Genome Analysis and Description of Three Novel Diazotrophs Geomonas Species Isolated From Paddy Soils

Five strictly anaerobic strains, designated RG2T, RG3, RG10T, RF4T, and RG29, were isolated from paddy soils in China. Strains RG2T, RF4T, RG10T, RG3, and RG29 grew at temperatures ranging 5–42°C and pH ranging 5.5–8.5. Strains RG2T, RF4T, RG3, and RG29 could tolerate NaCl up to 0–0.7% (w/v) while strain RG10T could tolerate NaCl up to 0–0.8% (w/v). The isolated strains showed the highest 16S rRNA gene sequence similarities to the type strains of Geomonas terrae Red111T and Geomonas paludis Red736T. In phylogenetic (based on 16S rRNA gene sequence) and phylogenomic trees, strains clustered with the members of the genus Geomonas. Menaquinone-8 was the predominant quinone present in all strains. The major fatty acid profiles of all strains were C15:1 ω6c, C16:0, iso-C15:0, and Summed Feature 3. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the isolated strains and the closely related Geomonas species were lower than the cutoff value (ANI 95–96% and dDDH 70%) for prokaryotic species delineation. Based on physiological, biochemical, and chemotaxonomic properties, strains RG2T, RG10T, and RF4T could easily be differentiated with the members of the genus Geomonas. Additionally, all the isolated strains possessed nifHDK clusters and catalytic compartments of nitrogenase. Based on the above results, the isolated five strains represent three novel species of the genus Geomonas, for which the names Geomonas oryzisoli sp. nov., Geomonas subterranea sp. nov., and Geomonas nitrogeniifigens sp. nov. are proposed. The type strains are RG10T (= GDMCC1.2537T = KCTC 26318T), RG2T (= GDMCC1.2536T = KCTC 25317T), and RF4T (= GDMCC 1.2547T = KCTC 25316T).


INTRODUCTION
The genus Geomonas was established by Xu et al. (2019). Members of the genus Geomonas are strictly anaerobic, rodshaped, and capable of reducing ferric iron to ferrous iron and form reddish colonies due to the presence of c-type cytochromes. At the time of writing, there were nine validly published Geomonas species 1 that were isolated from anoxic habitats, such as paddy soils and forest soils (Xu et al., 2019;Itoh et al., 2021).
Biological nitrogen fixation (BNF) is an essential chemical process that involves nitrogen-fixing bacteria, and these bacteria have the potential to minimize the use of N fertilizers in agriculture and reduce the negative impact of nitrogen fertilizers on the environment (Soumare et al., 2020). It has been confirmed that a large amount of nitrogen fertilizer utilization could inhibit and decrease the diversity and ability of nitrogen-fixing bacteria (Feng et al., 2018;Lin et al., 2018;Fan et al., 2019). The dominant diazotrophs in paddy soils were members of the Geobacteraceae family with iron reduction abilities . In the present study, we isolated five (designated, RG2 T , RG3, RG10 T , RF4 T , and RG29) nitrogen-fixing and iron-reducing strains from paddy soil samples obtained from a greenhouse in Fujian Province, China, where no fertilizer was administered for an extended period. The isolated five strains showed low 16S rRNA gene sequence similarity to members of the genus Geomonas; hence, the current investigation was carried out to determine their taxonomic position.

Isolation and Maintenance
Paddy soil samples were collected from a rice greenhouse belonging to Rice Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou City, Fujian Province, China (N26.1076 • , E119.3014 • ) in January 2021. All strains were isolated based on the soil slurry incubation method according to the description by Xu et al. (2019). Briefly, 10 g fresh paddy soil samples were added aseptically into 120 ml bottles containing 90 ml autoclaved oxygen-free water and shaken in the incubator for half an hour with a speed of 100 rpm at 30 • C. The anaerobic and sterile water was obtained by inflating with N 2 :CO 2 (80:20, v/v) gas mixture for at least 1 h and then autoclaved. Under sterile and oxygen-free conditions, soil suspensions were serially diluted in oxygenfree water at a concentration ranging 10 −1 to 10 −3 . Then, 200 µl soil suspension from each dilution was spread on R2A agar plates (Hopebio, Qingdao, China) containing 40 mM disodium fumarate (Macklin, Shanghai, China) (modified R2A medium). The plates were then incubated at 30 • C for 10 days in Whitley DG250 Anaerobic Workstation (Don Whitley Scientific, Bingley, United Kingdom). The red colonies were picked and purified, repeatedly re-streaked on modified R2A medium, and incubated anaerobically at 30 • C for 3 days until pure single 1 https://lpsn.dsmz.de/genus/geomonas colonies were obtained. The purified strains were preserved at −80 • C in modified R2A broth supplemented with 10% dimethyl sulfoxide (DMSO) and stored at −80 • C. In addition, the reference strains Geomonas terrae MCCC 1K04029 T , Geomonas paludis MCCC 1K03950 T , and Geomonas oryzae MCCC 1K03691 T (obtained from Marine Culture Collection of China through strains exchange) were cultured on the same medium mentioned as above.

