Genetic diversity into a novel free-living species of Bradyrhizobium from contaminated freshwater sediment

A free-living Bradyrhizobium strain isolated from a contaminated sediment sample collected at a water depth of 4 m from the Hongze Lake in China was characterized. Phylogenetic investigation of the 16S rRNA gene, concatenated housekeeping gene sequences, and phylogenomic analysis placed this strain in a lineage distinct from all previously described Bradyrhizobium species. The sequence similarities of the concatenated housekeeping genes support its distinctiveness with the type strains of the named species. The complete genome of strain S12-14-2 consists of a single chromosome of size 7.3M. The strain lacks both a symbiosis island and important nodulation genes. Based on the data presented here, the strain represents a new species, for which the name Bradyrhizobium roseus sp. nov. is proposed for the type strain S12-14-2T. Several functional differences between the isolate and other published genomes indicate that the genus Bradyrhizobium is extremely heterogeneous and has functions within the community, such as non-symbiotic nitrogen fixation. Functional denitrification and nitrogen fixation genes were identified on the genomes of strain S12-14-2T. Genes encoding proteins for sulfur oxidation, sulfonate transport, phosphonate degradation, and phosphonate production were also identified. Lastly, the B. roseus genome contained genes encoding ribulose 1,5-bisphosphate carboxylase/oxygenase, a trait that presumably enables autotrophic flexibility under varying environmental conditions. This study provides insights into the dynamics of a genome that could enhance our understanding of the metabolism and evolutionary characteristics of the genus Bradyrhizobium and a new genetic framework for future research.


Introduction
The genus Bradyrhizobium is a relatively well-studied rhizobial bacteria and well known for symbiosis and endophytic interaction with leguminous plants, where they exchange nutrients for survival.The genus includes a diverse collection of globally distributed bacteria that can nodulate a wide range of legumes (Salminen and Streeter, 1987;Van Rhijn and Vanderleyden, 1995;Okubo et al., 2012;Parker, 2015;Remigi et al., 2016;Sprent et al., 2017;Claassens et al., 2023;Lafay et al., 2023).It was widely recognized that the Bradyrhizobium species were capable of nitrogen fixation and the formation of symbiotic nodules in the roots of leguminous plants.The symbiotic species are crucial for soil health, plant growth, and food security, whereas nitrogen-fixing species are critical for the global nitrogen cycle (Jordan, 1982;Ladha and So, 1994;Stacey et al., 1995;Remigi et al., 2016;Gogoi et al., 2018).Members of Bradyrhizobium are characterized extensively for their symbiotic lifestyle with leguminous plants to form symbiotic nodules.Bradyrhizobium has biologically significant functions in soils, including photosynthesis, nitrogen fixation, denitrification, and degradation of aromatic compounds and others.Especially in the global nitrogen cycle, Bradyrhizobium removes nitrogen through heterotrophic denitrification and also has multiple roles in agriculture (Kaneko et al., 2002;Okubo et al., 2012).Nodulating Bradyrhizobium species typically contain nod and nif genes associated with nodulation and nitrogen fixation, respectively, the so-called symbiotic properties.Due to these characteristics, Bradyrhizobium has been regarded as a model of legume-rhizobia symbiosis and an ecologically significant microbe.However, nodulationdeficient isolates have been discovered and characterized, even though they possess genes for nitrogen fixation, and some Bradyrhizobium isolates recovered from forest soils have been found to lack both nodulation and nitrogen-fixing functions (VanInsberghe et al., 2015;Jones et al., 2016).A cultureindependent investigation has shown that the population of Bradyrhizobium in soil habitats is different from the rhizosphere of leguminous plants (VanInsberghe et al., 2015), suggesting that non-symbiotic Bradyrhizobia inhabit physically or functionally distinct niches.
Understanding the mechanisms of bradyrhizobial adaptation to independent existence in diverse environments is becoming increasingly important and urgent and may reveal the genetic potential of this globally significant genus.In the present study, we characterized a novel species of Bradyrhizobium isolated from freshwater sediment of the Hongze Lake in China.To reveal the genetic diversity and physiological features, here, we report the isolation, and the sequencing, assembly, taxonomic classification, and annotation of the genome of a new species of the genus Bradyrhizobium.These investigations will be extremely beneficial for elucidating its putative metabolism, contributing to a greater understanding of free-living Bradyrhizobium isolates, enhancing knowledge of the genes and enzymes involved in metabolic pathways, and possibly identifying genomic markers that can be utilized in future ecological studies.

