Characterization and Genomic Analysis of Fererhizobium litorale gen. nov., sp. nov., Isolated from the Sandy Sediments of the Sea of Japan Seashore

The taxonomic status of two gram-negative, whitish-pigmented motile bacteria KMM 9576T and KMM 9553 isolated from a sandy sediment sample from the Sea of Japan seashore was defined. Phylogenetic analysis revealed that strains KMM 9576T and KMM 9553 represent a distinct lineage within the family Rhizobiaceae, sharing 100% 16S rRNA sequence similarity and 99.5% average nucleotide identity (ANI) to each other. The strains showed the highest 16S rRNA sequence similarities of 97.4% to Sinorhizobium garamanticum LMG 24692T, 96.9% to Ensifer adhaerens NBRC 100388T, and 96.8% to Pararhizobium giardinii NBRC 107135T. The ANI values between strain KMM 9576T and Ensifer adhaerens NBRC 100388T, Sinorhizobium fredii USDA 205T, Pararhizobium giardinii NBRC 107135T, and Rhizobium leguminosarum NBRC 14778T were 79.9%, 79.6%, 79.4%, and 79.2%, respectively. The highest core-proteome average amino acid identity (cpAAI) values of 82.1% and 83.1% were estimated between strain KMM 9576T and Rhizobium leguminosarum NBRC 14778T and ‘Rhizobium album’ NS-104, respectively. The DNA GC contents were calculated from a genome sequence to be 61.5% (KMM 9576T) and 61.4% (KMM 9553). Both strains contained the major ubiquinone Q-10 and C18:1ω7c as the dominant fatty acid followed by 11-methyl C18:1ω7c and C19:0 cyclo, and polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, and two unidentified phospholipids. Based on phylogenetic and phylogenomic analyses, and phenotypic characterization, strains KMM 9576T and KMM 9553 are concluded to represent a novel genus and species, for which the name Fererhizobium litorale gen. nov., sp. nov. is proposed. The type strain of the type species is strain KMM 9576T (=NRIC 0957T).


Introduction
The genus Rhizobium, the type genus of the family Rhizobiaceae Conn 1938 [1], first described by Frank (1889) [2] and emended by Young et al. (2001) [3], has been the object of numerous taxonomic studies for a long time.It has been reported that the 16S rRNA gene sequencing has limitations in differentiating Rhizobiaceae members, whereas a multilocus sequence analysis (MLSA) of housekeeping genes proves to be more effective in resolving closely related species of the taxonomic position [4][5][6][7][8].The use of MLSA demonstrated high heterogeneity of the genera Rhizobium and Agrobacterium within the family Rhizobiaceae, which led to the reclassification of the species group "Rhizobium galegae" as a novel genus Neorhizobium [7].In addition, based on the MLSA results, the genus named Pararhizobium was proposed to accommodate the cluster of Rhizobium giardinii, R. herbae, "R.helanshanense," R. sphaerophysae, and Blastobacter capsulatus [7,8].Recently, in the phylogenomic study based on a whole genome sequencing analysis, Kuzmanovic et al. (2022) [9] have proposed a common approach for the genera delineation in the family Rhizobiaceae, applying a core-genome gene phylogeny and a pairwise core-proteome average amino acid identity (cpAAI) value of approximately 86%.The application of the proposed framework resulted in the reclassification of several Rhizobium species, including a proposal for the new genus Xaviernesmea and the retrieval of Ensifer and Sinorhizobium, which had previously been unified [10,11] as separate genera [9].Currently, the family Rhizobiaceae comprises 19 genera, including Rhizobium and related Ensifer, Sinorhizobium, Agrobacterium, Pararhizobium, Neorhizobium, Shinella, Ciceribacter, etc. [9].At the time of writing, the genus Rhizobium contains the largest number of validly described species; 88 as listed at http://lpsn.dsmz.de/genus/rhizobia,accessed on 12 September 2023, including Rhizobium leguminosarum as the type species of the genus [2,9,12,13].Most rhizobia are able to invade plants of the family Leguminosae and incite the production of tumors or/and root nodules where the bacteria act as intracellular nitrogen-fixing symbionts.The type species Ensifer adhaerens had been reported to be a predator of Micrococcus luteus [14].Members of the family Rhizobiaceae have been mainly originated from the rhizosphere and the roots of leguminous and other agricultural crops, as well as recovered from aquatic and marine ecosystems, including deep-sea and coastal sediments as non-symbiotic microorganisms [15][16][17][18][19][20][21].
The aim of the present study was to determine the taxonomic status of two aerobic, gram-negative, motile, whitish-pigmented bacteria designated KMM 9576 T and KMM 9553 isolated from a sandy sediment sample obtained from the Sea of Japan seashore.Based on molecular and phenotypic data obtained, a novel genus and species, Fererhizobium litorale gen.nov., sp.nov. is described.

