Isolation of Spirosoma foliorum sp. nov. from the fallen leaf of Acer palmatum by a novel cultivation technique

In the effort of isolating novel microbial species, the strain PL0132T was isolated from a fallen leaf under fresh water at a stream, which glided when grown on a tap water medium (without nutrients). The strain was determined to be Gram-negative, strictly aerobic, and rod-shaped, which grew optimally at 25 °C, pH 6–7, and the strain tolerates 1% (w/v) NaCl concentration. The complete genome of strain PL0132T comprises one contig with a sequencing depth of 76×, consisting of 8,853,064 base pairs and the genomic DNA G + C content was 46.7% (genome). 16S rRNA gene sequence analysis revealed that strain PL0132T represents a member of the phylum Bacteroidetes and is affiliated with the genus Spirosoma. Based on genomic, phenotypic, and chemotaxonomic characteristics, the strain PL0132T represents a novel species of the genus Spirosoma, for which the name Spirosoma foliorum sp. nov. is proposed (= KCTC 72228 T = InaCC B1447T).

The species of the genus Spirosoma have been isolated from soil, dust, air, water, and extreme conditions like Arctic glaciers [18][19][20] .Furthermore, the characteristics of the genus include Gram-stain-negative, strictly aerobic, non-spore-forming, yellow or orange pigmented bacteria which are characterized as menaquinone MK-7 as the respiratory quinone, phosphatidylethanolamine as the major polar lipid, and summed feature 3 (C 16: 1ω6C and/ or C 16: 1ω7C ) C 16:1ω5c , iso-C 15:0 , and C 16:0 as the major fatty acids 6 .In this paper, a gliding bacterium was isolated from a decaying leaf of Acer palmatum in a stream of fresh water.The distinctive characteristics led us to propose a novel species in the genus Spirosoma.
The complete genome sequence of strain PL0132 T comprised one contig with a sequencing depth of 76×; the contig was 8,853,064 nt long with a circular structure.The GC contents of the chromosome were 46.7%.Gene annotation by NCBI Prokaryotic Genome Annotation Pipeline identified 7,408 genes which include 7,126 genes coding protein, 9 rRNAs (5S, 3; 16S, 3; 23S, 3), and 44 tRNAs.The complete genome was deposited in the GenBank/EMBL/DDBJ under the accession number CP059732.The identification of secondary metabolite biosynthesis gene cluster was determined by using antiSMASH 21 .Secondary metabolite clusters annotated by antiSMASH included terpene synthase genes, polyketide synthase type I and III (T1PKS and T3PKS), unspecified ribosomally synthesized and post-translationally modified peptide product cluster (RiPP-like), RRE-element containing cluster and non-ribosomal peptide synthetase (NRPS) (Fig. S5).
The average nucleotide identity (ANI) scores between the genomic sequence of PL0132 T and Spirosoma arboris HMF4905 T , Spirosoma migulaei 15J9-8 T , and Spirosoma telluris HMF3257 T were 80.81, 79.13 and 79.72% 9 , Figure 1.Maximum-likelihood phylogenetic tree, based on 16S rRNA gene sequences, showing the phylogenetic position of Spirosoma foliorum PL0132 T among related strains of the genus Spirosoma.Closed circles indicate that the corresponding nodes were also recovered in the tree generated with the neighbor-joining and maximum parsimony algorithm.Bootstrap values in the order ML/NJ/MP are indicated as percentages of 1000 replications datasets, when greater than 70%.The tree was rooted using Bacteroides fragilis ATCC 25285 T (X83935) as an outgroup.Bar, 0.05 substitutions per nucleotide position., and Spirosoma telluris HMF3257 T , respectively.These values of ANI and dDDH were below the standard cut-off criteria for ANI (95-96%) 22 and dDDH (70%) 23 , indicating that strain PL0132 T represents a novel species of the genus Spirosoma.
To determine the taxonomic position of strain PL0132 T , a genome-based phylogenetic tree was reconstructed using an up-to-date bacterial core gene (UBCG) set consisting of 92 genes, as described by Na et al. 24 Briefly, the core genes identified (hmmsearch; v3.1b2) from predicted CDSs (Prodigal; v2.6.3) were concatenated, and aligned (MAFFT; v7.310), and subjected to construct a UBCG tree.The phylogenomic tree showed that PL0132 T formed a clade with S. arboris HMF4905 T within the genus Spirosoma (Fig. 2).
The strain PL0132 T was Gram-stain-negative, strictly aerobic, non-spore-forming, catalase and oxidasepositive, rod-shaped without flagella, approximately 0.8-1.0μm wide, 2-6 μm long (Fig. S3).The strain glided when grown on a tap water medium (without nutrients).Colonies grown on R2A for 48 h were circular, convex, and pale yellow.Growth occurred at 4-30 °C (optimum 25 °C), at pH 6.5-8.5 (optimum 7), and the strain did not require NaCl for its growth, but it tolerated at a concentration up to 1% (w/v) NaCl.The isolated strain grew on R2A and NA, and weakly on TSA but did not grow on LB agar.Physiological and biochemical characteristics of the isolated strain were described in the species description.Phenotypic and chemotaxonomic properties that differentiated strain PL0132 T from its closest relatives in the genus are listed in Table 1.
The quinone of strain PL0132 T was menaquinone-7, which is similar to in other members of the genus Spirosoma 5,6 .The polar lipid profile of PL0132 T showed the major lipids phosphatidylethanolamine (PE), two www.nature.com/scientificreports/aminophospholipids (APL1-2), and four unidentified lipids (L1-L4) (Fig. S4).The profile was similar to other strains in the genus with major polar lipids, and the profile of the novel isolate strain had more unidentified lipids and minor amounts of aminophospholipid.Strain PL0132 T had biochemical and physiological characteristics that differentiated strain PL0132 T from the other species of the genus Spirosoma.One difference was that Spirosoma foliorum PL0132 T and S. migulaei KCTC 52028 T could grow at 10 °C and on tryptic soy agar while the other closely species of genus Spirosoma do not.Second, S.foliorum PL0132 T and S. migulaei KCTC 52028 T could produce acid from gentiobiose and inulin while the type strains of S. arboris, S. litoris, and S. telluris could not (Table 1), however, S.foliorum PL0132 T could not produce acid from D-arabinose while the other close relative species could produce acid from D-arabinose.Third, it is important to note that glycolipid was found in other closely related species 9 but not in S. foliorum PL0132 T .