Sampling, isolation, and growth condition
Strain S12-14-2 was recovered from a contaminated sediment sample (33 • 14 26 N, 118 • 35 40 E) collected at a water depth of 4 m from Hongze Lake, the fourth largest freshwater lake in China.The Hongze Lake is located in the Jiangsu Province and surrounded by Suqian and Huai'an cities; it has a surface area of 1,960 km 2 , a mean water depth of 1.77 m, a maximum depth of 4.37 m, and a volume of 28 × 108 m 3 (Gao et al., 2009;Li et al., 2011).The ecological risk of the Hongze Lake sediments has been assessed.Heavy metals including copper (Cu), zinc (Zn), lead (Pb), cadmium (Cd), chromium (Cr), mercury (Hg), arsenic (As), iron (Fe), aluminum (Al), and manganese (Mn) have been identified in the lake sediments of the Hongze Lake, and antibiotics like atrazine and ofloxacin were also detected (Yao et al., 2016;Wu et al., 2023).One gram of a sediment sample was used to screen for bacterial strains with the serial dilution method.The strain was grown on modified R2A medium at 30 • C under dark conditions (Jin et al., 2019).

DNA extraction, PCR amplification, and genomic sequencing
Genomic DNA was extracted using the FastDNA TM SPIN kit following the manufacturer's instructions.The concentration and purity of the DNA were then checked on a ND-2000 spectrophotometer (Thermo Fisher Scientific).The 16S rRNA genes of strain S12-14-2 were amplified by PCR method using the universal primer sets 27F/1492R (Weisburg et al., 1991) and then identified using the EzBioCloud web service (Yoon et al., 2017).The use of multilocus sequence analysis (MLSA) has been proven useful for the classification and identification of several groups of rhizobia.In the genus Bradyrhizobium, the 16S rDNA sequences provide minimal taxonomic information due to the high degree of conservation among species and the relatively high sequence similarity.Consequently, MLSA studies of glnII, recA, dnaK, rpoB, and ITS genes were performed to assess the taxonomic position of the free-living strain among identified species (Martens et al., 2008;Menna et al., 2009;Rivas et al., 2009;Pérez-Yépez et al., 2014;Gnat et al., 2016).The partial sequences of the housekeeping genes, glnII, recA, dnaK, rpoB, and ITS genes, were amplified by PCR method.The primer sets and PCR amplification conditions were described in a previous study (Menna et al., 2009;Delamuta et al., 2013).Whole genome sequencing was done at Novogene Biotechnology, Beijing, China, using the Pacific Biosciences (PacBio) RS II single molecule real-time (SMRT) platform together with the Illumina HiSeq technology.The quality of the genome assemblies was assessed using SMRT Link version 5.0.1.

Phylogenetic analyses and genome annotation
For phylogenetic analysis of strain S12-14-2, the 16S rRNA sequences of Bradyrhizobium type strains were downloaded from the NCBI database.The sequences were edited and aligned with BIOEDIT and CLUSTAL X software, respectively (Thompson et al., 1997;Hall, 1999), and the phylogenetic tree was reconstructed using the MEGA7 software (Kumar et al., 2016).For genomic annotations, the draft genome sequence was applied to the RAST pipeline, and comparisons of the genome were performed in the SEED Viewer (Aziz et al., 2008(Aziz et al., , 2012)).The protein coding sequences (CDSs) were submitted to the COG (Clusters of Orthologous Groups) database1 for functional classification and summary statistics (Tatusov et al., 1997(Tatusov et al., , 2003)).For the pangenome analysis, we followed Delmont and Eren's description of the Anvi'o workflow (Delmont and Eren, 2018), a community-driven, open-source analysis and visualization platform for microbial-omics, which is available at http://merenlab.org/software/anvio/.The structures of protein monomeric units were predicted using the SWISS-MODEL (Bienert et al., 2017;Waterhouse et al., 2018).Biosynthetic gene clusters (BGC) were identified and analyzed using the secondary metabolite databases antiSMASH database. 2The average nucleotide identity (ANI) was determined using the OrthoANI tool in the EZBioCloud server, and the digital DNA-DNA hybridization (dDDH) value was calculated with the Genome-to-Genome Distance Calculator (GGDC 2.1) (Meier-Kolthoff et al., 2013;Yoon et al., 2017).The average amino acid identity (AAI) was calculated with the AAI web-based calculator developed by Konstantinidis Lab.3

