variations in the composition and characteristics of the culture medium and the bacterial growth time led to subtly different spectral profiles for the same strains, these distinctions did not significantly affect their overall identification. Consequently, there was no need for additional steps such as culture broth handling or protein extraction methods, as mentioned in other studies [49].\u003c/p\u003e\n\u003cp\u003eMALDI-TOF MS analysis generated distinct mass spectra of whole-cell proteins for all the studied strains, revealing 39\u0026ndash;41 well-defined peaks of high quality (Fig. 5; Fig. S3). These peaks vary from 3,097.9 to 11,352.3 in mass. Using these protein mass profiles, a cladogram was constructed, which revealed that all strains shared similarity among no more than 85% (case of the strains BR 11662\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, and BR 11752\u003csup\u003eT\u003c/sup\u003e) or less than 75% in the case of other strains, including BR 11142\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN. amazonense\u003c/em\u003e and BR 11140\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eN. viridazoti\u003c/em\u003e (Fig. 5). The observed similarity level confirms that the strains represent new taxa, [50] and the result corroborated the positioning of the strains in different groups in the phylogenetic 16S rRNA and \u003cem\u003erecA\u003c/em\u003e tree and the phylogenomic tree (Fig. 1 and 2).\u003c/p\u003e\n\u003cp\u003eThe fatty acid profiles of our strains showed notable similarity of new strains with the strain BR 11140ᵀ, which was analyzed simultaneously. Summed Feature 8, which includes C\u003csub\u003e18:1\u003c/sub\u003e \u003cem\u003e\u0026omega;\u003c/em\u003e7c and/or C\u003csub\u003e18:1\u003c/sub\u003e \u003cem\u003e\u0026omega;\u003c/em\u003e6c, represented the dominant fraction in the profiles of all strains (ranging from 40.2% to 44.6%) (Table S99), like what has been previously reported for DSM 22198ᵀ (\u003cem\u003eN. iridis\u003c/em\u003e) and BR 11142ᵀ (\u003cem\u003eN. amazonense\u003c/em\u003e) [7]. Furthermore, C\u003csub\u003e18:1\u003c/sub\u003e 2OH was consistently present in all strains, as were C\u003csub\u003e16:1\u003c/sub\u003e \u003cem\u003e\u0026omega;\u003c/em\u003e5c (5.0%\u0026ndash;6.9%) and C\u003csub\u003e16:0\u003c/sub\u003e 3OH (5.2%\u0026ndash;10.3%). Notably, strain BR 11865ᵀ exhibited the most divergent profile, with a substantially higher percentage of C\u003csub\u003e16:1\u003c/sub\u003e \u003cem\u003e\u0026omega;\u003c/em\u003e5c (11.28%) and C\u003csub\u003e16:0\u0026nbsp;\u003c/sub\u003e3OH (10.31%), and a lower level of C\u003csub\u003e16:0\u003c/sub\u003e (3.14%) compared to the other strains (Table S1). Altogether, the chemotaxonomic results clearly indicate that the proposed type strains for the new species exhibit a fatty acid composition consistent with that observed for members of the genus \u003cem\u003eNitrospirillum\u003c/em\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMorphology of colonies, cells, and physiological assays\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll six candidate type strains of the new species grew at the tested pH levels in solid LGI medium, although more abundant growth was observed between pH 5 and 8. We did not thoroughly test different temperatures for the growth of these strains, but previous studies have shown that strains affiliated with the genus \u003cem\u003eNitrospirillum\u003c/em\u003e have an optimal growth temperature between 30 and 32 \u0026deg;C, and they can grow often at higher temperatures [15]. Analysis of the colony morphology on homemade potato agar medium [8] revealed that the strains formed white, circular, large, and flat colonies with raised margins. Most of the strains exhibited agglutinated, wrinkled colonies, a characteristic similar to that observed in \u003cem\u003eN. amazonense\u003c/em\u003e (Fig. S5). However, this characteristic was not observed in the strains BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11752\u003csup\u003eT\u003c/sup\u003e, and BR 1182\u003csup\u003eT\u003c/sup\u003e, which displayed slightly wet and elevated colonies.\u003c/p\u003e\n\u003cp\u003eWhen the strains were grown on potato agar medium supplemented with 10% sucrose, strains BR 11164\u003csup\u003eT\u003c/sup\u003e and BR 11752\u003csup\u003eT\u003c/sup\u003e appeared to be less affected by the high sugar concentrations (Fig. S5). In contrast, strains BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, BR 11828\u003csup\u003eT\u003c/sup\u003e, and BR 11865\u003csup\u003eT\u003c/sup\u003e exhibited moderate growth but formed smaller colonies. Strain BR 11140\u003csup\u003eT\u003c/sup\u003e showed poor growth, with small colonies, whereas strain BR 11142\u003csup\u003eT\u003c/sup\u003e produced only a few small colonies in the presence of 10% sugar, as previously observed [12].\u003c/p\u003e\n\u003cp\u003eComparative analysis of cell morphology revealed that most of the new strains were curved or highly curved, spiral, or vibroid, and accumulated significant amounts of polyhydroxybutyrate (PHB) when grown in nitrogen-free LGI semi-solid medium (Fig. S6). BR 11865\u003csup\u003eT\u003c/sup\u003e had longer spiral cells, whereas BR 11163\u003csup\u003eT\u003c/sup\u003e had larger spiral cells. Transmission electron micrography also revealed a large accumulation of PHB in the cells of strains BR 11163ᵀ, BR 11164ᵀ, BR 11622ᵀ, BR 11752ᵀ, BR 11828ᵀ, and BR 11865ᵀ, as was also observed for strains BR 11142ᵀ and BR 11140ᵀ (Fig. S8). All analyzed strains exhibited polar monotrichous flagella and cell sizes ranging from 1.3 to 1.8 \u0026mu;m, which were generally slightly smaller than the sizes observed for strains BR 11142ᵀ and BR 11140ᵀ (Fig. S8).\u003c/p\u003e\n\u003cp\u003eOverall, the new strains formed a characteristic veil-like pellicle on the subsurface of the semisolid LGI medium. Strains BR 11163\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, and BR 11142\u003csup\u003eT\u003c/sup\u003e slightly acidified the medium, whereas strain BR 11865\u003csup\u003eT\u003c/sup\u003e caused strong acidification unlike the other strains, which maintained a neutral pH (Fig. S7). The pellicle thickness varied among the strains, with strain BR 11164\u003csup\u003eT\u003c/sup\u003e and BR 11865\u003csup\u003eT\u003c/sup\u003e producing thicker pellicles than BR 11140\u003csup\u003eT\u003c/sup\u003e. In contrast, the other strains, including strain BR 11142\u003csup\u003eT\u003c/sup\u003e, showed thinner veil-like pellicles on the subsurface of the medium (Fig. S7).\u003c/p\u003e\n\u003cp\u003eThe formation of a pellicle on the subsurface of the semi-solid medium is reportedly an indicator of the capacity for BNF through nitrogenase activity [51]. To confirm this activity in the new \u003cem\u003eNitrospirillum\u003c/em\u003e strains, an assay was conducted to evaluate the ability to reduce acetylene, given that nitrogenase can reduce acetylene to ethylene [51]. It was observed that all six new candidate type strains, in addition to the strains BR 11140\u003csup\u003eT\u003c/sup\u003e, BR 11142\u003csup\u003eT\u003c/sup\u003e, and DSM 22198\u003csup\u003eT\u003c/sup\u003e, produced between 496.2 and 648.6 nmol of C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e mL\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e (Table S4), confirming the diazotrophic capacity of all strains. The amount of ethylene produced was similar to previously reported values for this type of assay with diazotrophic associative bacteria [52].