Introduction

Flavobacteriia, a class in the phylum Bacteroidota, contains eight bacterial families, of which 182 genera have been identified. Among them, 155 genera are members of the family Flavobacteriaceae1. Species in Flavobacteriaceae have been isolated from diverse environments, including the ocean, freshwater, and soil. Their lifestyles range widely, encompassing roles as plankton, nutrient recyclers, commensals, and even pathogens. A wide range of different habitats among the taxa is one of the most intriguing characteristics of the family. Genomic comparison of bacteria in the genus Flavobacterium revealed that the niche adaptation to aquatic or terrestrial environments correlates with genomic characteristics such as genome size and the number of genes to utilize peptides or carbohydrates2.

The genus Mariniflexile3,4 belongs to Flavobacteriaceae5, and the type strain KCTC 12570T of the type species Mariniflexile gromovii was first isolated from sea urchins in Troitsa Bay, Russia3. Currently, the type strains of eight validly named Mariniflexile species have been isolated from marine environments3,4,6,7,8,9. Specifically, Mariniflexile aquimaris HWR-17T was isolated from seawater9 and M. gromovii KCTC 12570T and Mariniflexile ostreae TYO-10T from marine Animalia3,4. Mariniflexile jejuense SSK2-3T and Mariniflexile soesokkakense RSSK-9T were isolated from the junction between seawater and freshwater6,8. Mariniflexile fucanivorans SW5T was isolated from a water-treatment facility and was able to degrade sulfated fucans7,10. Mariniflexile litorale KMM 9835T was isolated from shallow marine sediments11. Mariniflexile maritimum M5A1MT was isolated from seawater collected from the South Sea of the Republic of Korea12.

In our previous work13, we reconstructed a draft microbial genome, TRG1 (tomato rhizosphere genome 1), from the metagenomic sequence data, that was more abundant in the rhizosphere of Hawaii 7996, which is a tomato cultivar resistant to bacterial wilt caused by Ralstonia solanacearum14 than in the rhizosphere of Moneymaker, which is susceptible to the disease15. Using the genetic information in the reconstructed genome, we successfully isolated 22 bacterial strains from the Hawaii 7996 rhizospheric soil and designated them TRM1-1 through TRM1-22, collectively referred to as the TRM1 strains13. Moreover, the inoculation of TRM1-10 to Moneymaker significantly reduced both the incidence and severity of bacterial wilt, indicating that this bacterium plays a role in suppressing disease development13. Genomic characterization of the TRM1 strains originating from soil, may expand our knowledge about habitats and lifestyles of the TRM1 strains and the genus Mariniflexile, whose members have been known to reside in the marine environment.

In this study, we characterized TRM1-10T using phylogenetic, phenotypic, and chemotaxonomic approaches to propose it as the type strain of a novel species in Mariniflexile. We also determined the genome sequences of TRM1-10T, TRM1-13, TRM1-18, TRM1-20, and TRM1-22, to support the polyphasic taxonomy and elucidate the unique lifestyle of this species. Finally, comparative genomic analysis of the taxon and other phylogenetically close flavobacteria demonstrated that carbohydrate metabolism could be a major factor for this species in adaptation to the rhizosphere environment.

Results

Morphological, phylogenetic, and phylogenomic characterization of TRM1-10T

Strain TRM1-10T is a Gram-negative, rod-shaped, and obligate aerobic bacterium. The cells are 0.3–0.5 μm in diameter and 3.0–4.0 μm in length, forming yellow colonies of 0.5–2.0 mm in size on marine agar within three days (Fig. 1a). The optimum growth of the strain TRM1-10T was observed at 25–30 °C (Fig. 1b), with a range from 4 to 40 °C. In the genome of TRM1-10T, two copies of the 16S rRNA gene were detected and their nucleotide sequences indicated that they are 100% identical. The full length of the 16 S rRNA genes in TRM1-10T is 1,510 nucleotides and it shows high sequence similarities with those of M. soesokkakense RSSK-9T (96.9%), M. fucanivorans SW5T (96.5%), M. litorale KMM 9835T (96.4%), and Mariniflexile gromovii KCTC 12570T (96.2%). M. maritimum M5A1MT belongs to the same genus but shows a lower identity (93.9%) than other Mariniflexile type strains.

To explore the evolutionary affiliation of TRM1-10T to related bacteria, phylogenetic and phylogenomic trees were reconstructed and compared. According to the 16S rRNA gene tree, TRM1-10T is most closely related to M. litorale KMM 9835T and the genus Mariniflexile appears paraphyletic with three species of Confluentibacter in the same clade (Fig. 1c). Fifteen strains with available genome sequences, among the close relatives of TRM1-10T identified through 16S rRNA gene phylogeny, were then selected to construct a phylogenomic tree based on core genes. These strains are in the genera Mariniflexile, Aestuariibaculum, Confluentibacter, Flaviramulus, Gelidibacter, Siansivirga, and Yeosuana, which belong to Flavobacteriaceae (Supplementary Table S1). Additionally, four TRM1 strains, TRM1-13, TRM1-18, TRM1-20, and TRM1-22, isolated from the tomato rhizosphere, exhibiting different phenotypes in growth rate and colony morphology compared to TRM1-10T, were also included (Supplementary Fig. S1). A phylogenomic tree based on 1,347 core genes (after removal of duplicated ones) of 20 Flavobacteriaceae strains shows that the strain TRM1-10T belongs to the genus Mariniflexile (Fig. 1d), which is monophyletic.

Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values were calculated to assess the genomic similarity of TRM1-10ᵀ to Flavobacteriaceae species. (Table 1 and Supplementary Fig. S2). In addition to other TRM1 strains, TRM1-10ᵀ exhibited the high ANI values with M. soesokkakense RSSK-9ᵀ (85.86%) and M. litorale KMM 9835ᵀ (85.42%). The corresponding dDDH values were also relatively high at 27.8% and 27.0%, respectively. The ANI and dDDH values support the delineation of TRM1-10ᵀ as a novel species within the genus Mariniflexile.

Fig. 1
figure 1

Physiology and phylogenetic relationship of strain TRM1-10T. (a) A colony and a scanning electron micrograph of TRM1-10T grown on a marine agar plate at 30 °C for 4 days. (b) Growth curve of TRM1-10T in marine broth. (c) A phylogenetic tree based on the 16S rRNA gene of type species in the family Flavobacteriaceae. Maximum likelihood method was used for tree construction using Kimura 2-parameter method, and bootstrap values (percentages of 1,000 replications) greater than 50% are shown. Filled circles indicate that the corresponding nodes were recovered by all three tree construction methods: maximum likelihood, neighbor-joining, and maximum parsimony, while open circles represent nodes recovered by any two of these methods. Accession numbers of the 16S rRNA genes of each type strain were shown in parentheses. (d) A phylogenomic tree based on 1,347 core genes of the analyzed strains. Maximum likelihood was used for tree construction using the Jones-Taylor-Thornton model for the calculation of evolutionary distances. Bootstrap values (percentages of 1,000 replications) greater than 50% are shown. Accession numbers of the analyzed genomes are shown in Supplementary Table S1.

Full size image
Table 1 Average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH), and G + C content difference between TRM1-10T and related species in the family Flavobacteriaceae. The first ANI value represents the comparison of TRM1-10ᵀ against the reference genome, and the second indicates the reverse direction. The 95% confidence interval (C.I.) of dDDH is also shown. G + C difference represents the absolute percentage difference in G + C content between each genome and that of TRM1-10T.
Full size table

Physiological, biochemical, and chemotaxonomic characteristics

Differential physiological, biochemical, and chemotaxonomic characteristics of TRM1-10T and other Mariniflexile species are provided in Supplementary Table S2. Additionally, the optimal pH range for the growth of TRM1-10T was 7.0–8.0, while the overall growth range was 6.0–8.0. TRM1-10ᵀ was able to grow at NaCl concentrations of 0–4% (w/v) (Supplementary Fig. S3). TRM1-10ᵀ has six copies of the Na⁺/H⁺ antiporter (nha) gene (Supplementary Table S3) that is commonly associated with osmoregulation, whereas M. fucanivorans harbors five copies, M. gromovii five copies, M. maritimum six copies, M. litorale six copies, and M. soesokkakense four copies. These results suggest that TRM1-10ᵀ, while adapted to the soil environment, may retain genomic features that contribute to salt tolerance.

TRM1-10T was negative for H₂S production but positive for catalase activity. Flexirubin-type pigment was not observed. Enzyme activity tests using API ZYM and API 20NE indicated positive results for α-galactosidase, acid phosphatase, alkaline phosphatase, β-galactosidase, esterase (C4), esterase lipase (C8), leucine arylamidase, N-acetyl-β-glucosaminidase, naphthol-AS-BI-phosphohydrolase, and valine arylamidase, while negative results were observed for α-fucosidase, α-glucosidase, α-mannosidase, α-chymotrypsin, arginine dihydrolase, β-glucosidase, β-glucuronidase, cystine arylamidase, lipase (C14), protease, trypsin, and urease. As for carbon source utilization, D-raffinose, lactose, and melibiose were utilized, whereas D-mannitol, D-ribose, and L-rhamnose were not. The strain was susceptible to ampicillin, carbenicillin, lincomycin, novobiocin, rifampicin, and tetracycline, but resistant to cephalothin, chloramphenicol, gentamicin, kanamycin, neomycin, penicillin G, polymyxin B, streptomycin, and sulfamethoxazole.

The major isoprenoid quinone detected in TRM1-10T was menaquinone-6 (MK-6), a common feature of the genera Mariniflexile. The composition of the cellular fatty acids of TRM1-10T was analyzed and compared with the fatty acids of M. soesokkakense RSSK-9T, M. aquimaris HWR-17T, and M. gromovii KCTC 12570T. The major fatty acids of TRM1-10T were iso-C15:0, iso-C17:0 3-OH, and iso-C15:0 Ga, and its proportions were 15.1, 13.3, and 10.4%, respectively (Supplementary Table S4). The major fatty acids of RSSK-9T that have the highest 16S rDNA sequence similarity to TRM1-10T were iso-C15:0 (19.06%), iso-C15:0 3-OH (14.36%), iso-C17:0 3-OH (12.81%), while iso-C15:0 Ga is missing. The contents of the polar lipid of TRM1-10T, RSSK-9T, HWR-17T, and KCTC 12570T were analyzed and compared with each other. The polar lipid of TRM1-10T consisted of phosphatidylethanolamine, unidentified aminolipids, and unidentified lipids (Supplementary Fig. S4). The G + C content of TRM1-10T is 34.56% based on its genome sequence, which is similar to other species in the genera Mariniflexile.