Genomic analysis: the taxonomic status
The phylogenetic analysis based on 16S rRNA gene sequences revealed that strain S12-14-2 should taxonomically be classified to the genus Bradyrhizobium.The strain shared 99.36-99.69%pairwise similarity with B. lablabi CCBAU 23086 T , B. jicamae PAC68 T , B. erythrophlei CCBAU 53325 T , and B. algeriense RST89 T and less than 99.3% with the other species within the genus Bradyrhizobium (Table 1).The 16S rRNA gene sequence neighbor-joining tree revealed that strain S12-14-2 unambiguously clustered with members of Bradyrhizobium.Due to its high level of conservation within the genus Bradyrhizobium, the 16S rRNA gene is an inadequate molecular marker for distinguishing species (Germano et al., 2006;Rivas et al., 2009).Therefore, a genome-based phylogenetic analysis was used.The topology of the phylogenetic and phylogenomic trees is consistent with the sequence similarities of the 16S rRNA gene between the novel strain and the type strains of the Bradyrhizobium species (Figure 1 and Supplementary Figure 2).

No.
Type Phylogenomic tree based on genome sequences of strain S12-14-2 T and its closely related type strains of Bradyrhizobium in the TYGS (https://tygs.dsmz.de/)and iTOL.
is a powerful tool for discriminating species (Rivas et al., 2009;Tang et al., 2012;Duran et al., 2014).The phylogenetic tree of five concatenated housekeeping gene sequences (ITS, dnaK, glnII, recA, and rpoB) located the novel strain in a clearly distinct lineage from the named Bradyrhizobium species.The phylogenetic tree of the concatenated ITS-dnaK-glnII-recA-rpoB gene sequences confirmed the placement of the novel strain in a lineage distinct from the named Bradyrhizobium species (Supplementary Figure 3).ANI, AAI, and dDDH analyses based on whole-genome sequences were implemented for additional characterization.We estimated the values for the complete genome sequence of S12-14-2 by conducting pair-wise comparisons with genome sequences of the type strains of Bradyrhizobium species obtained from public databases.The ANI values of the named species ranged from 79.2% (Bradyrhizobium daqingense) to 86.6% (B.algeriense), which is significantly lower than the 95-96% threshold value for circumscription of bacterial species (Konstantinidis and Tiedje, 2005;Rivas et al., 2009;Chun et al., 2018;Ciufo et al., 2018).The AAI (dDDH) values for all pairwise comparisons between the novel strain and related Bradyrhizobium species ranged from 71.6 to 86.5% (22.8-32.5%)(Table 1), which is also under the borderline of 95% (ANI) and 70% (dDDH) species delineation (Goris et al., 2007;Richter and Rossello-Mora, 2009;Luo C. et al., 2014;Konstantinidis et al., 2017).

General genome properties
The genome of isolate S12-14-2 has a size of 7,319,411 base pairs, a GC content of 63.3%, three rRNA genes, 62 tRNA genes, and 6893 CDS, of which 5,838 genes were allocated to COG (Supplementary Table 3).Bradyrhizobium spp.have relatively large genomes compared to other groups, ranging between 5 and 11M, whereas the smallest genome size found to date is approximately 5M in the free-living strain S2-11-2 (Ormeño-Orrillo and Martínez-Romero, 2019; Jin et al., 2022b;Klepa et al., 2022;Zhang et al., 2023).The GC content is consistent with other Bradyrhizobium type strains (Figure 1 and Supplementary Table 2).The abundant genes were associated with amino acid transport and metabolism (COG category E), inorganic ion transport and metabolism (COG category P), energy production and conversion (COG category C), transcription (COG category K), signal transduction mechanisms (COG category T), and cell wall/membrane/envelope biogenesis (COG category M), and lipid transport and metabolism (COG category I), and approximately 32.5% of the genes were assigned to the unknown function COG category (Supplementary Table 4).