\u003c/p\u003e\n\u003cp\u003eAll tested strains tolerated chloramphenicol, azithromycin, tetracycline, and vancomycin and were sensitive to penicillin. The strain BR11865\u003csup\u003eT\u003c/sup\u003e was sensitive to kanamycin, gentamicin, neomycin, and streptomycin, whereas strains BR 11140\u003csup\u003eT\u003c/sup\u003e and BR 11142\u003csup\u003eT\u003c/sup\u003e were not. The strains BR 11163\u003csup\u003eT\u003c/sup\u003e and BR 11164\u003csup\u003eT\u003c/sup\u003e were the most tolerant and differed from each other because BR 11163\u003csup\u003eT\u003c/sup\u003e was tolerant to azithromycin. The strains BR 11828\u003csup\u003eT\u003c/sup\u003e, BR 11622\u003csup\u003eT\u003c/sup\u003e, and BR 11752\u003csup\u003e\u0026nbsp;T\u003c/sup\u003e were the most sensitive strains, with only the strain BR 11622\u003csup\u003eT\u003c/sup\u003e and BR 11752\u003csup\u003eT\u003c/sup\u003e being sensitive to amoxicillin, and only the latter being sensitive to ampicillin (Table S5).\u003c/p\u003e\n\u003cp\u003eBacterial antibiotic resistance investigated through the k-mer-based approach showed that only the gene sequences corresponding to the antimicrobial resistance (AMR)mechanism for efflux pumps varied depending on the genome analyzed. Other genes were present in all the strains, indicating antibiotic resistance targets (Table S6). The presence of AMR-related genes (even full-length genes) in each genome does not directly imply an antibiotic-resistant phenotype. Transmembrane proteins expel a wide range of toxic substances, including antibiotics, from the cell, thereby reducing the intracellular concentration of the drug and consequently its effectiveness [53]. In bacteria of the genus \u003cem\u003eNitrospirillum\u003c/em\u003e, which are important in terrestrial environments, particularly in nitrogen fixation, antibiotic resistance mediated by efflux pumps can be a critical adaptation that allows them to survive in soils contaminated with antimicrobial compounds [54].\u003c/p\u003e\n\u003cp\u003eRegarding source usage/tolerance, all strains showed a positive reaction in 25 sources tested by the API and Biolog kits, indicating that they could assimilate the carbon source or other nutrients, or were not inhibited by them. In contrast, none of the strains reacted in 16 tests. Thus, variability in responses was observed for more than 50 tests using the Biolog kit. Strain BR 11162\u003csup\u003eT\u003c/sup\u003e showed the highest number of positive reactions (69 in total), followed by strain BR 11164\u003csup\u003eT\u003c/sup\u003e. The other strains showed between 38 and 61 positive reactions. All strains were able to grow at pH 5\u0026ndash;8, tolerate 1% sodium lactate, and only strains BR 11865\u003csup\u003eT\u003c/sup\u003e and BR 11164\u003csup\u003eT\u003c/sup\u003e did not grow in the presence of 1% NaCl; none of them showed a positive reaction at concentrations of 4 and 8% NaCl (Fig. S5).\u003c/p\u003e\n\u003cp\u003eThe heatmap generated using the Biolog test reaction data indicated that the tested strains had different phenotypic fingerprints (Fig. S8). Strains BR 11865\u003csup\u003eT\u003c/sup\u003e, BR 11164\u003csup\u003eT\u003c/sup\u003e, and BR 11622\u003csup\u003eT\u003c/sup\u003e formed a cluster further away from BR 11142\u003csup\u003eT\u003c/sup\u003e and BR 11140\u003csup\u003e\u0026nbsp;T\u003c/sup\u003e. In contrast, strains BR 11828\u003csup\u003eT\u003c/sup\u003e, BR 11163\u003csup\u003eT\u003c/sup\u003e, and BR 11752\u003csup\u003eT\u003c/sup\u003e also formed a cluster. However, with greater similarity to strains typical of \u003cem\u003eN.\u0026nbsp;amazonense\u003c/em\u003e and \u003cem\u003eN. viridazoti\u003c/em\u003e (Fig. S8). This analysis showed a slight discrepancy between the 16S rRNA and \u003cem\u003erecA\u003c/em\u003e analyses. Strain BR 11865\u003csup\u003eT\u003c/sup\u003e and BR 11164\u003csup\u003eT\u003c/sup\u003e showed greater similarity to BR 11142\u003csup\u003eT\u003c/sup\u003e and BR 11140\u003csup\u003eT\u003c/sup\u003e than to BR 11828\u003csup\u003eT\u003c/sup\u003e and BR 11752\u003csup\u003eT\u003c/sup\u003e (Fig. S8).\u003c/p\u003e\n\u003cp\u003eOverall, the enzymatic activities evaluated by the API kit were very similar among all new strains, with the only difference being strains BR 11164\u003csup\u003eT\u003c/sup\u003e and BR 11865\u003csup\u003eT\u003c/sup\u003e, which were negative for arginine dihydrolase. In contrast, the other strains were positive (Table S5). All new strains were positive for urease, catalase, and oxidase, which are typical enzymes for bacterial phenotypic characterization studies, whereas only strain BR 11622\u003csup\u003eT\u003c/sup\u003e tested positive for cellulases (Table S5).\u003c/p\u003e\n\u003cp\u003eNone of the proposed type strains solubilized calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, or zinc phosphate. However, all strains exhibited siderophore production capacity, a trait indicated by halos in the culture medium, including strains BR 11142\u003csup\u003eT\u003c/sup\u003e and BR 11140\u003csup\u003eT\u003c/sup\u003e of the \u003cem\u003eN. amazonense\u003c/em\u003e, and \u003cem\u003eN. viridazoti\u003c/em\u003e, respectivelly It was also observed that all strains showed indole acetic production, as observed for strains BR\u0026nbsp;11142\u003csup\u003eT\u003c/sup\u003e and BR 11140\u003csup\u003eT\u003c/sup\u003e. In this characteristic, strain BR 11164\u003csup\u003eT\u003c/sup\u003e stood out, producing more than 17 \u0026micro;g/mL indole compounds in 72 h (Table S5). Notably, in the API test analysis, indole production (L-tryptophan test) was negative for all strains, indicating the need for further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study contributes to our understanding of bacterial diversity within the genus\u003cem\u003e\u0026nbsp;Nitrospirillum\u003c/em\u003e. The taxonomic framework based on phylogenetic and phylogenomic analyses provides a comprehensive view of the genus \u003cem\u003eNitrospirillum\u003c/em\u003e evolution. This genus is distributed across diverse biomes, including the Amazon, Atlantic Forest, and Cerrado, and possesses the ability to colonize dozens of plant species, with a particular focus on Poaceae, which is the most studied genus to date. ANI and dDDH support the proposition of six new species within the genus \u003cem\u003eNitrospirillum\u003c/em\u003e: \u003cem\u003eN. bahiense\u003c/em\u003e, \u003cem\u003eN. guanabarense\u003c/em\u003e, \u003cem\u003eN. guaranorum\u003c/em\u003e, \u003cem\u003eN. karajorum\u003c/em\u003e, \u003cem\u003eN. guiasense\u003c/em\u003e and \u003cem\u003eN. pindoramense\u003c/em\u003e. Analyses of \u003cem\u003enifHDK\u003c/em\u003e genes, mass spectrometry profiles, FAME and physiological tests confirmed the distinctiveness of the proposed species and its ability to fix nitrogen \u003cem\u003ein vitro\u003c/em\u003e. These findings expand our systematic understanding of bacterial classification and demonstrate the critical role of preserving and investigating microbial repositories to elucidate prokaryotic diversity. The biotechnological potential of this genetic resource is elucidated based on the results presented here.