Isolation and Phenotypic Characterization of Bacteria
A sandy sediment sample was collected from offshore of the Sea of Japan, Russia, (42 •  54.8416 N 131 • 43.0430 E) at a water depth of 0.3 m on 25 October, 2012.Strains KMM 9576 T and KMM 9553 were isolated using the dilution plating technique on tryptic soya agar (TSA) and the seawater medium (SWM), as described in a previous paper [22].The strains were grown aerobically on Marine Agar 2216 (MA 2216) or Marine Broth 2216 (MB 2216), Tryptic Soya Agar (TSA) or Tryptic Soya Broth (TSB), and R2A agar (all BD Difco), yeast extract-mannitol agar (YMA) or broth (YMB), containing 10 g of mannitol; 0.5 g of KH 2 PO 4 ; 0.2 g of MgSO 4 7H 2 O; 0.1 g of NaCl; 4 g of CaCO 3 ; 0.4 g of yeast extract; 15 g of agar, distilled water of 1 l, and stored at −70 • C in the MB supplemented with 20% (v/v) glycerol.No growth was observed when they were tested for the anaerobic growth on MA 2216 for a week using an AnaeroPack TM (Mitsubishi Gas Chemical America, Inc., New York, NY, USA).Strains KMM 9553 and KMM 9576 T have been deposited to the Collection of Marine Microorganisms (KMM), G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia, and the type strain KMM 9576 T was deposited to the NODAI Culture Collection Center (NRIC), Tokyo University of Agriculture, Japan, under number of NRIC 0957 T .The type strains Rhizobium leguminosarum NBRC 14778 T , Ensifer adhaerens NBRC 100388 T , Sinorhizobium garamanticum LMG 24692 T , Rhizobium rhizogenes NBRC 13257 T , Agrobacterium radiobacter NBRC 13532 T , Pararhizobium giardinii NBRC 107135 T , and Mesorhizobium mediterraneum JCM 21565 T were kindly provided by the respective culture collections.All strains used in this study for phenotypic tests and lipid analyses were grown on/in YMA and YMB, if not stated otherwise.Gram-staining, oxidase, catalase, and motility (the hanging drop method) tests were examined according to the standard methods described by Smibert and Krieg (1994) [23].The morphology of cells negatively stained with a 1% phosphotungstic acid was examined by electronic transmission microscopy Libra 120 (Carl Zeiss, Oberkochen, Germany), provided by the Far Eastern Centre of electronic microscopy, A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, using cells grown in diluted (1/10) TSB on carbon-coated 200-mesh copper grids.The tests, including the hydrolysis of starch, gelatin, L-tyrosine, chitin, casein, DNA, and nitrate reduction (sulfanilic acid/α-naphthylamine test), as well as the growth at different salinities (0-10% NaCl), temperatures (5-40 • C), and pH values (4.0-11.5)were conducted as described by Smibert and Krieg (1994) [23].The medium YMA (or YMB) was used as a basal, while mannitol and CaCO 3 were omitted for determination of substrate hydrolysis and pH, respectively.Formation of H 2 S from thiosulfate was tested in the YMB using a lead acetate paper strip.Biochemical tests for all studied using API 20E, and strains KMM 9576 T and KMM 9553 using API ID32 GN (bioMérieux, Marcy-l'Étoile, France), were performed as described by the manufacturer.Carbon source utilization was performed with the API 50 CHB/E tests (bioMérieux, Marcy-l'Étoile, France) according to the manufacturer's instructions.