Pangenomics and secondary metabolites
To investigate the genomic diversity of Bradyrhizobium sp.S12-14-2 and related Bradyrhizobium spp., we compiled a total of 44 complete whole genomes (including 7 free-living and 34 symbiotics) downloaded from the NCBI.This dataset contains genomes representing 41 species validly published.Across the entire dataset, the genome size (GS) varied between 5.5 and 11.5M (mean, 8.5M); the GC content varied between 61.4 and 65.1% (mean, 64.9), and the number of predicted genes ranged from 5,220 to 11,061 (mean, 8,111).
Following the workflow, pangenomic analysis of 37 Bradyrhizobium strains was performed using Anvi'o (version 7.1).Single copy gene clusters (SCGs) are shown in purple color on the outside, which are drawn by 1,235 gene clusters from 43,916 splits and 44,526 objects.Bradyrhizobium sp.S12-14-2 T exhibited a similar pattern of blue-colored single copy gene clusters as the other three free-living microorganisms Bradyrhizobium sediminis S2-20-1 T , B. sediminis S2-11-2, and B. sediminis S2-11-4.SCGs were also the same with the core genes, and others not included in the SCGs were accessory genes.B. erythrophlei GAS138 T (LT670817) has the largest number of singleton gene clusters which is almost equal to 2,046 (Figure 2).BGC from 44 Bradyrhizobium genomes from the free-living and symbiotic groups were identified using antiSMASH, and a comprehensive network analysis was performed in BiG-SCAPE using all similar BGC sequences from the MIBiG databases.The average number of BGCs from the 44 genomes is 11, and the network analysis distributed 557 BGCs in different types, including 99 terpenes, 84 NRPS (non-ribosomal peptides), 93 hserlactones (homoserine lactones), 44 redox-cofactors (redox-cofactors such as PQQ), 47 RiPP-like (other unspecified ribosomally synthesized and post-translationally modified peptide products), 35 NRPS-like (NRPS-like fragmenta), and 37 TPKS (type I, II, and III PKSs), and generally, the number of secondary metabolites from free living bacteria are less than symbiotic bacteria (Supplementary Figure 4).We identified common BGCFs shared by all strains, such as terpenes and redox-cofactors and nine BGCFs not found in free-living strains, such as Bradyrhizobium sp.S12-14-2 (Supplementary Figure 4).Additionally, Bradyrhizobium sp.S12-14-2 possesses a phosphonate-producing gene cluster, confirmed by phosphorous metabolism-related genetic analysis.

Genome size of Bradyrhizobium
Genome size variation is a biological trait, although its evolutionary sources and effects are debated.GS's neutrality, selective pressures, and strength are unknown.The genetic segments responsible for this diversity directly affect evolutionary outcomes and are targeted by diverse evolutionary pressures.Bradyrhizobium isolates have rather big genomes ranging from 5 to 11M, with an average size of 8.6M (Ormeño-Orrillo and Martínez-Romero, 2019; Avontuur et al., 2022;Jin et al., 2022b;Claassens et al., 2023;Lafay et al., 2023;Zhang et al., 2023).Nevertheless, the range of GSs within the genus remains unknown.The smallest genome was 5.5M in length and related to strain S12-14-2 recovered from freshwater sediment.The largest full assembly (11.7M) was discovered from the soil isolate GAS478 (FSRD01000000).Notably, strains S12-14-2 (7.3M), S2-20-1 T (5.6M), S2-11-2 (5.5M), S2-11-4 (5.5M), and GAS478 (11.7M) lacked symbiosis genes, suggesting that GS in Bradyrhizobium is unrelated to its capacity to interact with legumes.In the Nitrobacteraceae family, Bradyrhizobium has the largest genome, followed by Tardiphaga and Bosea.Bradyrhizobium, Tardiphaga, and Bosea, three taxa with large genomes, interact with plants (De Meyer et al., 2012;Ormeño-Orrillo and Martínez-Romero, 2019).And taxa with small genomes, including Nitrobacter, Oligotropha, and Variibacter, were discovered to be metabolically constrained, indicating that the Nitrobacteraceae GS appears to be associated with lifestyle.Understanding the GS variation and its causes and effects requires trustworthy methodologies; however, there are still obstacles.From genome annotation and content analysis to laboratory-and sequence-based GS measurement accuracy, there are problems and recommended practices.Even among big consortia building high-quality genome Pangenome analysis of Bradyrhizobium species including Bradyrhizobium sp.S12-14-2 T and closely related strains calculated with Anvi'o version 7.1.Dark regions in purple color (SCGs) represent genes (black) found in each genome at least one time.
assemblies, gold standards and best practices are continually changing.