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eNitrospirillum bahiense\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003esp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNitrospirillum bahiense\u003c/em\u003e (ba.hi.en\u0026rsquo;se. N.L. neut. adj. \u003cem\u003ebahiense\u003c/em\u003e, in reference to the state of Bahia, Brazil, in honor of its cultural and historical importance).\u003c/p\u003e\n\u003cp\u003eThis species displays major characteristics of the genus. The cells are Gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. Optimum temperature for growth is approximately 30 \u0026deg;C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11865\u003csup\u003eT\u003c/sup\u003e (UCCCB 233\u003csup\u003eT\u003c/sup\u003e) is resistant to amoxicillin, ampicillin, chloramphenicol, azithromycin, penicillin, tetracycline, and vancomycin and sensitive to rifampicin, kanamycin, gentamicin, neomycin, cephalexin, streptomycin, and erythromycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, sucrose, \u003cem\u003e\u0026alpha;\u003c/em\u003e-D-glucose, D-mannose, D-galactose, L-fucose, and others, but does not grow in D-raffinose, D-serine, and D-sorbitol. The enzymatic tests for the type strain showed positive reaction to glucokinase, urease, \u0026beta;-glucosidase, catalase, and oxidase, and negative reactions to nitrate reduction. The ability to produce ethylene, indoles, and siderophores was observed, but the capability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, and zinc phosphate was not observed. The cell size of strain BR 11865\u003csup\u003eT\u003c/sup\u003e ranges from 1.7 to 1.8 \u0026mu;m. Strains of \u003cem\u003eN. bahiense\u003c/em\u003e were isolated from the roots \u003cem\u003eBrachiaria humidicola\u003c/em\u003e, \u003cem\u003eB. brizantha,\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e. The species were distinguishable by molecular and genomic approaches, and via MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11865\u003csup\u003eT\u003c/sup\u003e was isolated from the roots of \u003cem\u003eB. humidicola\u003c/em\u003e in Bahia, Brazil. The genome of the type strain has a size of 7.0 Mbp with a DNA G+C content of 67.5%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eNitrospirillum guanabarense\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003esp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNitrospirillum guanabarense\u003c/em\u003e (gua.na.bar.en\u0026rsquo;se. N.L. neut. adj. \u003cem\u003eguanabarense\u003c/em\u003e, named after the indigenous word for the city of Rio de Janeiro, Brazil, honors the cultural and historical significance of the term).\u003c/p\u003e\n\u003cp\u003eThis species displays major characteristics of the genus. The cells are Gram-negative and non-sporulating. All strains grow well on LGI and DYGS media. Optimum temperature for growth is approximately 30 \u0026deg;C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11163\u003csup\u003eT\u003c/sup\u003e (UCCCB 228\u003csup\u003eT\u003c/sup\u003e) is resistant to amoxicillin, ampicillin, rifampicin, chloramphenicol, cephalexin, penicillin, and erythromycin and is sensitive to azithromycin, kanamycin, gentamicin, neomycin, vancomycin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, sucrose, \u003cem\u003e\u0026alpha;\u003c/em\u003e-D-glucose, D-mannose, D-galactose, L-fucose, and D-fructose, and tolerate 1% NaCl. The enzymatic tests for the type strain showed a positive reaction to glucokinase, urease, \u0026beta;-glucosidase, catalase, and oxidase, and a negative reaction to nitrate reduction. The ability to produce ethylene, indoles, and siderophores was observed for the type strain but not to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, or zinc phosphate. The cell size of strain BR 111163\u003csup\u003eT\u003c/sup\u003e ranges from 1.2 to 1.5 \u0026mu;m. Strains of \u003cem\u003eN. guanabara\u003c/em\u003e were isolated from the roots of \u003cem\u003eSorghum vulgare\u003c/em\u003e. The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. Type strain BR 11163\u003csup\u003eT\u003c/sup\u003e was isolated from the roots of \u003cem\u003eSorghum vulgare\u003c/em\u003e in Rio de Janeiro, Brazil. The genome of the type strain has a size of 7.1 Mbp with a DNA G+C content of 67.6%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eNitrospirillum guaranorum\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003esp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNitrospirillum guaranorum\u003c/em\u003e (\u003cu\u003egua.ra.no\u003c/u\u003e\u0026rsquo;rum. N.L. gen. pl. n. \u003cem\u003eguaranorum\u003c/em\u003e, named in honor of the Guarani people, the principal indigenous population originating from the Southeast of Brazil).\u003c/p\u003e\n\u003cp\u003eThis species displays major characteristics of the genus. The cells are Gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. Optimum temperature for growth is approximately 30 \u0026deg;C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11164\u003csup\u003eT\u003c/sup\u003e (UCCCB 229\u003csup\u003eT\u003c/sup\u003e) is resistant to amoxicillin, ampicillin, rifampicin, azithromycin, chloramphenicol, cephalexin, penicillin, and erythromycin and sensitive to kanamycin, gentamicin, neomycin, vancomycin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, sucrose, \u003cem\u003e\u0026alpha;\u003c/em\u003e-D-glucose, D-mannose, D-galactose, L-fucose, D-fructose, \u003cem\u003e\u0026alpha;\u003c/em\u003e-D-lactose, and D-turanose. The enzymatic tests for the type strain show a positive reaction to glucokinase, urease, \u0026beta;-glucosidase, catalase, and oxidase, and a negative reaction to nitrate reduction and cellulase. The ability to produce ethylene, indoles, and siderophores was observed, but not the capability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, and zinc phosphate. The cell size of strain BR 11164\u003csup\u003eT\u003c/sup\u003e ranges from 1.5 to 1.7 \u0026mu;m. Strains of \u003cem\u003eN. guaiasensis\u003c/em\u003e have been isolated from the roots \u003cem\u003eOryza sativa\u003c/em\u003e. The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11164\u003csup\u003eT\u003c/sup\u003e was isolated from the roots of \u003cem\u003eOryza sativa\u0026nbsp;\u003c/em\u003ein Rio de Janeiro, Brazil. The genome of the type strain has a size of 7.0 Mbp with a DNA G+C content of 68.2%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eNitrospirillum karajorum\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003esp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNitrospirillum karajorum\u003c/em\u003e (ka.ra.jo\u0026rsquo;rum. N.L. gen. pl. n. \u003cem\u003ekarajorum\u003c/em\u003e of the Karaj\u0026aacute; people, in reference to the indigenous Karaj\u0026aacute; people, longstanding inhabitants along the Araguaia River with territorial occupations across the states of Goi\u0026aacute;s, Tocantins, Mato Grosso, and Par\u0026aacute;).