Introduction

The genus Dyadobacter, a member of the family Cytophagaceae, was proposed by Chelius and Triplett (2000) to accommodate Gram-negative, rod-shaped bacteria within the phylum Bacteroidetes, occurred in pairs in young cultures but in chains of coccoid cells in old cultures and produced a nondiffusible, yellow-like pigment (Reddy and Garcia-Pichel 2005). At the time of writing, 13 species of this genus have been included in list of prokaryotic names with standing in nomenclature (LPSN, https://www.bacterio.net). The genus Dyadobacter has been isolated from various sources, such as soil, plants, freshwater, seawater, desert sand, glacial samples and subterranean sediment samples (Chelius and Triplett 2000; Reddy and Garcia-Pichel 2005; Chaturvedi et al. 2005; Liu et al. 2006; Baik et al. 2007; Dong et al. 2007; Tang et al. 2009; Zhang et al. 2010; Lee et al. 2010; Chen et al. 2012; Chun et al. 2013; Shen et al. 2013; Wang et al. 2015; Tian et al. 2015; Gao et al. 2016; Dahal and Kim 2018; Song et al. 2019). To investigate the rhizobacteria of roses, a novel strain, designated RS19T, was obtained.

Materials and methods

Isolation, cultivation, and maintenance

Strain RS19T was isolated from rose rhizosphere soil in the Mentougou District of Beijing, People’s Republic of China (39°57′48″N 116°05′00″E), with Luria–Bertani (LB) agar plates in May 2016. D. psychrophilus CGMCC 1.8951T and D. alkalitolerans CGMCC 1.8973T were obtained from the China General Microbiological Culture Collection Center and were used as related type strains.

Morphology

The purified colonies were observed on LB medium. The cellular morphology was observed by scanning electron microscopy. Gram stain was performed using the bioMérieux Gram stain kit according to the manufacturer’s instructions.

Phylogenetic analysis

DNA was extracted and purified using a commercial kit (TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0). The 16S rRNA gene was amplified by PCR with the universal primers 27F and 1492R. The 16S rRNA gene sequence of strain RS19T was submitted to GenBank and the EzBioCloud server (EzTaxon-e database) to search for similar sequences. The 16S rRNA gene sequence of Dyadobacter luteus RS19T was compared with the sequences available in the National Center for Biotechnology Information (NCBI) GenBank database and EzTaxon-e database. Multiple alignments were performed using CLUSTAL_X software (Thompson et al. 1997). The phylogenetic trees were reconstructed using the neighbor-joining (NJ), maximum likelihood (ML) and minimum-evolution (ME) analysis with the MEGA 7 program (Kumar et al. 2016). The evolutionary distances were calculated using the Maximum Composite Likelihood Method (Tamura et al. 2004). Bootstrap values were calculated based on 1000 replications in each case.

Physiological and biochemical tests

The growth temperature range and optimal growth temperature were tested in tryptic soy broth (TSB) liquid medium at 1, 4, 15, 20, 25, 30, 37, and 45 ℃. Bacterial growth was measured by an increase in turbidity at 600 nm using a spectrophotometer. The bacterium was grown in TSB liquid medium at pH 3, 4, 5, 6, 7, 8, 9, and 10 and at 0, 1, 2, 3, 5, 7, and 10% (w/v) NaCl to determine the pH range and NaCl tolerance for bacterial growth.

Oxidase activity was tested using 1% (w/v) tetra-methyl-p-phenylene diamine. Catalase activity was measured by bubble production in 3% (v/v) H2O2. Carbon source utilization tests, enzyme activity tests, and additional physiological and biochemical tests were performed using Biolog GEN III microplates, API ZYM and API 20NE kits (bioMérieux) according to the manufacturer's instructions.

Chemotaxonomic characterization

For measurement of cellular fatty acid composition, D. luteus RS19T, D. psychrophilus CGMCC 1.8951T and D. alkalitolerans CGMCC 1.8973T were incubated on trypticase soy agar (TSA) at 28 ℃ for 2–3 days. The fatty acids were analyzed by a 6890N gas chromatograph (Agilent) using the Sherlock Microbial Identification System with standard MIS Library Generation Software (version 6.0 and date 4; Microbial ID Inc., Newark, DE, USA) (Sasser 2001).