General features of the complete TRM1-10T genome

Basic features of the complete genome of the strain TRM1-10T are described in Supplementary Table S1. The genome of TRM1-10T is composed of a single circular chromosome of 4,858,325 bp with a G + C content of 34.56%. The sequence encodes 4,071 genes containing 4,023 coding sequences (CDSs), 41 transfer RNA (tRNA), six ribosomal RNA (rRNA), and a transfer-messenger RNA (tmRNA). Among the 4,023 CDSs, more than 60% of them were putatively assigned with functions and also assigned to the COG categories. In the genome of TRM1-10T, 11 genomic islands ranging from 4.4 to 50 kb were predicted.

Comparison of the TRM1-10T genome and metagenome-assembled TRG1

Previously, we reconstructed the metagenome-assembled genome (MAG) of an uncultured Flavobacteriaceae strain from tomato rhizosphere samples and designated it as TRG1. Using the genomic information derived from this MAG, we were subsequently able to selectively isolate the corresponding strains, which we named TRM113. When compared to the complete genome sequence of the isolated strain TRM1-10ᵀ, the draft genome sequence of TRG1 was 4.1 Mb in size, consisting of 57 contigs13 and is approximately 0.7 Mb shorter. Results from synteny analysis between the draft TRG1 and the complete genome of TRM1-10T showed that the 57 contigs of TRG1 can be fully aligned to the TRM1-10T genome (Supplementary Fig. S5 and Supplementary Table S5). Based on this, we ascertain that there were no contaminated sequences in the reconstructed genome of TRG1.

Genomic comparison between TRM1-10T and other Mariniflexile species

Genomic and metabolic features of TRM1-10T were investigated by conducting genomic comparisons with the TRM1-10T genome and those of M. fucanivorans SW5T, M. gromovii KCTC 12570T, M. litorale KMM 9835T, M. maritimum M5A1MT, and M. soesokkakense RSSK-9T. The relative abundance of each functional category was calculated based on the total number of COG assignments16. Analysis of the COG distribution indicated that genes assigned to COG categories “cell wall/membrane/envelope biogenesis (M)”, “carbohydrate transport and metabolism (G)”, and “general function prediction only (R)” were most abundant in Mariniflexile. TRM1-10T exhibited a higher abundance of genes associated with “cell motility (N)”, “intracellular trafficking, secretion, and vesicular transport (U)”, and “extracellular structures (W)” rather than in other Mariniflexile species. Additionally, genes in the “transcription (K)” COG category were also abundant in TRM1-10T (Fig. 2 and Supplementary Table S6).

Fig. 2
figure 2

Comparison of the relative abundance of the COG assignments. (a) M, cell wall/membrane/envelope biogenesis; G, carbohydrate transport and metabolism; R, general function prediction only; J, translation, ribosomal structure and biogenesis; P, inorganic ion transport and metabolism; E, amino acid transport and metabolism; K, transcription; O, posttranslational modification, protein turnover, chaperones; T, signal transduction mechanisms; H, coenzyme transport and metabolism; I, lipid transport and metabolism; C, energy production and conversion; L, replication, recombination and repair; S, function unknown; V, defense mechanisms; F, nucleotide transport and metabolism; D, cell cycle control, cell division, chromosome partitioning; U, intracellular trafficking, secretion, and vesicular transport; Q, secondary metabolites biosynthesis, transport and catabolism; X, mobilome: prophages, transposons; N, cell motility; Z, cytoskeleton; W, extracellular structures; B, chromatin structure and dynamics. (b) The COG categories with the higher relative abundance of genes in TRM1-10ᵀ are shown separately from (a): U (intracellular trafficking, secretion, and vesicular transport), N (cell motility), and W (extracellular structures).

Full size image

In the category of “cell wall/membrane/envelope biogenesis (M)”, TRM1-10T and other Mariniflexile species exhibited a high number of orthologous genes associated with glycosyltransferase activity, multidrug efflux pump subunits, outer membrane proteins, and related peptidoglycan-associated proteins (COG0438, COG0463, COG0845, and COG2885). In the category of “carbohydrate transport and metabolism (G)”, they showed a significant prevalence of genes associated with β-galactosidase/β-glucuronidase, β-xylosidase, and pectate lyase functions (COG3250, COG3507, and COG3866), respectively. Significantly, TRM1-10T had 20 β-xylosidase genes (COG3507) surpassing the counts observed in other Mariniflexile species (Supplementary Table S7).

In TRM1-10T, genes categorized under “cell motility (N)” included a slightly higher number of genes involved in ssDNA/RNA exonuclease activity, flagellar biosynthesis, type II secretion system/type IV pilus alignment, and type IV pilus assembly pilotin (COG0084, COG1298, COG3167, and COG3063), respectively. Notably, TRM1-10T had 12 genes identified as the CelD/BcsL family acetyltransferases (COG5653) involved in cellulose biosynthesis, representing the highest count among Mariniflexile species (Supplementary Table S7). In a previous study13, eight acetyltransferase genes were identified, and four additional genes were found in the complete genome sequence.