\u003c/p\u003e\n\u003cp\u003eThis species displays major characteristics of the genus. The cells were Gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. Optimum temperature for growth is approximately 30 \u0026deg;C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11752\u003csup\u003eT\u003c/sup\u003e (UCCCB 231\u003csup\u003eT\u003c/sup\u003e) was resistant at tested levels to penicillin, tetracycline, vancomycin, and erythromycin and sensitive to amoxicillin, ampicillin, rifampicin, chloramphenicol, azithromycin, kanamycin, gentamicin, neomycin, cephalexin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, \u003cem\u003e\u0026alpha;\u003c/em\u003e-D-glucose, D-mannose, D-galactose, L-fucose, D-fructose, acetic acid, and pectin, and tolerate 1% NaCl. The enzymatic tests for the type strain showed positive reaction to nitrate reduction, glucokinase, urease, \u0026beta;-glucosidase, arginase, citrase, \u0026beta;-galactosidase, and a negative reaction to cellulase. The ability to produce ethylene, indoles, and siderophores was observed for the strain BR 11752\u003csup\u003eT\u003c/sup\u003e, but its ability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, and zinc phosphate was not observed. The cell size of strain BR 11752\u003csup\u003eT\u003c/sup\u003e ranges from 1.4 to 1.6 \u0026mu;m. Strains of \u003cem\u003eN. karajorum\u003c/em\u003e have been isolated from the roots \u003cem\u003eOryza sativa\u003c/em\u003e. The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11752\u003csup\u003eT\u003c/sup\u003e was isolated from the roots of \u003cem\u003eOryza sativa\u0026nbsp;\u003c/em\u003ein Rio de Janeiro, Brazil. The genome of the type strain has a size of 6.5 Mbp and DNA G+C content of 67.3l%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eNitrospirillum guiasense\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003esp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNitrospirillum guiasense\u003c/em\u003e (gu.ias.en\u0026rsquo;se. N.L. neut. adj. \u003cem\u003eguiasense\u003c/em\u003e about the Guai\u0026aacute;s indigenous tribe, which originated from the name Goi\u0026aacute;s state, Brazil, in honor of the cultural and historical significance of this term).\u003c/p\u003e\n\u003cp\u003eThis species displays major characteristics of the genus. The cells were Gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. Optimum temperature for growth is approximately 30 \u0026deg;C, and optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11828\u003csup\u003eT\u003c/sup\u003e (UCCCB 232\u003csup\u003eT\u003c/sup\u003e) was resistant to ampicillin, penicillin, and amoxicillin, and sensitive to tetracycline, vancomycin, erythromycin, rifampicin, chloramphenicol, azithromycin, kanamycin, gentamicin, neomycin, cephalexin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, \u003cem\u003e\u0026alpha;\u003c/em\u003e-D-glucose, D-mannose, D-galactose, L-fucose, D-fructose and D-glucuronic acid, and tolerate 1% NaCl. The enzymatic tests for the type strain showed positive reaction to nitrate reduction, glucocinase, urease, \u0026beta;-glucosidase, arginase, citrase, \u0026beta;-galactosidase, and a negative reaction to cellulase. The ability to produce ethylene, indoles, and siderophore was observed, but not the capability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, or zinc phosphate. The cell size of strain BR 11828\u003csup\u003eT\u003c/sup\u003e ranges from 1.5 to 1.8 \u0026mu;m. Strains of \u003cem\u003eN. guaiasensis\u003c/em\u003e have been isolated from the roots \u003cem\u003eOryza sativa\u003c/em\u003e. The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11828\u003csup\u003eT\u003c/sup\u003e was isolated from the roots of \u003cem\u003eOryza sativa\u0026nbsp;\u003c/em\u003ein Rio de Janeiro, Brazil. The genome of the type strain has a size of 6.3 Mbp with a DNA G+C content of 67.7%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eNitrospirillum pindoramense\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003esp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNitrospirillum pindoramense\u0026nbsp;\u003c/em\u003e(pin.do.ra.men\u0026rsquo;se. N.L. neut. adj. \u003cem\u003epindoramense\u003c/em\u003e, derived from the term \u0026ldquo;Pindorama\u0026rdquo;, which translates to \u0026quot;Land of Palm Trees\u0026quot; in reference to how indigenous people referred to the lands of Brazil before the arrival of the Portuguese)\u003c/p\u003e\n\u003cp\u003eThis species displays major characteristics of the genus. The cells were gram-negative and non-sporulating. All strains grew well on LGI and DYGS media. The optimum temperature for growth is approximately 30 \u0026deg;C, and the optimum pH for growth ranges from 5 to 8 in LGI medium. The type strain BR 11622\u003csup\u003eT\u003c/sup\u003e (UCCCB 230\u003csup\u003eT\u003c/sup\u003e)\u003csup\u003e\u0026nbsp;\u003c/sup\u003eis resistant to penicillin, ampicillin, tetracycline, vancomycin, and erythromycin and sensitive to vancomycin, erythromycin, amoxicillin, rifampicin, chloramphenicol, azithromycin, kanamycin, gentamicin, neomycin, cephalexin, and streptomycin. The strain can grow in different carbon sources, typically D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, \u003cem\u003e\u0026alpha;\u003c/em\u003e-D-glucose, D-mannose, D-galactose, L-fucose, D-fructose, and acetic acid, and tolerate 1% NaCl. The enzymatic tests for the type strain showed a positive reaction to nitrate reduction, glucocinase, urease, \u0026beta;-glucosidase, citrase, \u0026beta;-galactosidase, arginase, and a negative reaction to cellulase. The ability to produce ethylene and indoles was observed, but not the capability to solubilize calcium phosphate, aluminum phosphate, zinc oxide, zinc carbonate, or zinc phosphate. Strains of \u003cem\u003eN. pindoramensis\u003c/em\u003e have been isolated from the roots \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eZea mays,\u003c/em\u003e and \u003cem\u003eBrachiaria brizantha\u003c/em\u003e. The cell size of strain BR 11622\u003csup\u003eT\u003c/sup\u003e ranges from 1.4 to 1.6 \u0026mu;m. The species were distinguishable by molecular and genomic approaches, and by using MALDI-TOF MS analysis of whole-cell mass spectra. The type strain BR 11622\u003csup\u003eT\u003c/sup\u003e was isolated from the roots of \u003cem\u003eZea mays\u0026nbsp;\u003c/em\u003ein Rio de Janeiro, Brazil. The genome of the type strain has a size of 6.6 Mbp with a DNA G+C content of 67.2%.