16S rRNA Gene Sequence and Phylogenetic Analysis
Genomic DNAs of strains KMM 9576 T and KMM 9553 extracted according to the method of Saito and Miura [28] were used to determine 16S rRNA gene sequences as described by Shida et al. (1997) [29].The 16S rRNA gene sequences were compared with those of the closest relatives using EzBioCloud service [30].Phylogenies were performed on the GGDC web server (http://ggdc.dsmz.de/,accessed on 12 September 2023) [31] using the DSMZ phylogenomics pipeline [32] adapted to single genes.Maximum likelihood (ML) and maximum parsimony (MP) trees were inferred from the alignment with RAxML [33] and TNT [34], respectively.The robustness of phylogenetic trees was estimated by the bootstrap analysis of 1000 replicates.

Whole-Genome Sequencing, Phylogenomic, and Comparative Analyses
The genomic DNAs were obtained from the strains KMM 9576 T and KMM 9553 using the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland).The quantity and quality of the genomic DNAs were measured using DNA gel electrophoresis and the Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Singapore, Singapore).Preparation of the DNAs sequencing libraries was conducted using Nextera DNA Flex kits (Illumina, San Diego, CA, USA) and whole-genome sequencing were performed subsequently using paired-end runs on an Illumina MiSeq platform with a 150-bp read length.The reads were trimmed using Trimmomatic version 0.39 [35], and their quality was assessed using FastQC version 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 21 August 2021).Filtered reads were assembled into contigs with SPAdes version 3.15.3[36], and genome metrics were calculated with the help of QUAST version 5.0.2 [37].The genome completeness and contamination were estimated by CheckM version 1.1.3based on the taxonomic-specific workflow (family Rhizobiaceae) [38].
The draft genome assemblies were annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotation using Subsystem Technology (RAST) [39,40].Comparisons of the Average Nucleotide Identity (ANI), Average Amino Acid Identity (AAI), and digital DNA-DNA hybridization (dDDH) values of the strains KMM 9576 T and KMM 9553 and their closest neighbors were performed with the online server ANI/AAI-Matrix [41] and TYGS platform [31], respectively.Core-proteome average amino acid identity (cpAAI) values between the strains and representatives of the family Rhizobiaceae were computed using a pipeline proposed by Kuzmanović et al. (https://github.com/flass/cpAAI_Rhizobiaceae, accessed on 10 January 2023) [9].The core-proteome phylogeny based on 170 non-recombining core protein markers, including various members of Rhizobium genus, was inferred using IQ-TREE software under the LG + F + I + I + R8 model with bootstrapping using 100 replicates [42].