Polar lipids of strain RS19T and related strains were extracted from 200 mg of freeze-dried cell material according to the method of Minnikin et al. (1977, 1984) and separated by two-dimensional silica gel thin-layer chromatography (Macherey-Nagel Art. No. 818 135). It was developed in chloroform:methanol:water (65:25:4, v/v/v) for the first direction and in chloroform:methanol:acetic acid:water (80:12:15:4, v/v/v/v) for the second direction. Total lipid material was detected using staining reagents (ninhydrin, molybdenum blue and molybdophosphoric acid) specific for defined functional groups. The polar lipid analysis was performed by the identification service of the Agricultural Culture Collection of China.

Respiratory quinones were extracted from lyophilized cells according to the method of Collins et al. (1977), purified by TLC and analyzed by means of HPLC as reported by Xie and Yokota (2003).

Genome sequencing and genotypic characterization

For genome sequencing of strain RS19T, Illumina MiSeq sequencing was performed at Shanghai Personal Biotechnology Co., Ltd., China. The raw data were filtered and trimmed by PRINSEQ v0.20.4 (Schmieder and Edwards 2011). The trimmed reads were assembled using A5-miseq v20150522 with default parameters (Coil et al. 2015). CheckM v1.0.3 was used to estimate the completeness of the genome. Protein-coding open-reading frames (ORFs) were predicted by Glimmer v3.02 (Delcher et al. 2007).

The relatedness of the genome sequence of Dyadobacter luteus RS19T to the whole genome sequences of related type strains was determined based on the average nucleotide identities (ANI) (Goris et al. 2007; Meier-Kolthoff et al. 2014; Richter and Rossello-Mora 2009). Genome sequences in a pairwise comparison were split into 1000 bp windows and aligned with nucmer in MUMmer v3.23 (ANIm) (Kurtz et al. 2004). ANI were calculated using JSpecies v1.2.1 (Meier-Kolthoff et al. 2014).

Results and discussion

Morphological and physiological characteristics

Cells are rod-shaped, Gram-stain negative and aerobic. Colonies on TSB agar are circular, smooth, mucoid, convex with clear edges, yellow and 1.0–2.0 mm in diameter after 24 h of incubation at 28 °C.

Phylogenetic analysis

The 16S rRNA gene sequence (1398 bp) of strain RS19T was deposited in GenBank under the accession number MH558673. The 16S rRNA gene sequences in the GenBank database revealed that strain RS19T belongs to the genus Dyadobacter. Based on the analysis of the EzBioCloud database, the Dyadobacter psychrophilus strain showed the highest pairwise similarity of 97.4%. The following highly related species were Dyadobacter alkalitolerans, Dyadobacter sediminis and Dyadobacter crusticola, with pairwise similarities of 97.1%, 96.8% and 96.6%, respectively.

The NJ tree, ME tree and ML tree for the 16S rRNA are presented in Fig. 1, Fig. S1 and Fig. S2. The phylogenetic tree indicated that strain RS19T was clustered with D. psychrophilus CGMCC 1.8951T and D. alkalitolerans CGMCC 1.8973T.

Fig. 1
figure 1

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain Dyadobacter luteus RS19T and related taxa. Bootstrap values were determined based on 1000 replications. Only values > 50% are shown. Bar, 0.01 substitutions per nucleotide position. GenBank accession numbers are indicated for each strain

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Chemotaxonomic characteristics

Strain RS19T grew with d-trehalose, gentiobiose, stachyose, l-fucose, 1% sodium lactate, pectin, d-galacturonic acid, l-galactonic acid lactone and d-glucuronic acid, which are negative for D. psychrophilus CGMCC 1.8951T and D. alkalitolerans CGMCC 1.8973T (Table 1). All strains were positive for utilization of d-glucose. All strains were negative for hydrolysis of gelatin and assimilation of d-mannitol, potassium gluconate, decanoic acid, adipic acid, malic acid, citric acid and phenylacetic acid.

Table 1 Differential characteristics of Dyadobacter luteus RS19T and the type strains of closely related species
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The fatty acid analysis revealed that all strains contained iso-C15:1, iso-C15:0, C16:1ω5c, C16:0, iso-C15:0 3-OH, iso-C17:0 3-OH and summed feature 3 (C16:1ω7c and/or C16:1ω6c) as the major components. The major cellular fatty acid profile (> 5% of total) of strain RS19T was summed feature 3 (C16:1ω7c and/or C16:1ω6c), iso-C15:0, C16:1ω5c, iso-C17:0 3-OH and iso-C15:0 3-OH (Table 2).