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no ethical issues exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in the National Center for Biotechnology - NCBI. Information and Data is provided within the supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmbrapa (Brazilian Agricultural Research Corporation; INCT Plant Growth\u0026ndash;Promoting Microorganisms for Agricultural Sustainability and Environmental Responsibility (CNPq 465133/2014-2, Funda\u0026ccedil;\u0026atilde;o Arauc\u0026aacute;ria-STI-043/2019, CAPES); CNPq Productivity Grant for some of researchers and FAPERJ (Funda\u0026ccedil;\u0026atilde;o Carlos Chagas Filho de Amparo\u0026nbsp;\u0026agrave;\u0026nbsp;Pesquisa do Estado\u0026nbsp;do\u0026nbsp;Rio de Janeiro), projects E-26/201.074/2022 and E-26/210.303/2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJEZ coordinated the project, analyzed, and wrote the paper; SS, AFO, and EB performed the genomic analyses and assisted in writing the manuscript; NSF collaborated on DNA sequencing; JLSA, BJRA, ALS, and VMR collaborated on the nitrogen fixation test; LFWR collaborated on genome sequencing; FSD and KMOL collaborated on DNA sequencing and MALDI-TOF MS analyses; CNR collaborated on the phenotypic analysis; LHBS collaborated on MALDI-TOF MS analyses; and JIB collaborated on the phenotypic analysis and writing review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank CNPq (Brazilian National Council for Scientific and Technological Development) for productivity grants awarded to\u0026nbsp;some authors, CAPES (Coordination of Superior Level Staff Improvement) for student grants, and FAPERJ for Grant Program Cientista do Nosso Estado. We also thank Dr. Aharon Oren and Dr. Bernhard Schink for reviewing and suggesting new species names, Dr. Alane Vermelho for helping with the transmission electronic images and Dr. Paula Morais for helping with FAME analyzes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSoumare A, Diedhiou AG, Thuita M, Hafidi M, Ouhdouch Y, Gopalakrishnan S, et al. Exploiting biological nitrogen fixation: A route towards a sustainable agriculture. Plants. 2020;9. https://doi.org/.3390/plants9081011 \u003c/li\u003e\n\u003cli\u003eMohanty P, Singh PK, Chakraborty D, Mishra S, Pattnaik R. Insight into the role of PGPR in sustainable agriculture and environment. Frontiers in Sustainable Food Systems. 2021;5. https://doi.org/10.3389/fsufs.2021.667150.\u003c/li\u003e\n\u003cli\u003eNam JH, Thibodeau A, Qian YL, Qian MC, Park SH. Multidisciplinary evaluation of plant growth promoting rhizobacteria on soil microbiome and strawberry quality. AMB Express. 2023;13. https://doi.org/10.1186/s13568-023-01524-z.\u003c/li\u003e\n\u003cli\u003eSantos MS, Nogueira MA, Hungria M. Outstanding impact of \u003cem\u003eAzospirillum brasilense\u003c/em\u003e strains ab-v5 and ab-v6 on the Brazilian agriculture: Lessons that farmers are receptive to adopt new microbial inoculants. Rev Bras Cienc Solo. 2021;45. https://doi.org/10.36783/18069657RBCS20200128.\u003c/li\u003e\n\u003cli\u003eReis VM, Rios FA, Braz GBP, et al. Agronomic performance of sugarcane inoculated with \u003cem\u003eNitrospirillum amazonense\u003c/em\u003e (BR 11145). Revista Caatinga. 2020;33. https://doi.org/10.1590/1983-21252020v33n406rc.\u003c/li\u003e\n\u003cli\u003eMagalh\u0026atilde;es FM, Baldani JI, Souto SM, Kuykendall JR, D\u0026ouml;bereiner J. A new acid-tolerant \u003cem\u003eAzospirillum\u003c/em\u003e species. An Acad Bras Cienc. 1983;55:417\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eLin SY, Hameed A, Shen FT, Liu YC, Hsu YH, Shahina M, Lai WA, Young CC. Description of \u003cem\u003eNiveispirillum fermenti\u003c/em\u003e gen. nov., sp. nov., isolated from a fermentor in Taiwan, transfer of \u003cem\u003eAzospirillum irakense\u003c/em\u003e (1989) as \u003cem\u003eNiveispirillum irakense\u003c/em\u003e comb. nov., and reclassification of \u003cem\u003eAzospirillum amazonense\u003c/em\u003e (1983) as \u003cem\u003eNitrospirillum amazonense\u003c/em\u003e gen. nov. A Van Leeuwenhoek. 2014; 105. https://doi.org/10.1007/s10482-014-0176-6. \u003c/li\u003e\n\u003cli\u003eBaldani JI, Reis VM, Videira SS, Boddey LH, Baldani VLD. The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists. Plant Soil. 2014;384. https://doi.org/10.1007/s11104-014-2186-6.\u003c/li\u003e\n\u003cli\u003eMagalh\u0026atilde;es FMM. Nitrogen fixing bacteria isolated from diverse soils and grass roots in Amaz\u0026ocirc;nia. In: Vose P., Ruschel A. P., editors. Associative N\u003csub\u003e2\u003c/sub\u003e-Fixation. New York: CRC Press; 1981. p. 39\u0026ndash;48.\u003c/li\u003e\n\u003cli\u003eReis FB, Silva MF, Tekeira KRS, Urquiaga S, Reis VM. Identification of \u003cem\u003eAzospirillum amazonense\u003c/em\u003e isolates associated to Brachiaria spp. at different stages and growth conditions, and bacterial plant hormone production. Rev Bras Cienc Solo. 2004;28. https://doi.org/10.1590/s0100-06832004000100011.\u003c/li\u003e\n\u003cli\u003eHartmann A, Baldani JI. The Genus Azospirillum. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes: Volume 5: Proteobacteria: Alpha and Beta Subclasses. New York, NY: Springer New York; 2006. p. 115\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eBaldani JI, dos Santos Ferreira N, Shwab S, Reis VM, de Barros Soares LH, Sim\u0026otilde;es-Araujo JL, et al. \u003cem\u003eNitrospirillum viridazoti\u003c/em\u003e sp. nov., an Efficient nitrogen-fixing species isolated from grasses. Curr Microbiol. 2024;81:144. https://doi.org/10.1007/s00284-024-03665-1.\u003c/li\u003e\n\u003cli\u003eReis VM, Rios FA, Braz GBP, Constantin J, Hirata ES, Biffe DF. Agronomic performance of sugarcane inoculated with \u003cem\u003eNitrospirillum amazonense\u003c/em\u003e (BR 11145). Revista Caatinga. 2020;33. https://doi.org/10.1080/07352680091139213.\u003c/li\u003e\n\u003cli\u003eMagalh\u0026atilde;es FMM, D\u0026ouml;bereiner J. Ocorr\u0026ecirc;ncia de \u003cem\u003eAzospirillum amazonense\u003c/em\u003e em alguns ecossistemas da Amaz\u0026ocirc;nia. Rev Microbiol. 1984;15:246\u0026ndash;52.\u003c/li\u003e\n\u003cli\u003eBaldani JI, Baldani VLD. History on the biological nitrogen fixation research in graminaceous plants: Special emphasis on the Brazilian experience. Anais da Academia Brasileira de Ciencias. 2005;77. https://doi.org/10.1590/S0001-37652005000300014.\u003c/li\u003e\n\u003cli\u003eGaunt MW, Turner SL, Rigottier-Gois L, Lloyd-Macgilp SA, Young JPW. Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int J Syst Evol Microbiol. 2001;51. https://doi.org/10.1099/00207713-51-6-2037.\u003c/li\u003e\n\u003cli\u003eTamura Koichiro, Glen Stecher, Kumar Sudhir. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38, 3022-3027. https://doi.org/10.1093/molbev/msab120.\u003c/li\u003e\n\u003cli\u003eJukes TH, Cantor CR. Evolution of Protein Molecules. New York: Academic Press. 1969. pp. 21\u0026ndash;132.\u003c/li\u003e\n\u003cli\u003eTamura K. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol Biol Evol. 1992;9. https://doi.org/10.1093/oxfordjournals.molbev.a040752.\u003c/li\u003e\n\u003cli\u003eTajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989;123. https://doi.org/10.1093/genetics/123.3.585.\u003c/li\u003e\n\u003cli\u003eDe Coster W, D\u0026rsquo;Hert S, Schultz DT, Cruts M, Van Broeckhoven C. NanoPack: Visualizing and processing long-read sequencing data. Bioinformatics. 2018;34. https://doi.org/10.1093/bioinformatics/bty149.\u003c/li\u003e\n\u003cli\u003eKolmogorov M, Bickhart DM, Behsaz B, Gurevich A, Rayko M, Shin SB, et al. metaFlye: scalable long-read metagenome assembly using repeat graphs. Nat Methods. 2020;17. https://doi.org/10.1038/s41592-020-00971-x.\u003c/li\u003e\n\u003cli\u003eOlson RD, Assaf R, Brettin T, Conrad N, Cucinell C, Davis JJ, et al. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res. 2023;51 1 D. https://doi.org/10.1093/nar/gkac1003. \u003c/li\u003e\n\u003cli\u003eStamatakis A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30. https://doi.org/10.1093/bioinformatics/btu033.\u003c/li\u003e\n\u003cli\u003eOverbeek R, Begley T, Butler RM, Choudhuri J V., Chuang HY, Cohoon M, et al. The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 2005;33. https://doi.org/10.1093/nar/gki866.