Phylogenetic and Phylogenomic Analyses
The 16S rRNA gene sequences of strains KMM 9576 T and KMM 9553 submitted to the GenBank under accession numbers LC126306 and LC126307 were 100% identical to the 16S rRNA gene sequences ON040664 and ON040663, respectively, and retracted from their genomic sequences.Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains KMM 9576 T and KMM 9553 are close to each other (100% identity), representing a distinct lineage within the family Rhizobiaceae (Figure 1).In the phylogenetic trees, Hoeflea anabaenae WH2K T was a sister clade.However, the strains shared the highest sequence similarity to S. garamanticum LMG 24692 T (97.7%), followed by E. adhaerens NBRC 100388 T (96.9%) and P. giardinii NBRC 107135 T (96.8%).The similarity to other Rhizobiaceae members did not exceed 96.8%.adhaerens NBRC 100388 T (96.9%) and P. giardinii NBRC 107135 T (96.8%).The similarity to other Rhizobiaceae members did not exceed 96.8%.The ANI values between strain KMM 9576 T and E. adhaerens NBRC 100388 T , S. fredii USDA 205 T , P. giardinii NBRC 107135 T , and R. leguminosarum NBRC 14778 T were 79.9%, 79.6%, 79.4%, and 79.2%, respectively.The AAI values between strains KMM 9576 T and KMM 9553, and members of related genera, varied from 66.7 to 69.8% (Table S2).The AAI values fell into the range of 60-80% [47] and did not exceed a cut-off value of 75% accepted for the delineation of Rhizobiaceae genera [48].Genome sequences of strains KMM 9576 T and KMM 9553 exhibited ANI/AAI values of the 99.5%/ 99.7%, and the dDDH values of 99.6%, confirming their non-clonal origin (Table S1).
In addition, the highest cpAAI values of 82.1% and 83.1% were estimated between strain KMM 9576 T and R. leguminosarum NBRC 14778 T and "Rhizobium album" NS-104 T [49], respectively, which is below a threshold value of 86% proposed by Kuzmanovic et al. (2022) [9] for the genera discrimination in the family Rhizobiaceae (Figure 2, Table S2).The core-proteome phylogeny provided by the cpAAI pipeline based on 97 strains of Rhizobiaceae genera (github.com/flass/cpAAI_Rhizobiaceae)showed that the strains KMM 9576 T and KMM 9553 were more closely related to "R. album" NS-104 T and "leguminosarumrhizogene" clades (Figure 3).The position of strain "R.album" NS-104 on the phylogenetic tree (Figure 3), as well as its pairwise cpAAI values (Table S2), which indicate that it was incorrectly named and should be designated as a new Rhizobiaceae genus.Phylogenomic analysis confirmed that the novel strains form a distinct lineage adjacent to R. leguminosarum NBRC 14778 T , which indicated that they may represent a novel genus of the family Rhizobiaceae.
Microorganisms 2023, 11, x FOR PEER REVIEW 6 of 16 phylogenetic tree (Figure 3), as well as its pairwise cpAAI values (Table S2), which indicate that it was incorrectly named and should be designated as a new Rhizobiaceae genus.Phylogenomic analysis confirmed that the novel strains form a distinct lineage adjacent to R. leguminosarum NBRC 14778 T , which indicated that they may represent a novel genus of the family Rhizobiaceae.Thus, these phylogenetic and phylogenomic data suggested that strains KMM 9576 T and KMM 9553 represent a novel species and a novel genus within the family Rhizobiaceae.Thus, these phylogenetic and phylogenomic data suggested that strains KMM 9576 T and KMM 9553 represent a novel species and a novel genus within the family Rhizobiaceae.