Motility
The genome of strain S12-14-2 contains 192 genes for motility via flagella and genes for chemotaxis (data not shown).At least 132 genes related to flagellar motility (components of the flagellar apparatus or specific regulators) are grouped in two clusters in the genome separated by unknown genes.Notably, the cultured S12-14-2 cells lack any discernible flagella or pili (Supplementary Figure 1).The reason could be that twitching motility may be a more efficient mode of locomotion in environments devoid of conditions that require or prohibit pilus-facilitated or flagellumpowered movement.

Genes involved in carbon metabolism and photosynthesis
Free-living Bradyrhizobium organisms also perform vital roles in soil ecology, nutrient cycling, and carbon metabolism.As free-living microbes, they must acquire carbon from other environmental sources.Bradyrhizobium can metabolize a vast array of carbon sources, including simple carbohydrates, organic acids, and amino acids, via diverse biochemical pathways (VanInsberghe et al., 2015;Tao et al., 2021).The TCA (tricarboxylic acid) cycle, 10.3389/fmicb.2023.1295854also known as the Krebs cycle, is one of the primary pathways involved in carbon metabolism in free-living Bradyrhizobium.This pathway is essential for the growth and survival of microorganisms as it is involved in producing energy from carbon sources.The complete gene sets of the TCA cycle, Entner-Doudoroff pathway, and glycolysis and gluconeogenesis were observed in the genome of strain S12-14-2.Furthermore, genes for encoding phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31)and pentose phosphate pathway were also observed.The genome of S12-14-2 contained genes for encoding RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) or the light-harvesting complex (Figure 3A), which means that strain S12-14-2 may fix CO 2 and potentially have photosynthetic function.
The gene cluster responsible for photosynthesis in Bradyrhizobium encompasses several genes that have a crucial role in the production of pigments and proteins necessary for the process of photosynthesis.The genes in question encode various proteins, such as bacteriochlorophyll, carotenoids, and reaction center proteins (Gregor and Klug, 1999;Igarashi et al., 2001;Hata et al., 2023).The presence of genes associated with photosynthesis in the genome of S12-14-2 has been verified.The genetic components in this set are as follows: bacteriochlorophyll genes (bchCXYZGFPF), genes for light-independent protochlorophyllide reductase (chlIDNBL), carotenoid genes (crtCBICDEF), genes for light harvesting polypeptides (pucCAB), and genes for reaction center subunits (puhBA/pufLM) (Figure 3B).Several species of Bradyrhizobium have the ability to utilize carbon dioxide through the Calvin cycle, also known as the Calvin-Benson-Bassham cycle.In this metabolic pathway, the enzyme ribulose-1, 5-bisphosphate carboxylase oxygenase (RuBisCo) has a crucial role.The strain S12-14-2 was found to possess all gene sets associated with the Calvin cycle except for a gene responsible for encoding sedoheptulose-1,7-bisphosphatase (SBP) (Figure 3A).The Calvin cycle involves the execution of distinct tasks by RuBisCo (RbcL), phosphoribulokinase (PRK), and SBP.The combined utilization of RuBisCo and PRK can be used as reliable indicators for the presence of the cycle within a particular organism.However, the enzymatic activity of SBP in bacteria is commonly facilitated by fructose-1,6-bisphosphatase (FBP) with a dual sugar (Jiang et al., 2012).
The genes encoding NifH 1 , 2 DK are located upstream of the cluster, while the genes encoding NifNE are adjacent to nifKDH.In contrast to symbiotic strains, free-living Bradyrhizobium bacteria do not have nif or nod genes, and some nod-independent strains contain just nif genes (Ferrieres et al., 2004;Bobik et al., 2006;Meilhoc et al., 2010;VanInsberghe et al., 2015).No nodulation genes were identified in the genome, indicating that strain S12-14-2 could fix atmospheric nitrogen; as for nodulation, it is uncertain whether strain S12-14-2 forms nodules at this time because of nod-factorindependence of symbiosis.
Denitrification is a prominent contributor to nitrous oxide (N 2 O) emission in soil, serving as a substantial constituent of the worldwide nitrogen cycle.This process takes place in both terrestrial and marine ecosystems.Certain bacteria can adjust their metabolic processes in response to oxygen-depleted settings, wherein the concentration of dissolved and accessible oxygen is diminished.In such conditions, these bacteria use nitrate as a respiratory substrate for the process of denitrification (Coates et al., 2001;Luo F. et al., 2014;Zhang et al., 2020).The strain S12-14-2 contains a periplasmic nitrate reductase (Nar), a copper (Cu)containing nitrite reductase (Nir), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the narGHJI, nirKV, and nosRZDFYLX genes, respectively (Figure 4B).Nitrite produced by NarGHJI will be released to the periplasm through a membrane transporter and reduced to nitric oxide (NO) by Cu-containing nitrite reductase (NirK) (Richardson, 2000;Simon and Klotz, 2013).Due of its high cytotoxicity, NO will immediately reduce to N 2 O by Nor (nitric oxide reductase).Typically, nor enzymes are usually periplasmic cytochrome c (cNor) or quinones (qNor) (Nordling et al., 1990;Pearson et al., 2003;Rinaldo and Cutruzzola, 2007;Simon and Klotz, 2013).Notably, the genome does not contain NO reductase genes for the reduction stage.The apparent absence of Nor-encoding genes in strain S12-14-2 is of special interest, but it is speculated that some cNor or qNor-like proteins are encoded by some other unknown genes.