The COG category “intracellular trafficking, secretion, and vesicular transport (U)” includes diverse bacterial secretion systems. We analyzed the type and distribution of secretion system genes in the Mariniflexile genomes. Secretion systems were identified with rules defined by the TXSScan model in MacSyFinder v217. The analysis revealed that Mariniflexile species commonly possess Gld, Por, and Spr components of the type IX secretion system (T9SS). TRM1-10T contained some genes related to the type IV secretion system (T4SS), such as vib, t4cp2, and MOBP1, whereas SW5T contained a broader array of T4SS genes. Additionally, SW5T, KMM9835T, and RSSK-9T possessed genes related to the type I secretion system (T1SS), while SW5T and KCTC 12570T harbored genes related to the type Va secretion system (T5aSS) (Supplementary Fig. S6). T9SS is a secretion system primarily observed in Bacteroidota, facilitating the export of proteins across the outer membrane and playing a role in gliding motility18. Additionally, aside from genes related to type secretion systems, TRM1-10T exhibited a relatively higher number of the genes in the COG category “intracellular trafficking, secretion, and vesicular transport (U)” compared to most Mariniflexile species. These genes are associated with large exoproteins involved in heme utilization or adhesion, autotransporter adhesins, and the periplasmic component TolB of the Tol biopolymer transport system (COG3210, COG3468, and COG0823), respectively (Supplementary Table S7).

In the COG category “extracellular structure (W)”, TRM1-10T exhibited a higher abundance of the genes compared to other Mariniflexile species, related to type Vc autotransporter adhesin of Ata/Hia family and P pilus assembly chaperone (COG5295 and COG3121), respectively (Supplementary Table S7).

For the genes assigned to “transcription (K)”, RNA polymerase sigma factor genes were the most abundant with 38 genes, 33 of which were classified as extracytoplasmic function (ECF) sigma factors in TRM1-10T (Supplementary Table S7). ECF sigma factors are phylogenetically diversified and known to regulate bacterial responses to changing environmental cues and threats, which may contribute to rhizosphere adaptation19. When a phylogenetic tree was constructed with the ECF sigma factor genes of TRM1-10T and the five Mariniflexile species, a unique clade of ECF sigma factors specific to TRM1-10T was identified (Fig. 3a). 15 ECF sigma factor genes, including those in the clade, were classified within the ECF240 group, that is one of the ECF groups categorized in the ECF Hub database20. ECF240 is a sigma factor frequently found in Bacteroidota, derived from the ECF10 group, which is known to harbor a gene encoding a FecR-like anti-sigma factor, genes encoding outer membrane proteins, glycosyl hydrolases, and carbohydrate metabolism enzymes in its adjacent genomic regions. ECF240, which retains the characteristics of the ECF10 group, is also predicted to be associated with carbohydrate degradation19,20. An investigation of the regions surrounding the 11 ECF factor genes classified within ECF240 revealed that consistent with reported characteristics, most of these genes were located near FecR-like anti-sigma factors, SusC family proteins, SusD family proteins, and glycoside hydrolase proteins (Fig. 3b). The other four ECF sigma factors assigned to ECF240 (CJ739_237, CJ739_1332, CJ739_1386, and CJ739_3076) exhibited distinct gene contexts. Downstream of these ECF sigma factors, genes involved in metal ion transport and metabolism, membrane transport, stress response, and resistance to antibiotics and toxic substances were located (Supplementary Fig. S7).

Fig. 3
figure 3

A phylogenetic tree of ECF RNA polymerase sigma factor genes and the genetic contexts of members of ECF240 (a) A neighbor-joining tree was constructed with the Jones-Taylor-Thornton model using ECF sigma factors from TRM1-10T, Mariniflexile soesokkakense RSSK-9T, Mariniflexile litorale KMM 9835T, Mariniflexile fucanivorans SW5T, Mariniflexile gromovii KCTC 12570T, and Mariniflexile maritimum M5A1MT. Bootstrap values (percentages of 1,000 replications) greater than 50% are shown. RpoD and RpoD homologs were used as the outgroup, and the 33 ECF sigma factors of TRM1-10T were labeled. The genes classified under ECF240 are marked with orange circles. (b) In the gene context surrounding the ECF sigma factors classified under ECF240, the anti-sigma factor gene fecR was identified, along with genes encoding SusD family proteins and TonB-dependent receptors involved in the transport of external substances, including polysaccharides. Additionally, glycoside hydrolase proteins were found downstream and are indicated in green.

Full size image

When comparing the Mariniflexile genomes to identify TRM1-10T strain-specific genes, 141 genes within 43 orthologous groups were found exclusively in TRM1-10T (Fig. 4a and Supplementary Table S8). These TRM1-10T genes were highly clustered and some of them were located in genomic islands (Fig. 4b). Among these, 88 genes encoded hypothetical proteins, 13 encoded transposases, and eight encoded glycoside hydrolases (GH) family proteins. Additionally, as observed in COG category K, five genes encoding ECF sigma factors were also found to be specific to TRM1-10T.