Materials and methods
All experiments were carried out in accordance with relevant institutional, national, and international guidelines and legislation.
Sampling sites and isolation.The strain PL0132 T was isolated from a fallen leaf of Acer palmatum under fresh water at a stream in Naejang mountain, in Jeongeup city, South Korea (35°28′48.3′′N126°53′21.6′′E) in July 2018.The leaf was cut into a small piece of 5 × 5 mm and placed on a tap water agar medium without nutrients and incubated at 20 °C.After gliding motility was observed by a stereoscopic microscope, one strain was purified by transferring on Reasoner's 2A (R2A) (BD Difco) agar.Finally, a bacterium with yellow color, designated PL0132 T , was collected.Then, the strain was preserved in 15% (v/v) glycerol suspension at − 80 °C.www.nature.com/scientificreports/16S rRNA gene phylogeny.The genomic DNA of the strain PL0132 T was extracted from cells grown on R2A at 25 °C for 48 h.The 16S rRNA gene was amplified with two primers 27F and 1492R 25 .The sequence analysis was carried out by Sanger's sequencing with an ABI3730XL automated sequencer (Applied Biosystems, USA).Then, the 16S rRNA gene sequences were uploaded to the EzBiocloud server 26 to collect sequence information.All sequences of corresponding species obtained from the EzBiocloud were aligned and edited by BioEdit 27 and CLUSTAL X software 28 .The phylogenetic trees were reconstructed by using neighbor-joining (NJ) 29 , maximumlikelihood (ML) 30 , and maximum parsimony (MP) 31 methods in the MEGA 7 program 32 with 1000 bootstrap replications.
Genome sequencing and annotation.Whole-genome sequence of strain PL0132 T was determined by Nanopore technology.High molecular weight DNA was prepared and libraries were constructed following the native barcoding genomic DNA protocol (with EXP-NBD104, and SGK-LSK109, version NBE_9065_v109_ revV_14Aug2019), after which sequencing was carried out.The library was loaded onto a MinION flow cell model FLO-MIN 106 (version R10.3).Then, sequencing was performed on MinKNOW platform (Oxford Nanopore Technologies).The resulting raw reads were quality assessed using pycoQC (2.5.2) 33 , and preprocessing was performed with porechop (0.2.4) to obtain 2.4 GB of reads.These reads were then processed using a custom pipeline based on Canu 2.0 34 , which was improved by referring to the CCBGpipe (Consensus Circular Bacterial Genome pipeline) 35 .Sequencing reads were produced through base calling on ONT Guppy software (version 3.2.10,Oxford Nanopore Technologies, Ltd., Oxford, UK), for 4 h.Assembling was done for reads which had good quality of quality scores ≥ 7 and sequence length ≥ 3000 bp.Before filtering, the mean read length was about 3,420 bp, and the read count was 675,410.After filtering, the mean read length was about 16,821 bp and the read count was 39,426.Subsequently, The assembled contigs were corrected and polishing was performed with Medaka 36 (version 1.3.2,bacteria_odb10 1.3.2,https:// github.com/ nanop orete ch/ medaka) to improve genome quality and the completeness of the Nanopore assembly was evaluated and the quality of the assembled genome was confirmed with BUSCO using BUSCO 37 (version 5.1.2,(https:// busco.ezlab.org/, score: 94.4) and CheckM (version 1.1.3,score: 99.7) 38 , and annotations were added using PROKKA (1.1.2) 39 .
Morphological and phenotypic analyses.Gram-staining was determined with a BD Gram-staining kit.Cells of the strain PL0132 T incubated 48 h on R2A (Difco) at 25 °C were used to examine the morphology using a FEI Quanta 250 FEG scanning electron microscope (FEI).Gliding motility was tested by microscopic hanging drop 16 .The phenotypic features were determined as follows: the optimal temperature for growth was investigated at 4, 10, 20, 25, 30, 37, and 45 °C on R2A agar after incubation for 7 days.Salt tolerance was examined using R2A broth containing 0, 0.5, 1, 2, 3, 4, 5 and 10% (w/v) NaCl.Growth at pH 5 to 10 (at intervals of 0.5 unit) was determined in R2A broth adjusted with various buffers, the concentration of 100 mM, acetate for pH 5-6, phosphate for pH 6.5-8, Tris for pH 8.5-9, and carbonate for pH 9.5-10 9 .Growth in the broth media was observed using OD 600 .Catalase and oxidase activity were carried out as described by Han et al. 40 Growth Chemotaxonomic analyses.To determine the fatty acid composition, cells of strain PL0132 T and the reference strains were collected from R2A agar at the same physiological age, by using the method of Sasser 42 .Fatty acid analyses were analyzed by the Sherlock Microbial Identification System (TSBA; library version 6.0) 43 .
The isolated strain was cultured in R2A broth (Difco, BD) for 48 h to collect cell mass for isoprenoid quinone and polar lipid analysis.The isoprenoid quinone was extracted according to the method of Komagata and Suzuki 44 from 100 mg freeze-dried cells using the solution of chloroform/methanol (2:1, v/v).The crude compound was purified by using a preparative TLC (20 mm × 20 mm, silica gel 60 F254 plates, Merck) with petroleum benzene/ diethyl ether (9:1, v/v); then the compound was analyzed by using reverse-phase HPLC with a mixture of methanol and isopropyl ether (3:1, v/v), and detected by a UV detector.Polar lipids were extracted by a chloroform/ methanol/water system, and then they were developed and separated using two-dimensional TLC 45 .The polar lipids were identified as described by Han et al. 46 .