\u003c/li\u003e\n\u003cli\u003eKanehisa M, Goto S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Research. 2000;28. https://doi.org/10.1093/nar/28.1.27.\u003c/li\u003e\n\u003cli\u003ePritchard L, Glover RH, Humphris S, Elphinstone JG, Toth IK. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. Anal Methods. 2016;8:12\u0026ndash;24. doi:10.1039/c5ay02550h.\u003c/li\u003e\n\u003cli\u003eGoris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 2007;57. https://doi.org/10.1099/ijs.0.64483-0.\u003c/li\u003e\n\u003cli\u003eMeier-Kolthoff JP, Auch AF, Klenk HP, G\u0026ouml;ker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013;14. https://doi.org/10.1186/1471-2105-14-60.\u003c/li\u003e\n\u003cli\u003eHayashi Sant\u0026rsquo;Anna F, Bach E, Porto RZ, Guella F, Hayashi Sant\u0026rsquo;Anna E, Passaglia LMP. Genomic metrics made easy: what to do and where to go in the new era of bacterial taxonomy. Critical Reviews in Microbiology. 2019;45. https://doi.org/10.1080/1040841X.2019.1569587.\u003c/li\u003e\n\u003cli\u003eYamano R, Yu J, Jiang C, Harjuno Condro Haditomo A, Mino S, Sakai Y, Sawabe T. Taxonomic revision of the genus \u003cem\u003eAmphritea\u003c/em\u003e supported by genomic and \u003cem\u003ein silico\u003c/em\u003e chemotaxonomic analyses, and the proposal of \u003cem\u003eAliamphritea\u003c/em\u003e gen. nov. PloS one. 2022; 17:e0271174. https://doi.org/10.1371/journal.pone.0271174. \u003c/li\u003e\n\u003cli\u003eVolodina E, Steinb\u0026uuml;chel A. (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB\u0026apos;) from fatty acid degradation operon of \u003cem\u003eRalstonia eutropha\u003c/em\u003e H16. AMB Express. 2014;28: 4-69. https://doi:0.1186/s13568-014-0069-0.\u003c/li\u003e\n\u003cli\u003eAlvarez-Santullano Villegas NP, Mardones MS, Dur\u0026aacute;n RE, Donoso R, Gonz\u0026aacute;lez A, Sanhueza C, Navia R, Acevedo F, P\u0026eacute;rez-Pantoja D, et al. Genome-wide metabolic reconstruction of the synthesis of Polyhydroxyalkanoates from sugars and fatty acids by \u003cem\u003eBurkholderia\u003c/em\u003e \u003cem\u003esensu sato\u003c/em\u003e species. Microorganisms. 202;9:1290. https://doi.org/10.3390/microorganisms9061290.\u003c/li\u003e\n\u003cli\u003eSastre DE, Pulschen AA, Basso LGM, Benites Pariente JS, Marques Netto CGC, Machinandiarena F, Albanesi D, Navarro MVAS, de Mendoza D, Gueiros-Filho FJ. The phosphatidic acid pathway enzyme \u003cem\u003ePlsX\u003c/em\u003e plays both catalytic and channeling roles in bacterial phospholipid synthesis. J Biol Chem. 2020;295:2148-2159. https://doi:10.1074/jbc.RA119.011147. \u003c/li\u003e\n\u003cli\u003eSuzuki K, Okada K, Kamiya Y, Zhu XF, Nakagawa T, Kawamukai M, Matsuda H (1997) Analysis of the decaprenyl diphosphate synthase (\u003cem\u003edps\u003c/em\u003e) gene in fission yeast suggests a role of ubiquinone as an antioxidant. J Biochem 121:496\u0026ndash;505. https://doi.org/10.1093/oxfordjournals.jbchem.a021614.\u003c/li\u003e\n\u003cli\u003eWaterhouse AM, Procter JB, Martin DMA, et al. Jalview Version 2-A multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25: https://doi.org/10.1093/bioinformatics/btp033.\u003c/li\u003e\n\u003cli\u003eLarkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23: https://doi.org/10.1093/bioinformatics/btm404. \u003c/li\u003e\n\u003cli\u003eTavar\u0026eacute; S. Some probabilistic and statistical problems in the analysis of DNA sequences. American Mathematical Society: Lect. Math. Life Sci. 1986; 17, 57\u0026ndash;86.\u003c/li\u003e\n\u003cli\u003eSasser M. Identification of bacteria by gas chromatography of cellular fatty acids. In: MIDI Technical Note 101. Newark, Delaware, USA: MIDI Inc; 1990.\u003c/li\u003e\n\u003cli\u003eReasoner DJ, Geldreich EE. A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol. 1985;49: https://doi.org/10.1128/aem.49.1.1-7.1985.\u003c/li\u003e\n\u003cli\u003eLindhout T, Lau PCY, Brewer D, Lam JS. Truncation in the core oligosaccharide of lipopolysaccharide affects flagella-mediated motility in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1 via modulation of cell surface attachment. Microbiology (Reading). 2009;155: https://doi.org/10.1099/mic.0.030510-0.\u003c/li\u003e\n\u003cli\u003eRodrigues Neto J, Malavolta J\u0026uacute;nior VA, Victor O. Meio simples para o isolamento e cultivo de \u003cem\u003eXanthomonas campestris\u003c/em\u003e pv. citri Tipo B. Sum Phyt. 1986;12\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003eRibeiro NV da S, Vidal MS, Barrios SCL, et al. Genetic diversity and growth promoting characteristics of diazotrophic bacteria isolated from 20 genotypes of Brachiaria spp. Plant Soil. 2020;451: https://doi.org/10.1007/s11104-019-04263-y.\u003c/li\u003e\n\u003cli\u003eOliveira A, Teixeira P, Azevedo M, et al. Corynebacterium pseudotuberculosis may be under anagenesis and biovar Equi forms biovar Ovis: A phylogenic inference from sequence and structural analysis. BMC Microbiol. 2016;16: https://doi.org/10.1186/s12866-016-0717-4.\u003c/li\u003e\n\u003cli\u003eVaux F, Trewick SA, Morgan-Richards M. Lineages, splits and divergence challenge whether the terms anagenesis and cladogenesis are necessary. Biol. J. Linn. Soc\u003cem\u003e.\u003c/em\u003e 2016;117\u003c/li\u003e\n\u003cli\u003eChung EJ, Park TS, Kim KH, Jeon CO, Lee HI, Chang WS, Aslam Z, Chung YR. \u003cem\u003eNitrospirillum irinus\u003c/em\u003e sp. nov., a diazotrophic bacterium isolated from the rhizosphere soil of Iris and emended description of the genus \u003cem\u003eNitrospirillum\u003c/em\u003e. A van Leeuwenhoek. 2015;108(3):721\u0026ndash;729. https://doi.org/10.1007/s10482-015-0528-x.\u003c/li\u003e\n\u003cli\u003eChun J, Oren A, Ventosa A, et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol. 2018;68: https://doi.org/10.1099/ijsem.0.002516.\u003c/li\u003e\n\u003cli\u003eDe Lajudie PM, Andrews M, Ardley J, et al. Minimal standards for the description of new genera and species of rhizobia and agrobacteria. Int J Syst Evol Microbiol. 2019;69: https://doi.org/10.1099/ijsem.0.003426.\u003c/li\u003e\n\u003cli\u003eWang J, Wang H, Cai K, et al. Evaluation of three sample preparation methods for the identification of clinical strains by using two MALDI-TOF MS systems. J Mass Spect. 2021;56: https://doi.org/10.1002/jms.4696.\u003c/li\u003e\n\u003cli\u003eGaia V, Casati S, Tonolla M. Rapid identification of \u003cem\u003eLegionella\u003c/em\u003e spp. by MALDI-TOF MS based protein mass fingerprinting. Syst Appl Microbiol. 2011;34: https://doi.org/10.1016/j.syapm.2010.11.007.\u003c/li\u003e\n\u003cli\u003eReis VM, Olivares FL, D\u0026ouml;bereiner. Improved methodology for isolation of \u003cem\u003eAcetobacter diazotrophicus\u003c/em\u003e and confirmation of its endophytic habitat. World J Microbiol Biotechnol. 1994 ;10: https://doi.org/10.1007/BF00144460.\u003c/li\u003e\n\u003cli\u003eSanti C, Bogusz D, Franche C. Biological nitrogen fixation in non-legume plants, An Botany. 2013;111: https://doi.org/10.1093/aob/mct048.\u003c/li\u003e\n\u003cli\u003eHenderson PJF, Maher C, Elbourne, LDH, Eijkelkamp BA, Paulsen IT, Hassan KA. Physiological functions of bacterial \u0026ldquo;Multidrug\u0026rdquo; efflux pumps. Chem. Rev. 2021;121, 9, 5417\u0026ndash;5478. https://pubs.acs.org/doi/10.1021/acs.chemrev.0c01226.