Fig. 4
figure 4

Number of orthologous groups and circular representation of the complete genome of TRM1-10T. (a) A Venn diagram illustrating the numbers of orthologous groups among TRM1-10T, Mariniflexile soesokkakense RSSK-9T, Mariniflexile litorale KMM 9835T, Mariniflexile fucanivorans SW5T, Mariniflexile gromovii KCTC 12570T, and Mariniflexile maritimum M5A1MT (b) Circular representation of the genome of TRM1-10T. The first and second circles colored grey indicate the COG-assigned CDSs for forward and reverse strands. Next to the grey circle, blue and red-scattered spots indicate the tRNA and rRNA genes, respectively. The third purple circle indicates the CAZyme-assigned CDSs. The fourth red circle indicates the TRM1-10T specific CDSs. Inside the red circle, sky blue tiles indicate the CDSs in genomic islands. The fifth black circle represents the G + C content and the orange/yellow circle in the black circle is for the G + C skew. The COG-assigned CDSs, CAZyme-assigned CDSs, and genomic island-located CDSs among the TRM1-10ᵀ-specific genes are listed in Supplementary Table S8.

Full size image

Carbohydrate-active enzymes and polysaccharide utilization

The presence of glycoside hydrolase (GH) family genes specific to TRM1-10T, along with the high abundance of genes within the “carbohydrate transport and metabolism (G)” category in Mariniflexile (Supplementary Table S6) prompted an investigation into the unique carbohydrate metabolism of TRM1-10T. For this analysis, carbohydrate-active enzymes in TRM1-10T were compared with those in the five Mariniflexile species. Among the genes encoding carbohydrate-active enzymes, the glycoside hydrolase (GH) families that were more abundant in TRM1-10T include GH43, GH2, GH32, GH31, GH97, GH130, GH26, GH5, GH27, and GH33 (Fig. 5). Notably, GH27 (α-galactosidase) was observed exclusively in TRM1-10T. Additionally, TRM1-10T specific genes included those in the GH26 (β-mannosidase) and GH32 (levanase, β-fructosidase B) families (Supplementary Table S8 and Supplementary Table S9).

Fig. 5
figure 5

Comparison of the CAZyme-assigned CDSs among TRM1-10T, Mariniflexile soesokkakense RSSK-9T, Mariniflexile litorale KMM 9835T, Mariniflexile fucanivorans SW5T, Mariniflexile gromovii KCTC 12570T and Mariniflexile maritimum M5A1MT. Bar graphs show the number of CDSs assigned to the GH family, for which numbers are more abundant in the genome of TRM1-10T, and its relative abundance in the genome.

Full size image

SusC-like and SusD-like proteins, found in Bacteroidota, play an essential role in the recognition and uptake of carbohydrates. In Mariniflexile, pairs of SusC-like and SusD-like proteins were identified, with TRM1-10T possessing a total of 36 susC and susD pairs (Supplementary Fig. S8). SusC-like and SusD-like proteins are components of the starch utilization system (Sus), which is one of the polysaccharide utilization loci (PUL) in Bacteroidota, responsible for the degradation and absorption of complex carbohydrates21. Following the identification of SusC-like and SusD-like proteins, CAZyme gene clusters (CGCs) and PULs were analyzed to predict the substrates utilized by TRM1-10T and other Mariniflexile species (Supplementary Table S10). The results indicated that TRM1-10T contained more PULs related to xylan metabolism compared to other Mariniflexile species. It also possessed numerous PULs involved in pectin degradation, similar to those found in the species such as M. maritimum M5A1MT and M. litorale KMM 9835T. Notably, TRM1-10T was the only strain that contained PULs specific for the metabolism of arabinogalactan and β-mannan among the Mariniflexile species (Supplementary Fig. S9).

Description of M. rhizosphaerae sp. nov.

Mariniflexile rhizosphaerae (rhi.zo.sphae′rae. N.L. gen. fem. n. rhizosphaerae of the rhizosphere).

Cells are 0.3–0.5 μm in diameter and 3.0–4.0 μm in length. Cells form convex and yellow colonies on marine agar in 3 days at 30 °C and their diameters range from 0.5 to 2 mm. Anaerobic growth of the cells does not occur on marine agar for 10 days. The temperature range for growth is 4–40 °C and the optimum temperature is 30 °C. The pH range for growth is 6.0–8.0 and the optimum is 7.0–8.0. The range of NaCl concentration for growth is 0–4% (w/v) and the optimum is 0–1%. Cells were negative for nitrate reduction, H2S production, and oxidase activity and positive for catalase activity. Flexirubin-type pigment was not detected. Positive (API ZYM and 20NE) for α-galactosidase, acid phosphatase, alkaline phosphatase, β-galactosidase, esterase (C4), esterase lipase (C8), leucine arylamidase, N-acetyl-β-glucosaminidase, naphthol-AS-BI-phosphohydrolase, and valine arylamidase. Negative for α-fucosidase, α-glucosidase, α-mannosidase, α-chymotrypsin, arginine dihydrolase, β-glucosidase, β-glucuronidase, cystine arylamidase, lipase (C14), protease, trypsin, and urease. D-Cellobiose, D-mannose, D-raffinose, lactose, maltose, melibiose, succinate, and sucrose are utilized as carbon sources but not benzoate, D-galactose, D-mannitol, D-ribose, L-arabinose, and L-rhamnose. Cells are susceptible to ampicillin, carbenicillin, lincomycin, novobiocin, rifampicin, and tetracycline but not cephalothin, chloramphenicol, gentamicin, kanamycin, neomycin, penicillin G, polymyxin B, streptomycin, and sulfamethoxazole. The isoprenoid quinone is MK-6 and major fatty acids are iso-C15:0, iso-C17:0 3-OH, and iso-C15:0 G. Major polar lipids are phosphatidylethanolamine, unidentified aminolipids, and unidentified lipids.