16S rRNA Gene Sequencing and Phylogenetic Analysis
Genomic DNA was extracted using the TIANamp Bacterial DNA kit (TIANGEN, Beijing, China) according to the manufacturer's instruction. PCR amplification was performed as described by Dong et al. (2019). Pairwise 16S rRNA gene sequence similarities among the isolated five strains and against other type strains were calculated using the EzBioCloud platform (Yoon et al., 2017a). For phylogenetic analysis, 16S rRNA gene sequences of the closely related type strains were downloaded from the EzBioCloud platform. Phylogenetic trees were reconstructed based on the neighbor-joining (NJ) (Saitou and Nei, 1987), maximum likelihood (ML) (Felsenstein, 1981), and maximum-parsimony (Fitch, 1971) methods with the Kimura two-parameter model (Kimura, 1980) and 1,000 bootstrap replications (Felsenstein, 1985) using MEGA version X (Kumar et al., 2018).

Chemotaxonomic Characterization
For fatty acid detection, the biomass of the isolated five strains and their related type strains was collected from cultures grown for 3 days at 30 • C. The cellular fatty acids in the cell walls were extracted and analyzed according to the standard protocol of the Microbial Identification System (MIDI) tested by gas chromatography (model 7890, Agilent) (Sasser, 2001). The biomass of the isolated five strains for menaquinone analysis was collected from cultures grown for 7 days at 30 • C in modified R2A broth and then analyzed as described by Collins et al. (1977) using reverse-phase HPLC (Kroppenstedt, 1982).
Electron donor and acceptor utilization tests were performed using 10 ml of degassed modified freshwater medium (MFM) in a 20 ml serum tube under a N 2 /CO 2 (4:1, v/v) atmosphere at 30 • C without shaking. Acetate (10 mM) was used as an electron donor for all electron acceptor tests, while Fe (III)-NTA (5 mM) was used as an electron acceptor for all electron donor tests. The utilized electron donors were determined according to cell growth. The utilization of electron acceptors of Fe (III) compounds was determined by the color change from reddishbrown/green to colorless, whereas those of nitrate, fumarate, and sulfur were determined by cell growth. Fermentation growth in the absence of electron acceptors was evaluated with fumarate, lactate, glycerol, pyruvate, malate, and ethanol by checking the optical density at 600 nm.
was measured using a spectrophotometer (Shimadzu, Japan) at a wavelength of 562 nm. A standard curve was prepared with ferrous ammonium sulfate hexahydrate spanning a dilution concentration range of 0.1-0.8 mM ferrous ions.

16S rRNA Gene Sequencing and Phylogenetic Analysis
The isolated strains, RG2 T , RG3, RG10 T , RF4 T , and RG29 had 98.5-99.9% 16S rRNA gene sequence similarity to each other. Strains RG2 T , RG3, and RG29 showed the highest 16S rRNA gene sequence similarities to the type strain of G. terrae Red 111 T (98.2-98.5%) (Supplementary Table 1). These values were below the threshold (98.65%) for species delineation (Kim et al., 2014) but above the threshold (94.55-95.05%) for genus delineation (Yarza et al., 2014), suggesting that these strains were different at the species level within the genus Geomonas. Strains RF4 T and RG10 T had the highest similarity to the type strain of G. paludis Red 736 T (99.1-99.3%). Despite the fact that the sequence similarity of RF4 T and RG10 T was quite high, numerous earlier studies have revealed that two strains with 16S rRNA gene similarity greater than 99.0% were verified as separate species (Tohno et al., 2017;Hahn et al., 2018;Qin et al., 2020). A range of thresholds (98.2-99.0%) was also recommended by Meier-Kolthoff et al. (2013). Therefore, strains RG10 T and RF4 T might be novel species of the genus Geomonas.
The maximum likelihood phylogenetic tree based on the 16S rRNA gene sequences showed that strains RG2 T , RG3, RG10 T , RF4 T , and RG29 clade with Geomonas members (Figure 1). The phylogenetic position was also confirmed by the trees generated using neighbor-joining (Supplementary Figure 2) and maximum parsimony (Supplementary Figure 3) methods. In the phylogenomic tree, all strains clustered with the members of the genus Geomonas (Figure 2).