\u003c/li\u003e\n\u003cli\u003eHuang L, Wu C, Gao H, Xu C, Dai M, Huang L, Hao H, Wang X, Cheng G. Bacterial multidrug efflux pumps at the frontline of antimicrobial resistance: An overview. Antibiotics. 2022;13. https://doi.org/10.3390/antibiotics11040520. \u003c/li\u003e\n\u003cli\u003eMutanda I, Sun J, Jiang J, Zhu D. Bacterial membrane transporter systems for aromatic compounds: Regulation, engineering, and biotechnological applications. Biotechnol Adv. 2022;59. https://doi.org/10.1016/j.biotechadv.2022.107952.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 General features of the strains genomic sequence assemblies available in the NCBI genome database.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"898\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eStrains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003eGenome Length (Mb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003eGC Content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eNo. of contigs/scaffolds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003eContig L50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eContig N50 (Mb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eGenome coverage (x)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eNCBI RefSeq assembly\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eNCBI Accession number\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBR 11140\u003csup\u003eT\u0026nbsp;\u003c/sup\u003e(Y-2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e67.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF_007827975.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_007827975.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBR 11142\u003csup\u003eT\u0026nbsp;\u003c/sup\u003e(Y-1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e67.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF_029594735.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_029594735.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBR 11163\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e67.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF_034653355.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_034653355.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBR 11164\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e68.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF_034653275.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_034653275.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBR 11622\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e67.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF_007827955.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_007827955.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBR 11752\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e67.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF_036348415.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_036348415.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBR 11828\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e67.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF_034427435.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_034427435.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBR 11865\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e67.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF_007828025.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_007828025.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eDSM 22198\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e67.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCF 014205765.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGCA_014205765.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 Average nucleotide identity (ANIb) and digital DNA-DNA hybridization (dDDH) values between \u003cem\u003eN. iridis\u003c/em\u003e DSM 22198\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eN. viridazoti\u003c/em\u003e BR 111140\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eN. amazonense\u003c/em\u003e BR 11142\u003csup\u003eT\u003c/sup\u003e and the proposed new species.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"899\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003eStrain/Species\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eDSM 22198\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eBR 11142\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eBR 11140\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eBR 11865\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eBR 11163\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eBR 11164\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eBR 11828\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eBR 11752\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eBR 11622\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"bottom\" style=\"width: 899px;\"\u003e\n \u003cp\u003eANIb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eDSM 22198\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. iridis\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e85.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e84.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e84.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e85.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e85.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e84.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e84.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e84.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11142\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. amazonense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e85.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e93.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e94.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11140\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. viridazoti\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e84.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e93.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e94.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11865\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. bahiense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e84.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e94.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e94.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11163\u003csup\u003eT\u0026nbsp;\u003c/sup\u003e(\u003cem\u003eN. guanabarense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e85.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e90.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11164\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. guaranorum\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e85.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e91.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e90.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11828\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. guiasense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e84.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e92.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e92.