Genes involved in sulfur metabolism
Sulfur, an amino acid component and enzyme cofactor, is essential to all organisms.Sulfide is formed by the sulfate assimilation pathway in many bacteria and integrated into sulfurcontaining organic compounds.Under sulfur-limiting conditions, bacteria must receive sulfur from the environment because inorganic sulfate is not prevalent in nature.Sulfonates and sulfonate esters are natural or xenobiotic compounds (Kertesz, 2000;Van Der Ploeg et al., 2001b).Genes encoding a sulfur-oxidizing (Sox) function were discovered, and the sox gene cluster consists of 16 genes.soxR encodes a repressor protein, and soxSW encodes a periplasmic thioredoxin, which is essential for full expression of the sox genes.The subsequent soxXYZABCD genes encode four periplasmic proteins that reconstitute the Sox enzyme system: SoxXA, SoxYZ, SoxB, and SoxCD, which can convert a variety of reduced sulfur compounds to sulfates (Figure 5).Strain S12-14-2 also possess the soxF gene, which encodes flavoprotein SoxF with a sulfide dehydrogenase activity (Quentmeier et al., 2004).These sulfur oxidation Sox systems suggest that strain S12-14-2 might be capable of oxidizing thiosulfate to sulfate.
Under sulfur-limiting conditions, several bacteria express proteins to use organosulfonates or sulfonate esters as sulfur sources.Sulfate starvation-induced proteins ingest sulfonate, acquire sulfur from organic molecules, and defend against reactive oxygen species (Hummerjohann et al., 1998;Van Der Ploeg et al., 2001a;Ellis, 2011).A sulfonate transport system has been detected in the genome of Bradyrhizobium sp.S12-14-2, which includes sulfonate-binding protein (SsuA), ATP-binding protein (SsuB), and sulfonate permease protein (SsuC).The genes tauD and ssuD, which encode a taurine dioxygenase and an alkanesulfonate monooxygenase, respectively, were also found to be responsible for sulfonate and taurine metabolism.The tau operon encodes an ABC-type transporter and taurine dioxygenase (TauD), which oxidize taurine to hydroxytaurine.The unstable hydroxytaurine breaks down to yield the aminoacetaldehyde and sulfite products (Eichhorn et al., 1997).The SsuD monooxygenase enzyme catalyzes the desulfonation of a wide variety of organosulfonated products, producing the corresponding aldehyde and sulfonate (Eichhorn et al., 1999).
The structures of TauD and SsuD monomeric units were predicted using the SWISS-MODEL.The SsuD monomer consists of a total of 359 amino acid residues and belongs to the structural family of TIM-barrel proteins.SsuD is classified as a flavin-dependent oxidoreductase belonging to the LLM (luciferase-like monooxygenase) class.On the other hand, the TauD monomer consists of 297 amino acid residues and functions as an α-ketoglutarate-dependent dioxygenase (Figure 5).TauD prefers taurine but may also desulfonate alkanesulfonates.Although alkanesulfonate monooxygenase enzymes can use various organosulfate substrates, they have no enzymatic activity against taurine (Ellis, 2011).These TauD and SsuD enzymes possibly work together to ensure that the bacterial cell has sufficient 10.3389/fmicb.2023.1295854 Sulfur oxidation gene clusters and of structure of the monomeric unit of TauD and SsuD.The TauD and SsuD enzyme exist in the form of a TIM-barrel containing 297 and 359 amino acid residues, respectively.Color key: blue gray, sox genes; red, tau genes; orange, ssu genes (ac, ABC type nitrate/sulfonate/bicarbonate transport system, ATPase component; pc, ABC-type nitrate/sulfonate/bicarbonate transport systems, periplasmic components; prc, ABC-type nitrate/sulfonate/bicarbonate transport system, permease component.);gray, regulator genes.sulfur for biosynthetic processes.While most of these enzymes' kinetic properties are known, much remains to be learned about their detailed mechanistic properties.