Genomic Characteristics
According to an evaluation by the CheckM tool [38], the genome sequences of strains KMM 9576 T and KMM 9553 were obtained with high completeness (98.2% and 100%) at low values of contaminations (0.04% and 0.22%), respectively.The draft genomes of KMM 9576 T and KMM 9553 were de novo assembled into 67 and 94 contigs, with N 50 values of 350,002 and 211,989 bp, and L 50 values of 5 and 8, respectively.The genome sizes were estimated to be 5,252,096 and 5,494,465 bp in length with a coverage of 61× and 41×, respectively.The genome sequences were in accordance with the proposed minimal standards for bacterial taxonomy [50].The comparison of genome features with genomes of type strains of closely and distantly related Rhizobiaceae genera is presented in Table 1.The genome sequences contain from 4981 (KMM 9576 T ) to 7623 (R. leguminosarum USDA 2370 T ) genes, from 45 (KMM 9576 T ) to 64 (E.adhaerens ATCC 33212 T ) tRNAs, and from 1 up to 5 ribosomal RNA operon copies (E.adhaerens ATCC 33212 T ).
According to the RAST annotation, about 1400 genes for both strains KMM 9576 T and KMM 9553 are in subsystems, among which the largest number of genes was assigned to "Amino Acids and Derivatives," "Carbohydrates," and "Cofactors, Vitamins, Prosthetic Groups, Pigments."According to the KEGG annotation, both strains have genes encoding the Embden-Meyerhof pathway (except for phosphofructokinase and fructosebisphosphatase ones, but diphosphate-dependent phosphofructokinase is present), the pentose phosphate pathway, the Krebs cycle, and the glyoxylate cycle.In addition, there is a complete pathway for glycogen biosynthesis and degradation.According to the dbCAN3 server, KMM 9576 T and KMM 9553 encode a total of 109 and 113 CAZymes, respectively, with glycosyltransferases (GT) being more abundant in the GT2 and GT14 families.
To identify Fererhizobium specific genes, orthologous groups were analyzed using the translated proteomes of KMM 9576 T and KMM 9553 in comparison with those of the three closely related genera "R.album", R. leguminosarum, and R. rhizogenes (Figure 3).A total of 6487 orthologous clusters comprising 31,339 proteins were found.The core genome shared by the five compared strains was represented by 2547 orthologous clusters (Figure 4a).Among them, 1781 were single-copy gene clusters.
The distinctive feature of the Fererhizobium core genome was an excessive number of genes in the clusters of L-arabinose transport ATP-binding protein (GO:0015407; 11-12 genes versus 1-3), and putative adenylate cyclase 3 (GO: 0035556; 9-10 genes versus one).In pairwise comparison, the largest number of orthologous clusters of 963 was revealed for the pair KMM 9576 T /KMM 9553, following R. rhizogenes/R.leguminosarum (694), "R.album"/R.leguminosarum (248), and "R.album"/R.rhizogenes (145).Both Fererhizobium strains shared the most of clusters with "R.album" NS-104 T (246), followed by R. leguminosarum NBRC 13257 T (203) and R. rhizogenes NBRC 13257 T (87) (Figure 4a).Among 963 Fererhizobium-specific orthologous clusters, functions of the most were associated with biological processes, such as transcription (GO:0006351) and regulation of transcription (GO:0006355), transmembrane transport (GO:0055085), and sodium ion transport (GO:0006814).A number of specific clusters (Figure 4b) for every strain were 128 for "R.album" NS-104 T , 93 for R. leguminosarum NBRC 14778 T , 70 for R. rhizogenes NBRC 13257 T , and only 7 for KMM 9553.According to the RAST and eggNOG-mapper annotations, more than 50% of singletons were not annotated.Singletons for both strains KMM 9576 T and KMM 9553 were mostly enriched in genes encoding mobile or selfish elements, restriction-modification systems, transport systems, and regulatory elements.Both strains do not have key nitrogen-fixation (nifHDK) and nodulation (nodABC) genes in their genomes.In addition, genes that are involved in the biosynthesis of capsular polysaccharide, exopolysaccharide, and lipopolysaccharide were revealed in KMM 9576 T and KMM 9553.According to antiSMASH, the annotation of secondary metabolite biosynthetic gene clusters uncovered two homoserine lactone biosynthetic clusters, one redox-cofactor, such as PQQ (NC_021985:1458906-1494876), one thioamitide RiPP, as found in JOBF01000011, one other unspecified ribosomally synthesized and post-translationally modified peptide product (RiPP), one N-acyl amino acid, and one terpene biosynthetic cluster.shared by the five compared strains was represented by 2547 orthologous clusters (Figure 4a).Among them, 1781 were single-copy gene clusters.The distinctive feature of the Fererhizobium core genome was an excessive number of genes in the clusters of L-arabinose transport ATP-binding protein (GO:0015407; 11-12 genes versus 1-3), and putative adenylate cyclase 3 (GO: 0035556; 9-10 genes versus one).In pairwise comparison, the largest number of orthologous clusters of 963 was revealed for the pair KMM 9576 T /KMM 9553, following R. rhizogenes/R.leguminosarum (694), "R.album"/R.leguminosarum (248), and "R.album"/R.rhizogenes (145).Both Fererhizobium strains shared the most of clusters with "R.album" NS-104 T (246), followed by R. leguminosarum NBRC 13257 T (203) and R. rhizogenes NBRC 13257 T (87) (Figure 4a).Among 963 Fererhizobium-specific orthologous clusters, functions of the most were associated with biological processes, such as transcription (GO:0006351) and regulation of transcription (GO:0006355), transmembrane transport (GO:0055085), and sodium ion transport (GO:0006814).A number of specific clusters (Figure 4b) for every strain were 128 for "R.album" NS-104 T , 93 for R. leguminosarum NBRC 14778 T , 70 for R. rhizogenes NBRC 13257 T , and only 7 for KMM 9553.According to the RAST and eggNOG-mapper annotations, more than 50% of singletons were not annotated.Singletons for both strains KMM 9576 T and KMM 9553 were mostly enriched in genes encoding mobile or selfish elements, restriction-modification systems, transport systems, and regulatory elements.Both strains do not have key nitrogen-fixation (nifHDK) and nodulation (nodABC) genes in their