The type strain, TRM1-10T (= KCTC 18646PT = DSM 33122T), was isolated from the rhizospheric soil of the tomato cultivar Hawaii 7996. The DNA G + C content of the type strain is 34.56%.

Discussion

A group of bacteria collectively known as “flavobacteria” originates from the genus Flavobacterium22. They now have valid taxonomic ranks: the class Flavobacteriia, the order Flavobacteriales, and the family Flavobacteriaceae. A phylogenomic analysis indicated that the TRM1 strains belonged to the family Flavobacteriaceae, and were in the same clade as the flavobacterial genera, most of which reside in marine habitats: Mariniflexile3,6,7,11,12, Aestuariibaculum23,24, Confluentibacter25,26, Flaviramulus27, Gelidibacter28, Siansivirga29, and Yeosuana30,31. Phylogenetic comparisons suggested that ancestors of TRM1-10T could have been of marine origin, which then settled down at the rhizosphere of plants and its genome expanded to adapt to the terrestrial environment. TRM1-10T is the first taxon isolated from the rhizosphere in the clade of marine taxa.

Glycoside hydrolase families were abundantly encoded in TRM1-10T, with α-galactosidase, β-mannosidase, levanase, and β-fructosidase B identified as strain-specific enzymes. Additionally, glycoside hydrolase families including arabinanase, endo-β-1,4-galactanase, glucoamylase, sialidase, xylanase, α-glucosidase, α-L-fucosidase, α-L-iduronidase, α-L-rhamnosidase, α-mannosidase, β-1,4-mannosylglucose phosphorylase, and β-xylosidase were also present in high abundance. These enzymes are involved in the hydrolysis of oligo- or polysaccharides such as glycan, pectin, and cellulose. This suggests that the high abundance of the polysaccharide-hydrolyzing enzymes enables TRM1-10T to actively acquire the carbohydrate nutrients excreted from the plant root and to successfully adapt to the rhizosphere.

TRM1-10T contained a higher number of ECF sigma factors compared to other Mariniflexile species. ECF sigma factors regulate cellular responses to environmental stimuli, acting as regulatory elements that control the expression of specific genes to help the cell adapt to environmental changes or stress19. TRM1-10T was particularly enriched in the ECF240 group. Notably, the genes encoding ECF240 sigma factors were often located in proximity to membrane protein genes or carbohydrate-metabolism-related enzymes. Therefore, along with the abundance of carbohydrate utilization-related genes, these factors were predicted to play an important role in enabling TRM1-10T to adapt to the plant rhizosphere. Moreover, genes involved in metal ion transport and metabolism, membrane transport, stress response, and resistance to antibiotics and toxic substances were also identified downstream of the four ECF sigma factors assigned to ECF240. These ECF sigma factors were part of the five unique ECF sigma factors found in TRM1-10T. This suggests a possible role in the adaptation of TRM1-10T to the soil environment.

TRM1-10T possessed 12 genes encoding acetyltransferases from the CelD/BcsL family, which are involved in cellulose biosynthesis. In plant-associated bacteria, cellulose plays a significant role in biofilm formation and cell adherence, facilitating colonization in the rhizosphere and phyllosphere32. Consequently, the acetyltransferases involved in cellulose biosynthesis may support the establishment of TRM1-10T within the plant rhizosphere.

Our previous study showed that the TRM1 strains occupied the rhizosphere of disease-resistant Hawaii 799614 at more than 2%, and its abundance was more than two-fold higher in Hawaii 7996 than in disease-susceptible Moneymaker13. Furthermore, the incidence and progress of the disease were significantly suppressed when the rhizosphere of Moneymaker was pretreated with TRM1-10T13. Our analyses suggest that the genomic features associated with transcription, carbohydrate metabolism, and other functions are important for adaptation to the tomato rhizosphere that appears to be its prime niche, and for protecting the tomato plant against R. solanacearum, which is a devastating plant pathogen that causes bacterial wilt in hundreds of plant species33,34. The presence of specific monosaccharide transporters raises the tantalizing possibility that the preemptive intake of monosaccharides by the TRM1 strains reduces the sugars available to the Ralstonia pathogen for binding to its lectins, thus interfering with infection13. L-Fucose, L-galactose, D-arabinose, and D-mannose are known as lectin-binding sugars of R. solanacearum34. Additionally, the O-acetylation level in plant cell wall polysaccharides may impact pathogen resistance by hindering wall degradation by invading pathogens35.