Table 2 Cellular fatty acid compositions of strain RS19T and the type strains of closely related species
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The polar lipids of RS19T contained phosphatidylethanolamine (PE), unidentified phospholipid (PL), two aminolipids (AL), and eleven unidentified lipids (L) (Fig. S3). For the closely related species, the polar lipids were PE, PL, two AL, and nine L for D. psychrophilus CGMCC 1.8951T, and PE, PL, two AL, and seven L for D. alkalitolerans CGMCC 1.8973T.

Genotypic characteristics

The total genome sequence length of RS19T was 6.95 Mbp. 5857 ORFs, 38 tRNAs, 4 rRNAs and 14 ncRNAs were predicted in the genome sequence of RS19T. The G + C content was 43.1 mol%, which was lower than that of D. psychrophilus CGMCC 1.8951T (48.9 mol%) and D. alkalitolerans CGMCC 1.8973T (46.3 mol%). The ANIm values of D. psychrophilus CGMCC 1.8951T and D. alkalitolerans CGMCC 1.8973T versus D. luteus RS19T were 83.82% and 83.6%, respectively. Based on comparative analysis of the ANI values, the ANI threshold range for species demarcation was 95–96%, as suggested by Miller et al. (2016), Kim et al. (2014) and Richter and Rossello-Mora (2009). The ANIm values of Dyadobacter luteus RS19T to closely related type strains were lower than 84%, providing strong evidence in favor of recognizing RS19T as a novel species in the genus Dyadobacter.

Based on the genomic evidence, chemotaxonomic and physiological data, strain RS19T represents a novel species within the genus Dyadobacter, for which the name Dyadobacter luteus sp. nov. is proposed.

Description of Dyadobacter luteus sp. nov.

Dyadobacter luteus (lu'te.us. L. masc. adj. luteus yellow).

Cells are Gram stain negative, aerobic, nonmotile, and rod shaped with a length between 1.4 and 2.3 μm and a width between 0.5 and 0.6 μm. Cells occur in pairs in young cultures, but in chains of short rod shaped to coccoid cells in older cultures. Colonies on TSB agar are circular, smooth, yellow and 1.0–2.0 mm in diameter after 24-h incubation at 28 ℃. Growth occurs in liquid TSB medium at 1–37 ℃ (optimum at 15–30 ℃), at pH 6–8 and in the presence of up to 1% NaCl. Dyadobacter luteus sp. nov. is positive for catalase and oxidase. Esculin but not gelatin is hydrolyzed. Positive for nitrate reduction. Positive for assimilation of d-glucose, l-arabinose, d-mannose, N-acetyl glucosamine and d-maltose. The following substrates are utilized as sole carbon and energy sources: d-maltose, d-trehalose, d-cellobiose, gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, α-d-lactose, d-melibiose, β-methyl-d-glucoside, d-salicin, N-acetyl-d-glucosamine, α-d-glucose, d-mannose, d-fructose, d-galactose, l-fucose, l-rhamnose, 1% sodium lactate, pectin, d-galacturonic acid, l-galactonic acid lactone and d-glucuronic acid. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine-arylamidase, valine arylamidase, cystine arylamidase, acid phosphomonoesterase, naphthol-AS-BI-phosphoric acid, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminase, and α-mannosidase, but negative for P-nitroso-d-methyl galactose, lipase (C14), trypsin, chymotrypsin, β-glucuronidase, and β-fucosidase. The polar lipids are phosphatidylethanolamine (PE), unidentified phospholipid (PL), two aminolipids (AL), and eleven unidentified lipids (L). The major fatty acids are summed features 3 (C16:1ω7c and/or C16:1ω6c), iso-C15:0, C16:1ω5c and iso-C17:0 3-OH. The predominant respiratory quinone is menaquinone-7 (MK-7). The DNA G + C content of the type strain is 43.1 mol%.

The type strain RS19T (= CGMCC 1.13719T = ACCC 60381T = JCM 32940T) was isolated from rose rhizosphere soil in Mentougou District of Beijing, People’s Republic of China.