Phenotypic Characterization and Chemotaxonomy
Bacteria KMM 9576 T and KMM 9553 were small rod-or ovoid-shaped motile by one or two subpolar flagella, and cells containing more than two flagella were also observed (Figure 5a,c).Novel bacteria could grow in salinities from 0 to 4% and grew well on/in SWM, MA 2216, MB 2216, R2A agar, TSB, TSA, YMA, and YMB.On carbohydratecontaining media, novel strains produced copious extracellular material, particularly strain KMM 9553.Individual cells of strain KMM 9553 could be observed as non-motile probably due to the large amount of mucus the strain tends to produce (Figure 5b).
The novel strains were not identical in their phenotypic traits.Strain KMM 9576 T , unlike KMM 9553, demonstrated a weak reaction of DNA hydrolysis, which tested negative for the oxidation of L-arabinose in API 20E and proved to be resistant to cephazolin and oleandomycin (Table 2) and utilized different carbon sources in API 50 CHB/E tests (Table S3).Strains KMM 9576 T and KMM 9553 were similar to A. radiobacter NBRC 13532 T in being able to grow in 0-4% of NaCl, and to hydrolyze DNA.Unlike other relatives, strains KMM 9576 T and KMM 9553 and R. leguminosarum NBRC 14778 T were able to produce H 2 S and hydrolyze tyrosine (Table 2).Strains KMM 9576 T and KMM 9553 differed from E. adhaerens NBRC 100388 T and P. giardinii NBRC 107135 T in not being able to grow in 2-4% of NaCl and in their carbohydrate utilization patterns (Tables 2 and S3).
Bacteria KMM 9576 T and KMM 9553 were small rod-or ovoid-shaped motile by one or two subpolar flagella, and cells containing more than two flagella were also observed (Figures 5a,c).Novel bacteria could grow in salinities from 0 to 4% and grew well on/in SWM, MA 2216, MB 2216, R2A agar, TSB, TSA, YMA, and YMB.On carbohydrate-containing media, novel strains produced copious extracellular material, particularly strain KMM 9553.Individual cells of strain KMM 9553 could be observed as non-motile probably due to the large amount of mucus the strain tends to produce (Figure 5b).The novel strains were not identical in their phenotypic traits.Strain KMM 9576 T , unlike KMM 9553, demonstrated a weak reaction of DNA hydrolysis, which tested negative for the oxidation of L-arabinose in API 20E and proved to be resistant to cephazolin and oleandomycin (Table 2) and utilized different carbon sources in API 50 CHB/E tests (Table S3).Strains KMM 9576 T and KMM 9553 were similar to A. radiobacter NBRC 13532 T in being able to grow in 0-4% of NaCl, and to hydrolyze DNA.Unlike other relatives, strains KMM 9576 T and KMM 9553 and R. leguminosarum NBRC 14778 T were able to produce H2S and hydrolyze tyrosine (Table 2).Strains KMM 9576 T and KMM 9553 differed from E. adhaerens NBRC 100388 T and P. giardinii NBRC 107135 T in not being able to grow in 2-4% of NaCl and in their carbohydrate utilization patterns (Tables 2 and S3).Both strains KMM 9576 T and KMM 9553 contained Q-10 as the major ubiquinone; the predominant fatty acid was detected to be C 18:1 ω7c (52.47 and 46.16%), followed by 11-Methyl C 18:1 ω7c (14.91 and 14.11%), C 19:0 cyclo (13.01 and 15.27%), C 18:0 (10.98 and 7.09%), and C 14:0 3-OH (3.86 and 12.77%) in both strains, respectively (Table 3).The fatty acid profiles of strains KMM 9576 T and KMM 9553 were found to be similar to that of R. leguminosarum NBRC 14778 T .Strains E. adhaerens, NBRC 100388 T , and S. garamanticum LMG 24692 T differed from the novel strains in the presence of small amounts of C 16:0 3-OH, C 18:0 3-OH, and C 17:0 cyclo (in E. adhaerens NBRC 100388 T ), while P. giardinii NBRC 107135 T differed in the presence of C 18:0 3-OH and C 17:0 cyclo, as shown in Table 3. Strains M. mediterraneum JCM 21565 T did not contain C 14:0 3-OH and revealed a distinction in the presence of iso-C 17:0 as compared with other related strains (Table 3).R. rhizogenes NBRC 13257 T was significantly distinguished from all bacteria studied by the content of C 15:0 3-OH and C 19:0 .These findings corroborate the results reported by Tighe et al. [51].Unlike related type strains, R. rhizogenes NBRC 13257 T and A. radiobacter NBRC 13532 T contained high proportions of C 14:0 3-OH and C 16:0 3-OH, whereas novel strains contained a high amount of 11-Methyl C 18:1 ω7c (Table 3).