Chemotaxonomic Characters
The fatty acid profiles clearly distinguished the isolated five strains RG2 T , RG3, RG10 T , RF4 T , RG29, and their related type strains. All five strains contained C 15:1 ω6c, iso-C 15:0 , C 16:0 , and Summed Feature 3 as major fatty acid profiles, but their proportion was distinguished. Strains RG2 T , RG3, RG10 T , RF4 T , and RG29 had iso-C 15:0 and Summed Feature 3 levels more than 10% ( Table 2), but it was < 10% in G. paludis MCCC 1K03950 T and G. oryzae MCCC 1K03691 T . Fatty acid C 17:1 ω6c presented in strains RG2 T , RG3, and type strain G. paludis MCCC 1K03950 T was above 5% whereas it was much lower than 5% in other three strains and related type strains G. terrae MCCC 1K04029 T and G. oryzae MCCC 1K03691 T . C 15:1 ω6c was high in strain RG2 T and C 16:0 in RG10 T ; this value was less when compared with G. paludis MCCC 1K03950 T . The iso-C 15:0 value in G. terrae MCCC 1K04029 T and G. oryzae MCCC 1K03691 T was higher compared with other strains. Detailed comparative fatty acid profiles of strains RG2 T , RG3, RG10 T , RF4 T , RG29, G. paludis MCCC 1K03950 T , G. oryzae MCCC 1K03691 T , and G. terrae MCCC 1K04029 T are mentioned in Table 2.
The menaquinone composition was detected for all five isolated strains in the present study, menaquinone-8 (MK-8) was characterized as the predominant respiratory quinone, and small amounts of MK-7 and MK-9 were also identified for all strains, which was consistent with other type strains of the genus Geomonas (Xu et al., 2019;Itoh et al., 2021).
Functional annotation (Supplementary Table 4) analysis suggests that all strains encode key genes for glycolysis and tricarboxylic acid but only strains RG2 T and RG3 encode genes for the pentose phosphate pathway.
Autotrophic prokaryotic representatives play an important role in ecosystems by supplying organic carbon to heterotrophs (Hügler et al., 2005). The Calvin-Benson-Bassham cycle is the most significant autotrophic carbon fixation mechanism that exists today (Bassham et al., 1954;Hügler et al., 2005). Despite its worldwide importance, it is limited to species having a highenergy production from a chemotrophic or phototrophic lifestyle. Microorganisms found in extreme environments, such as high temperatures and anaerobic or acidic conditions, generally use different CO 2 fixation pathways, and three alternative pathways are currently known: the reductive tricarboxylic acid cycle, the reductive acetyl coenzyme A (CoA) pathway, and the 3hydroxypropionate cycle (Hügler et al., 2005). The key genes for the Calvin-Benson-Bassham and 3-hydroxypropionate cycle were not noticed in any strains, but the key genes related to the reductive tricarboxylic acid cycle were present in all strains.
BNF is the most common method of converting atmospheric nitrogen gas (N 2 ) to ammonia, and all known diazotrophs include at least one of three closely related nitrogenase subtypes: Nif, Vnf, and Anf. The nifHDK core set and catalytic compartment of nitrogenase (Dos Santos et al., 2012) were noticed in all strains (Supplementary Tables 2, 4). Furthermore, genes related to dissimilatory nitrate reduction (narGHI and nrfA) were also noticed in all strains.
Members of the genus Geomonas were known for metal reduction (Xu et al., 2019). It has been demonstrated that certain c-type cytochromes may form a conductive channel capable of crossing the inner membrane, periplasm, and outer membrane, allowing Geobacteraceae species to directly transport electrons from the quinone/quinol pool to metal oxides (Guo et al., 2021). The bacteria develop electrically conductive protein nanowires, known as e pili, which are built from the PilA pilin monomer to perform long-range extracellular electron transfer to metal oxide particles (Lovley and Walker, 2019;Guo et al., 2021). Furthermore, protein components such as CymA, MtrA, MtrB, MtrC, and OmcA have also been reported for Fe(III) oxide reduction (Shi et al., 2012). In the present study, all strains encode genes for iron reduction, iron gene regulation, iron acquisition-iron transport, and siderophore transport (Supplementary Figure 4). The details of the key    1, RG2 T ; 2, RG3; 3, RG10 T ; 4, RF4 T ; 5, RG29; 6, Geomonas paludis MCCC 1K03950 T ; 7, Geomonas terrae MCCC 1K04029 T ; 8, Geomonas oryzae MCCC 1K03691 T . All data were obtained from this study. Only content > 1% was shown. -, not detected. The values ≥ 5.0% were highlighted in bold. Summed Features are the fatty acids that cannot be separated using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed Feature 1, iso-C 15:1 H and/or C 13:0 3OH; Summed Feature 2, C 12:0 aldehyde and/or unknown 10.928; Summed Feature 3: C 16:1 ω7c and/or C 16:1 ω6c.
genes for iron reduction, iron gene regulation, iron acquisitioniron transport, and siderophore transport are mentioned in Supplementary Table 5. All strains harbored merA but not merB, indicating that they might be susceptible to mercury. Although previous studies indicated that certain Geomonas species contained kaiBC genes (encoding essential proteins in circadian rhythms) Itoh et al., 2021), we did not detect this in our study. Details of all the metabolic potential of the present study strains are mentioned in Supplementary Table 4.