In this study, we identified the strain TRM1-10T using phylogenetic and chemotaxonomic analyses and determined its complete genome sequence using the PacBio sequencing system. We also investigated its genetic features through a comparative genomic study with other Mariniflexile species. As some of the Mariniflexile genomes used for comparison are available only as drafts, it is possible that they do not contain the full complement of accessory genes. Nevertheless, we identified key genetic traits in TRM1-10T, including an enrichment of the glycoside hydrolase families, the ECF sigma factors, and the acetyltransferases, and we suggest that these features may contribute to its adaptation to the plant rhizosphere. Through these, we suggest that the strain TRM1-10T represents a novel species in the genus Mariniflexile, for which the name Mariniflexile rhizosphaerae sp. nov. is proposed.

Materials and methods

Tomato rhizosphere microbe TRM1

Strains TRM1-10T, TRM1-13, TRM1-18, TRM1-20, and TRM1-22 were isolated in 2016 from the rhizospheric soil of the tomato cultivar Hawaii 7996 that was cultivated in plastic houses at Dong-A University Agricultural Experimental Station, Busan, Republic of Korea (35.239° N, 128.978° E) since November 2011, by spreading onto 1/10 diluted marine agar 2216 (BD Difco) with 2% NaCl and incubating at 25 °C for 6 days13. Additionally, type strains of Mariniflexile species including M. aquimaris HWR-17T (KCTC 23346T), M. gromovii KCTC 12570T (KMM 6038T), and M. soesokkakense RSSK-9T (KCTC 32427T) were obtained from the Korean Collection for Type Culture (KCTC). They were routinely grown on marine agar or marine broth (BD Difco) at 30 °C.

Construction of a phylogenetic tree based on the 16S rRNA gene

The universal bacterial primer set 27F and 1492R36 was used for amplification of the 16S rRNA gene (PCR condition: annealing 55 °C, 30 s; extension 72 °C, 1.5 min; 30 cycles). The nucleotide sequence of the amplified gene was determined using Sanger sequencing (Macrogen Inc., Korea), and the taxonomic affiliation of the bacterium was identified using EzBioCloud’s taxon identification service37. Phylogenetic analysis was conducted using the maximum likelihood method with the Kimura 2-parameter, the neighbor-joining method with the Kimura 2-parameter, and the maximum-parsimony method with the subtree-pruning-regrafting method in MEGA X38.

Phenotypic analysis

For long-term preservation, cells were maintained as glycerol suspension (15%, w/v in distilled water) at -80 °C. Gram staining was performed using a Gram staining kit (YD Diagnostics Inc., Korea), and gliding motility was observed through the hanging drop method39 and then cell morphology and motility were observed using a light microscope (Axio Lab.A1, Carl Zeiss,

Germany). For observation of cell morphology of TRM1-10T, cells were fixed in 2.5% paraformaldehyde-glutaraldehyde mixture buffered with 0.1 M phosphate (pH 7.2) for 2 h, postfixed in 1% osmium tetroxide in the same buffer for 1 h, dehydrated in graded ethanol, and substituted by isoamyl acetate. Then they were dried at the critical point in CO2. Finally, the samples were sputtered with gold in a sputter coater (SC502, Polaron) and observed using the scanning electron microscope, FEI Quanta 250 FEG installed in Korea Research Institute of Bioscience and Biotechnology. Growth at temperatures from 4 to 40 °C was observed on marine agar for 7 days. Growth at pH 4.5–9 (0.5 pH unit intervals, buffered with sodium acetate and sodium carbonate) was observed on marine broth at 30 °C for 7 days. Growth at NaCl concentration at 0–10% (0, 0.5 and 1.0–10.0%, at increments of 1.0%) was observed on modified CPG broth (Peptone, 10 g/L; Casamino acid, 1 g/L; D-glucose, 5 g/L; MgCl2·6H2O, 5.9 g/L; MgSO4·7H2O, 3.24 g/L; CaCl2·2H2O, 1.8 g/L; KCl, 0.55 g/L) at 30 °C for 48 h. Anaerobic growth was tested through incubation on marine agar at 25 °C for 10 days in an anaerobic chamber. The presence of flexirubin-type pigment was determined by applying 20% KOH to the bacterial cells and observing the color change.

Enzymatic analysis

Enzyme activities for catalase and oxidase were examined using 3% H2O2 and oxidase reagent dropper (Becton Dickinson, USA), respectively. For investigation of other enzyme activities and assimilation of substrates, diluted cells in 0.85% NaCl were loaded in the capsules of API 20NE and ZYM kits (BioMérieux, France). Casein hydrolysis was tested on skim milk agar with 2% NaCl at 30 °C for 7 days. Hydrolysis of starch, xanthine, and hypoxanthine was tested on marine agar containing 0.2% starch, 0.4% xanthine, and 0.4% hypoxanthine, respectively. Hydrolysis of L-tyrosine was tested on agar plates containing 0.5% peptone, 0.3% beef extract, 0.5% tyrosine, and 2% NaCl at 30 °C for 7 days. Assimilation test of 95 kinds of carbon source was performed with Biolog GN2 microplates using diluted cells grown in Biolog fluid at 30 °C for 3 days. Antibiotic susceptibility was tested on marine agar using an antibiotic susceptibility test disc (Oxoid, Thermo Fisher Scientific Inc., USA): ampicillin (10 µg/disc), carbenicillin (100), cephalothin (30), chloramphenicol (50), gentamicin (30), kanamycin (30), lincomycin (15), neomycin (30), novobiocin (5), penicillin G (10 U), polymyxin B (300), rifampicin (5), streptomycin (25), sulfamethoxazole (25), and tetracycline (30).