The polar lipid compositions of strains KMM 9576 T , KMM 9553, and related bacteria tested did not significantly differ and included phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), an unidentified aminophospholipid (APL), and two unidentified phospholipids (PL and PL1) (Figure S1).The type strains R. rhizogenes NBRC 13257 T , S. garamanticum LMG 24692 T , E. adhaerens NBRC 100388 T , and A. radiobacter NBRC 13532 T differed from novel strains by the presence of additional unidentified phospholipids (PL2) in their lipid compositions.R. rhizogenes NBRC 13257 T and S. garamanticum LMG 24692 T contained additional unidentified lipids (L and/or L1).M. mediterraneum JCM 21565 T contained a minor amount of PE compared to those of novel strains and included an unidentified lipid (L2) (Figure S1).
The DNA GC contents of 61.4% and 61.5% were calculated from a genome sequence of strains KMM 9576 T and KMM 9553, respectively, which is close to the values of 49-68 mol% reported for the members of the family Rhizobiaceae [1].The phylogenetic distinctness of strains KMM 9576 T and KMM 9553 was supported by phenotypic differences in the temperature and salinity ranges that provided their growth, substrate hydrolysis ability, and carbohydrate utilization patterns.Differential phenotypic and physiological characteristics are indicated in Tables 2 and S2.Based on the combination of phylogenomic analyses and phenotypic characteristics, it is proposed to classify strains KMM 9576 T and KMM 9553 as a novel genus and species, Fererhizobium litorale.Gram-stain negative, aerobic non-spore-forming rod-shaped or ovoid cells.Oxidaseand catalase-positive.The major respiratory quinone is ubiquinone-10.The major fatty acid is C 18:1 ω7c, followed by 11-Methyl C 18:1 ω7c and C 19:0 cyclo.The polar lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, and two unidentified phospholipids.Phylogenetically related to members of the genera Rhizobium, Sinorhizobium, and Pararhizobium within the family Rhizobiaceae, class Alphaproteobacteria.The type species is Fererhizobium litorale sp.nov.
In addition to the properties described for the genus, the species is characterized as follows.Whitish-pigmented, rod-or ovoid-shaped cells, 0.5-0.7 µm in diameter and 0.8-1.4µm in length, encapsulated, motile by one or two subpolar flagella.Cells may be observed as non-motile, with growth on/in YMA, YMB, TSA, TSB, MA 2216, MB 2216, and SWM.On carbohydrate-containing media, extracellular material is produced abundantly.Growth occurs in 0-4% of NaCl (optimal is 0.5-1%), and at 5-38 • C (optimal is 28-30 • C); there is weak growth at 38 • C and no growth at 40 • C. The pH range for growth is 6.0-10.0,with an optimum of 7.0-8.0.It is negative for hydrolysis of gelatin, casein, starch, chitin, Tween 80, and nitrate reduction, and positive for DNA hydrolysis and H 2 S production in conventional tests.On the L-tyrosine-containing medium, it forms a transparent zone but does not produce black-brown pigments.
According to the API 20E, tests were positive for citrate utilization, PNPG test, urease production under anaerobic conditions, and oxidation of D-sucrose (weak reaction), while they were negative for gelatin hydrolysis, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, H 2 S production under anaerobic conditions and tryptophane deaminase, indole production, acetoin production (Voges-Proskauer reaction), and an oxidation of D-glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, D-melibiose, and amygdalin; oxidation of L-arabinose is strain-dependent (reaction of the type strain is negative).
The GenBank accession number for the whole-genome shotgun sequence of strain KMM 9576 T is JALDYZ010000000.