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11752\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. karajorum\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e84.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e92.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11622\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. pindoramense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e84.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e87.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e86.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e92.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"bottom\" style=\"width: 899px;\"\u003e\n \u003cp\u003edDDH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eDSM 22198\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. iridis\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e29.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e28.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e28.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e27.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e28.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11142\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. amazonense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e29.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e52.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e60.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e44.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e44.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11140\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. viridazoti\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e52.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e55.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e43.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e46.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11865\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. bahiense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e28.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e60.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e55.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e45.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e45.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11163\u003csup\u003eT\u0026nbsp;\u003c/sup\u003e(\u003cem\u003eN. guanabarense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e44.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e43.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e45.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e40.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11164\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. guaranorum\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e28.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e44.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e46.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e45.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e40.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11828\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. guiasense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e48.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e49.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11752\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. karajorum\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e27.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e48.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e66.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 214px;\"\u003e\n \u003cp\u003eBR 11622\u003csup\u003eT\u003c/sup\u003e (\u003cem\u003eN. pindoramense\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e28.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e49.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e66.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biological nitrogen fixation, genomic analysis, PGPR taxonomy, bacteria diversity, grasses, MALDI-TOF MS","lastPublishedDoi":"10.21203/rs.3.rs-5600752/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5600752/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, we characterized 86 plant growth-promoting bacterial strains belonging to the genus \u003cem\u003eNitrospirillum\u003c/em\u003e, isolated from diverse host plants and geographic regions. To investigate their evolutionary relationships, we employed phylogenetic analyses based on 16S rRNA and \u003cem\u003erecA\u003c/em\u003e genes, as well as phylogenomic approaches including average nucleotide identity (ANI) and digital DNA\u0026ndash;DNA hybridization (dDDH). The classification of type strains was further supported by \u003cem\u003ein silico\u003c/em\u003e analyses of chemotaxonomic markers, particularly genes involved in fatty acid biosynthesis and elongation, phospholipid and quinone production, and nitrogen fixation (\u003cem\u003enifHDK\u003c/em\u003e operon). Phenotypic and chemotaxonomic characterization was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid methyl ester (FAME) profiling, and physiological assays. These included evaluations of nitrogen fixation capacity, antibiotic resistance, carbon source utilization, and enzymatic activity. This integrative approach provided detailed insight into the characteristics and diversity of the studied strains. Phylogenetic and genomic analyses revealed six novel taxa within the genus \u003cem\u003eNitrospirillum\u003c/em\u003e, in addition to the previously described species \u003cem\u003eN. amazonense\u003c/em\u003e, \u003cem\u003eN. iridis\u003c/em\u003e, and \u003cem\u003eN. viridazoti\u003c/em\u003e. The distinctiveness of these new lineages was supported by both genomic metrics and phenotypic traits. All novel strains also exhibited ethylene production, confirming their nitrogen-fixing ability under \u003cem\u003ein vitro\u003c/em\u003e conditions. Based on these findings, we propose the formal description of six novel species: \u003cem\u003eNitrospirillum bahiense\u003c/em\u003e sp. nov. (=\u0026thinsp;BR 11865ᵀ, =UCCCB 233ᵀ), \u003cem\u003eNitrospirillum guanabarense\u003c/em\u003e sp. nov. (=\u0026thinsp;BR 11163ᵀ, =UCCCB 228ᵀ), \u003cem\u003eNitrospirillum guaranorum\u003c/em\u003e sp. nov. (=\u0026thinsp;BR 11164ᵀ, =UCCCB 229ᵀ), \u003cem\u003eNitrospirillum karajorum\u003c/em\u003e sp. nov. (=\u0026thinsp;BR 11752ᵀ, =UCCCB 231ᵀ), \u003cem\u003eNitrospirillum guiasense\u003c/em\u003e sp. nov. (=\u0026thinsp;BR 11828ᵀ, =UCCCB 232ᵀ), and \u003cem\u003eNitrospirillum pindoramense\u003c/em\u003e sp. nov. (=\u0026thinsp;BR 11622ᵀ, =UCCCB 230ᵀ).\u003c/p\u003e","manuscriptTitle":"Exploring the Diversity of the Genus Nitrospirillum: Description of Six Novel Nitrogen-Fixing Bacteria Species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 09:01:13","doi":"10.21203/rs.3.rs-5600752/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ce287b4f-954b-4599-9a0c-065ea4fdcb77","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-24T12:23:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-30 09:01:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5600752","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5600752","identity":"rs-5600752","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}