Genes involved in phosphate metabolism
Phosphorus is an essential nutrient for all organisms and has crucial roles in respiration, energy production and storage, the biosynthesis of nucleic acids, ATP, and phospholipids and other physiological and biochemical processes in specific environments (Balemi and Negisho, 2012;Nath et al., 2017).Phosphorus availability and plant assimilation are governed by microorganisms, and phosphorus-solubilizing microorganisms are capable of dissolving insoluble phosphorus in soil and converting it into soluble phosphorus for plant absorption and use (Cao, 2014;Moro et al., 2021).Phosphonate compounds, which contain a direct C-P bond instead C-O-P ester linkage, are prevalent in the environment, where they are a significant source of organic phosphorus and a common pollutant (Horsman and Zechel, 2017;Rott et al., 2018).The degradation of phosphonates is especially advantageous for microorganisms, where the bioavailability of phosphorus is frequently a growth-limiting factor (Sosa et al., 2019).The genome of Bradyrhizobium sp.S12-14-2 contains all the predicted C-P lyase genes necessary for phosphonate degradation and the additional putative auxiliary functions (Figure 6A).These genes included a TetR family transcriptional regulator, phosphonate ABC transporters phnCDE 1 E 2 , purine ribonucleoside triphosphate phosphorylase components phnGHIJKL, a chloramphenicol acetyltransferase, a triphosphoribosyl 1-phosphonate diphosphohydrolase phnM, and a phosphoribosyl cyclic phosphodiesterase phnP.
Phosphonates are a type of phosphorus-based metabolites distinguished by a highly stable C-P bond.They comprise a significant portion of the dissolved organic phosphorus reservoir in the oceans and are widely distributed among more primitive life forms (Villarreal-Chiu et al., 2012;Acker et al., 2022).Almost all known pathways of phosphonate biosynthesis involve a central C-P bond-forming reaction in which the enzyme PEP phosphomutase (Ppm) intramolecularly rearranges the intermediary metabolite phosphoenolpyruvate (PEP) to form phosphonopyruvate (PnPy) (Metcalf and Van Der Donk, 2009;Acker et al., 2022).The genome of Bradyrhizobium sp.S12-14-2 contains the genes pepM which encodes Ppm, ppd encoding phosphonopyruvate decarboxylase (Ppd) which subsequently converts PnPy to phosphonoacetaldehyde (PnAa) in next reaction, and pdh encoding phosphonoacetaldehyde dehydrogenase (Pdh) which dehydrogenates of PnAa to 2-hydroxyethylphosphonic acid (HEP) (Figure 6B).

Conclusion
To summarize our investigations, the strain S12-14-2 T from a contaminated freshwater sediment belonging to the genus Bradyrhizobium was studied through genome analysis and a polyphasic approach.The phylogenetic analysis indicated that the strain S12-14-2 T was affiliated closely with the species of Bradyrhizobium, based on the phylogenetic, genomic, and physiological differences; thus, we propose strain S12-14-2 T as a novel species, Bradyrhizobium roseus sp.nov., in the family Nitrobacteraceae (Table 2).The B. roseus pan-genome is considered to be in an open state, genetically diverse, and with a flexible gene repertoire associated with multiple functions.B. roseus contained genes encoding all enzymes of the Calvin-Benson cycle and phototrophic systems with carotenoids synthesizing genes and possesses complete genes for nitrogen fixing and denitrification.B. roseus S12-14-2 T also contains genes for sulfur oxidation and the essential genes encoding for the sulfonate transport system.Moreover, strain S12-14-2 T contains all the necessary genes for phosphonate degradation and phosphate biosynthesis.The physiological characteristics and comparative genome analyses of B. roseus S12-14-2 T help us to understand the metabolism and evolutionary features of the genus Bradyrhizobium and provide understanding of this microorganism and a new genetic framework for future studies.