Chemotaxonomic analysis

The type strains of the Mariniflexile species were grown on marine agar at 30 °C for 3 days. For the fatty acid analysis, cellular fatty acids were extracted from cells in the active growth stage according to Miller’s method40 and identified using Agilent 6890 gas chromatograph (Korean Culture Center of Microorganisms, KCCM, Korea). Polar lipids were extracted according to Minikin et al.41 and analyzed by two-dimensional thin-layer chromatography with molybdophosphoric acid staining and ninhydrin staining (KCCM, Korea). Respiratory isoprenoid quinones were extracted using chloroform-methanol (2:1, v/v) and identified by high-performance liquid chromatography42 (KCCM).

Genome sequencing, assembly, gene prediction, and annotation

Genomic DNA was extracted using Wizard Genomic DNA Purification Kit (Promega, USA). The complete genome sequence of TRM1-10T was determined with a hybrid approach using PacBio RS II (DNA Link Inc., Korea) and HiSeq 2000 sequencer of Illumina platforms (DNA Link Inc.). A 20-kb library and SMRT Cell with P6-C4 chemistry were used for the PacBio RS II sequencing. Following sequencing and quality trimming, a total of 1,404,921,581 bp of continuous long reads43 were generated from the PacBio system. De novo assembly and scaffolding were performed using HGAP and AHA in SMRTpipe44, respectively. The final complete genome sequence of TRM1-10T was generated with SMRTpipe Quiver44. For synteny analysis, the complete genome sequence of TRM1-10ᵀ was compared with the metagenome-assembled draft genome of TRG1 using MUMmer445 and Mauve 2.4.046. For TRM1-13, TRM1-18, TRM1-20, and TRM1-22, shotgun sequencing was performed on the Illumina NovaSeq 6000 platform (DNA Link Inc.). The raw reads were trimmed using Trimmomatic 0.3947. De novo assembly of the reads from each strain was carried out with SPAdes 4.0.048. Structural gene prediction and functional assignment of the predicted genes were conducted with Prokka49.

Comparative sequence analysis

To investigate the genomic features of TRM1-10T, a comparative genome analysis was conducted with the genome sequences of several related species: TRM1-13, TRM1-18, TRM1-20, TRM1-22, Aestuariibaculum marinum IP7T, Aestuariibaculum suncheonense SC17T, Confluentibacter citreus XJNY T, Confluentibacter lentus HJM-3T, Confluentibacter sediminis DSL-48T, Flaviramulus ichthyoenteri Th78T, Gelidibacter mesophilus DSM 14095T, M. fucanivorans SW5T, M. gromovii KCTC 12570T, M. litorale KMM 9835T, M. maritimum M5A1MT, M. soesokkakense RSSK-9T, Siansivirga zeaxanthinifaciens CC-SAMT-1T, Yeosuana aromativorans GW1-1T, and Yeosuana marina JLT21T. Accession numbers of each analyzed genome were provided in Supplementary Table S1. Nucleotide sequences of these genomes were retrieved from the GenBank genome database and the genomes. Gene prediction and annotation were generated using Prokka49 and served for comparative genome analysis among the genomes and TRM1-10T.

Identification of orthologous genes among the 16 Flavobacteriaceae strains was conducted using OrthoMCL 2.0.950 with the parameters of ≥ 20% identity, ≥ 20% coverage, and an e-value cutoff of ≤ 1e-0551. For the construction of a phylogenomic tree based on the core genes of 16 Flavobacteriaceae strains including TRM1-10T, duplicated genes were excluded from the orthologous genes. Alignment of the amino-acid sequences of the orthologous genes was conducted using MUSCLE52 and a maximum likelihood tree was constructed using the Jones-Taylor-Thornton model with IQ-TREE 253. Average Nucleotide Identity (ANI) values among the strains were calculated using the pyani 0.2.13.154, and digital DNA–DNA hybridization (dDDH) values were obtained using the Genome-to-Genome Distance Calculator (GGDC) 3.055.

For functional gene analyses, amino acid sequences were aligned with UniRef90, COG, and KEGG databases56,57,58 using DIAMOND, CD-Search, and RPS-BLAST software59,60,61. Carbohydrate-active enzymes (CAZyme) families, CAZyme gene clusters (CGCs), and Polysaccharide utilization loci (PULs) were analyzed using run_dbcan tool from dbCAN362. Extracytoplasmic function (ECF) sigma factors were categorized using the ecf_classify tool from ECF Hub20. A genome circle was constructed using Circos63. Type secretion systems were detected using MacSyFinder v2 with the TXSScan model17and genomic islands were predicted with IslandViewer 464.