Nitrogen Fixation and Electron Donors and Acceptors
In all strains, the nifH gene was amplified (Supplementary Figure 5A). Nitrogen fixation activity was also estimated based on acetylene reduction activity (ARA). The ARAs of strains RG2 T , RG3, RG10 T , RF4 T , and RG29 were (1.7 ± 0.2) × 10 −6 , (2.1 ± 0.4) × 10 −6 , (1.2 ± 0.7) × 10 −6 , (3.2 ± 0.2) × 10 −6 , and (2.8 ± 0.2) × 10 −6 nmol C 2 H 4 /h/cell, respectively (Supplementary Figure 5B). Therefore, it suggests that all the isolated five strains could fix nitrogen. Supplementary Figure 5B clearly shows that strain RF4 T had the highest nitrogen-fixing ability among all the strains. The availability of 10 electron donors for the isolated five strains was similar, while electron acceptors were different ( Table 1); for example, strains RG2 T , RG3, RF4 T , and RG29 could utilize Fe (III) citrate as an electron acceptor but strain RG10 T could not. For the fermentation growth, strains RF4 T , RG2 T , RG3, RG10 T , and RG29 showed weak growth using fumarate as a substrate in the absence of electron acceptors, but RF4 T and RG29 did not grow with malate and pyruvate. Strain RG10 T did not grow with lactate and glycerol. Strain RG3 grew with ethanol but not strain RG2 T .

Cytochromes and Ferric Reduction
Five isolated strains possessed ferric reducing ability, which reduced ferrihydrite to ferrous iron in 25 days. Although they had ferric reducing ability, their reduction rates were different (Supplementary Figure 6). Strain RF4 T showed the highest iron reduction ability among the five strains.
The spectra of whole cells of all the isolated five strains suggest they could produce c-type cytochromes (Supplementary Figure 7). The absorbance peaks of reduced cytochrome c of strains RG2 T , RG3, RG10 T , RF4 T ,and RG29 were 424,524,and 553 nm;424,524,and 553 nm;424,524,and 554 nm;425,523,and 554 nm;425,523, and 554 nm, respectively, which were quite similar to those of the type strains of Geomonas species (Table 1).

Taxonomic Conclusion
Based on the above results, three strains, RG10 T , RG2 T , and RF4 T represent three novel species of the genus Geomonas, for which the names Geomonas oryzisoli sp. nov., Geomonas subterranea sp. nov., and Geomonas nitrogeniifigens sp. nov. are proposed.
The type strain, RG10 T (= GDMCC1.2537 T = KCTC 26318 T ), was isolated from paddy soil collected from the greenhouse in Fujian Province, China. The 16S rRNA gene and wholegenome sequence of strain RG10 T have been deposited in the GenBank/ENA/DDBJ under the accession numbers MZ148503 and CP076723, respectively.
Description of Geomonas subterranea sp. nov.
The type strain, RG2 T (= GDMCC1.2536 T = KCTC 25317 T ), was isolated from paddy soil collected from the greenhouse in Fujian Province, China. The 16S rRNA gene and wholegenome sequence of strain RG2 T have been deposited in the GenBank/ENA/DDBJ under the accession numbers MZ148482 and CP077683, respectively.
The type strain, RF4 T (= GDMCC 1.2547 T = KCTC 25316 T ), was isolated from paddy soil collected from the greenhouse in Fujian Province, China. The 16S rRNA gene and wholegenome sequence of strain RF4 T have been deposited in GenBank/ENA/DDBJ under the accession numbers MZ148489 and CP077684, respectively.

AUTHOR CONTRIBUTIONS
G-HL and S-GZ designed the research and project outline. SY and RT performed the DNA extraction and chemotaxonomic analysis. C-JX performed the genome analysis. G-HL drafted the manuscript. All authors read and approved the final manuscript.

FUNDING
This work was financially supported by a grant from the Fujian Special Fund for Scientific Research Institutes in the Public Interest (2020R1034001).