Figure 1 .
Figure 1.ML tree based on 16S rRNA gene sequences available from the GenBank database showing relationships of the new strains KMM 9576 T , KMM 9553 (in bold), and related members of the family Rhizobiaceae.The tree was inferred under the GTR + GAMMA model and rooted by midpoint rooting.The branches are scaled in terms of the expected number of substitutions per site.The numbers above the branches are support values when larger than 60% from ML (left) and MP (right) bootstrapping (1000 replicates).

Figure 1 .
Figure 1.ML tree based on 16S rRNA gene sequences available from the GenBank database showing relationships of the new strains KMM 9576 T , KMM 9553 (in bold), and related members of the family Rhizobiaceae.The tree was inferred under the GTR + GAMMA model and rooted by midpoint rooting.The branches are scaled in terms of the expected number of substitutions per site.The numbers above the branches are support values when larger than 60% from ML (left) and MP (right) bootstrapping (1000 replicates).

Figure 2 .
Figure 2. Heatmap of pairwise cpAAI values between the strains KMM 9576 T and KMM 9553 and the members of Rhizobiaceae clades.The Mesorhizobium spp.strains and Georhizobium profindi WS11 were included as outgroups.The KMM 9576 T and KMM 9553 are marked in bold.

Figure 2 .
Figure 2. Heatmap of pairwise cpAAI values between the strains KMM 9576 T and KMM 9553 and the members of Rhizobiaceae clades.The Mesorhizobium spp.strains and Georhizobium profindi WS11 were included as outgroups.The KMM 9576 T and KMM 9553 are marked in bold.

Figure 3 .
Figure 3. ML tree based on concatenated 170 protein sequences showing a phylogenetic position of the novel strains KMM 9576 T , KMM 9553, and related members of the family Rhizobiaceae.The tree was inferred using IQ-TREE software under the LG + F + I + I + R8 model with bootstrapping of 100 replicates.The Mesorhizobium spp.strains and Georhizobium profindi WS11 were used as outgroups.Bar, 0.05 substitutions per amino acid position.Collapsed branches are shown in black triangles.

Figure 3 .
Figure 3. ML tree based on concatenated 170 protein sequences showing a phylogenetic position of the novel strains KMM 9576 T , KMM 9553, and related members of the family Rhizobiaceae.The tree was inferred using IQ-TREE software under the LG + F + I + I + R8 model with bootstrapping of 100 replicates.The Mesorhizobium spp.strains and Georhizobium profindi WS11 were used as outgroups.Bar, 0.05 substitutions per amino acid position.Collapsed branches are shown in black triangles.

Figure 4 .
Figure 4. Proteome comparison of Fererhizobium strains and type strains of closely related genera "R.album" NS-104 T , R. leguminosarum NBRC 14778 T , and R. rhizogenes NBRC 13257 T using Ortho-Venn3.UpSet-JS table displays common (a) and unique (b) orthologous clusters, and singletons (b).Horizontal bar chart on the left shows the number of orthologous clusters per strain.Right vertical bar chart indicates the number of orthologous clusters shared among the strains.The lines represent intersecting sets.Upper horizontal bar chart (b) shows the number of taxon-specific clusters including their proteins and singletons per strain.

Figure 4 .
Figure 4. Proteome comparison of Fererhizobium strains and type strains of closely related genera "R.album" NS-104 T , R. leguminosarum NBRC 14778 T , and R. rhizogenes NBRC 13257 T using Or-thoVenn3.UpSet-JS table displays common (a) and unique (b) orthologous clusters, and singletons (b).Horizontal bar chart on the left shows the number of orthologous clusters per strain.Right vertical bar chart indicates the number of orthologous clusters shared among the strains.The lines represent intersecting sets.Upper horizontal bar chart (b) shows the number of taxon-specific clusters including their proteins and singletons per strain.

Table 1 .
Genomic features of strains KMM 9576 T , KMM 9553, and type strains of closely related Rhizobiaceae genera.

Table 2 .
Differential characteristics of strains KMM 9576 T and KMM 9553 and type strains of related genera.

Table 2 .
Differential characteristics of strains KMM 9576 T and KMM 9553 and type strains of related genera.

Table 3 .
Cellular fatty acid composition (%) of strains KMM 9576 T and KMM 9553 and type strains of related Rhizobiaceae members.M. mediterraneum JCM 21565 T (all results were obtained from the present study).-, Not detected.Fatty acids representing <1% in all strains tested are not shown.Rhizobium a bacterial generic name; N.L. neut.n.Fererhizobium, a genus adjacent to Rhizobium).