Conclusion
Based on the data for phylogenetic analysis, phenotypic and chemotaxonomic characteristics, PL0132 T represents a novel species of the genus Spirosoma.However, several phenotypic differences between the isolated strain and its phylogenetically related strains were summarized in Table 1.Therefore, PL0132 T should be classified as a novel species of the genus Spirosoma, for which the name Spirosoma foliorum sp.nov. is proposed.

Description of Spirosoma foliorum sp. nov
Spirosoma foliorum sp.nov.(fo.li.o'rum.L. pl.gen.n. foliorum, of leaves, referring to the isolation of the type of strain from decaying leaves).The novel strain designated PL0132 T was isolated from decaying leaves under fresh water at a stream in Naejang mountain, in Jeongeup city, Republic of Korea.Cells are Gram-stain-negative, and strictly aerobic rods, 0.8-1.0μm wide, and 2-6 μm long.The strain glides when grown on a tap water medium (no nutrient).Colonies are convex, translucent, circular, and pale yellow.Growth occurs at occurred 4-30 °C (optimum 25 °C), at pH 6.5-8.5 (optimum 7), and the strain did not require NaCl for its growth.Tolerates 1% but not 2% (w/v) NaCl.The type of strain, PL0132 T (= KCTC 72228 T = InaCC B1447 T ), was isolated from decayed leaves in Naejang mountain, Jeongeup city, Republic of Korea.The complete genome sequence of Spirosoma foliorum PL0132 T comprised one contig with a sequencing depth of 76×; the contig was 8,853,064 nt long with a circular structure.The GC contents of the chromosome were 46.7%.

Figure 2 .
Figure 2. Genome-based phylogenetic tree of strain Spirosoma foliorum PL0132 T and other related type strains using UBCGs (concatenated alignment of 92 core genes).Bootstrap values are indicated at nodes.Scale bar, 0.05 substitutions per position; Predicted gene clusters of secondary metabolites biosynthesis annotated in antiSMASH against strain PL0132 T complete genome.Analyses provided the identification of six clusters involved in biosynthesis of terpene synthase genes, polyketide synthase type I and III (T1PKS and T3PKS), unspecified ribosomally synthesized and post-translationally modified peptide product cluster (RiPP-like), RRE-element containing cluster and non-ribosomal peptide synthetase (NRPS); Two-dimensional TLC patterns of the total polar lipids of strain Spirosoma foliorum PL0132 T with The following spray reagents were used for detection: (A) molybdatophosphoric acid (for total lipids); (B) molybdenum blue (for phospholipids); (C) ninhydrin (for amino lipids); (D) α-naphthol (for glycolipids).Phosphatidylethanolamine (PE), Amino lipid (AL), Amino Phospho Lipid (APL), and unidentified lipids (L1-L4).1st:first dimension; 2nd: second dimension; Scanning electron micrographs (SEM) of strain Spirosoma foliorum PL0132 T grown on R2A for 2 days at 25 °C.Bars 3 μm.