Introduction

The genus Luteimonas was described by Finkmann et al. [7] with a single species, Luteimonas mephitis, isolated from a biofilter. Phylogenetically, the genus is a member of phylum Proteobacteria and closely to the genera Lysobacter, Xanthomonas, Pseudoxanthomonas and Stenotrophomonas, which share some characteristics such as the presence of branched-chain fatty acids and ubiquinone-8 (Q-8). Members of the genus are Gram-negative, non-flagellated, non-spore-forming, aerobic, rod-shaped, and light yellow colored on R2A, TSA, or NA agar plates [7, 24]. Species of the genus contained iso-C15:0, iso-C15:1, iso-C17:1ω7c and iso-C17:0 as major fatty acids [7, 15]. Affiliates of this genus have been isolated from various sources including biofilter, tidal flat [16], fresh water, food waste, cucumber leaf, seawater, stratum water, sediment, and soil. At the time of writing, the genus Luteimonas comprises 19 recognized species (https://www.bacterio.net) with recently described species Luteimonas notoginsengisoli [3] Luteimonas terrae [14], Luteimonas dalianensis [24], and Luteimonas lutimaris [15].

Materials and Methods

Isolation of Bacterial Strain

In this study, a novel bacterial strain was isolated from the granules of wastewater treatment plant in Daejeon city, Republic of Korea. The samples of granules from wastewater treatment plant were collected and the strain Gr-4T was isolated on R2A agar medium at 30 °C after 2 weeks of incubation. Strain Gr-4T was cultured routinely on R2A agar plates at 30 °C and preserved as a suspension in R2A broth supplemented with 20% (v/v) glycerol at −80 °C.

The reference strains Luteimonas padinae KCTC 52403T; 3, Luteimonas terricola DSM 22344T; 4. Luteimonas arsenica KCTC 42824T were collected from two different cultures [Korean culture collection (KCTC) and German culture collection (DSM), which were used as a references for comparison with the novel isolated strain.

Phylogenetic Tree Construction

Genomic DNA of strain Gr-4T was extracted with a commercial genomic DNA extraction kit (Solgent, Korea) and PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to Kim et al. [11]. Nearly full-length sequence of the 16S rRNA gene was compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of 33 related taxa were obtained from GenBank database [https://www.ncbi.nlm.nih.gov/genbank] or [https://www.ezbiocloud.net/eztaxon]. Multiple alignments were performed by Clustal_X program [21]. Gaps were edited in the BioEdit program [9] and 1,375 nucleotides were used for phylogenetic tree construction. Evolutionary distances were calculated using the Kimura two-parameter model [12]. Phylogenetic trees were constructed using a neighbor-joining method [17], maximum-parsimony [8], and maximum-likelihood method [6] using the MEGA6 program [20] with bootstrap values based on 1000 replications [5].

Complete Genome Sequencing and DNA G+C Content Analysis

Complete genome sequence of strain Gr-4T was determined by Illumina HiSeq X Ten and was assembled by SOAPdenovo v. 3.10.1 de novo assembler. The genome annotation was performed by the NCBI prokaryotic genome annotation pipeline (PGAP). To estimate the degree of pairwise relatedness between Gr-4T and Luteimonas padinae CDR SL 15T, BLAST-based average nucleotide identity (ANI) and the genome-to-genome distance calculation (GGDC) were calculated as described previously [25].

The DNA G+C content (mol%) was analyzed via the complete genome sequence of the novel isolate using the online web server of NCBI.

DNA–DNA Hybridization

Using photobiotin-labeled DNA probes and micro-dilution wells plate method, the DNA–DNA hybridization experiment was performed between strain Gr-4T and two reference strains Luteimonas padinae KCTC 52403T and Luteimonas terricola DSM 22344T [4].

Physiological and Biochemical Characteristics

The Gram reaction was determined using the non-staining method, as described by Buck [1]. Cells motility was determined using hanging drop method, while cell morphology was examined with the scanning electron microscope (Hitachi SU-3500), using cells grown for 1 day at 30 °C on R2A agar medium. Catalase and oxidase tests were performed as outlined by Cappuccino and Sherman [2]. Biochemical tests were carried out using API 20NE, 32GN and API ZYM kits according to the instructions of the manufacturer (bioMérieux). Tests for degradation of DNA, casein, starch and carboxyl methyl cellulose were performed and evaluated after 7 days at 30 °C. Growth at different temperatures (4–45 °C, at intervals of 3 and 5 °C) and various pH values (pH 4–10 at intervals of 1 pH units) was assessed after 5 days incubation at 30 °C. Salt tolerance test was evaluated on R2A medium supplemented with 0–6% (w/v at intervals of 0.5% unit) NaCl and growth assessed after 7 days of incubation at 30 °C. Growth on different media were also tested using nutrient agar (NA, Diffco), trypticase soy agar (TSA, Diffco), LB agar (Diffco) and MacConkey agar (Diffco) at 30 °C. To determine the ginsenosides conversion ability of the novel isolate, the biotransformation of ginsenoside was carried out as described previously [19].

Chemotaxonomy

Analysis of Respiratory Quinone and Cellular Fatty Acids

Isoprenoid quinone was extracted with chloroform/methanol (2:1, v/v), evaporated under vacuum condition, and re-extracted in n-hexane/water (1:1, v/v). The crude n-hexane-quinone solution was purified using Sep-Pak Vac cartridges silica (Waters) and subsequently analyzed by HPLC as previously described [10]. Cellular fatty acids profiles were determined for strains grown on R2A agar for 2 days at 30 ºC. The cellular fatty acids were saponified, methylated, and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids analyzed by a gas chromatograph (Hewlett Packard 6890) were identified by the Microbial Identification software package based on Sherlock Aerobic Bacterial Database (TSBA60) [20].

Nucleotide Sequence Accession Numbers

The 16S rRNA gene sequence and complete genome accession number for strain Gr-4T is JQ349045, and CP042218, respectively.

Results and Discussion

Phylogenetic Assessment and DNA G+C Content

The nearly complete 16S rRNA gene sequence of strain Gr-4T (1469 nt) was determined and subjected to comparative analysis. Phylogenetic analysis using the maximum-likelihood method based on 16S rRNA gene sequences indicated that strain Gr-4T are clustered within the genus Luteimonas (Fig. 1). Moreover, this relationship was also evident in phylogenetic trees based on the neighbor-joining and maximum-parsimony methods.

Fig. 1
figure 1

Phylogenetic relationship of strain Gr-4T, recognized Luteimonas species and related species. The tree was constructed using the neighbor-joining method based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) greater than 60% are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with maximum-parsimony and maximum-likelihood algorithms. Bar, 0.005 substitutions per nucleotide position. Solimonas flava DSM 18980T (EF154515) was used as an outgroup

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Based on EzTaxon-e server (16S rRNA gene sequence similarity), strain Gr-4T show the highest sequence similarity with Luteimonas padinae KCTC 52403T (99.1), Luteimonas terricola DSM 22344T (98.5%) and Luteimonas arsenica KCTC 42824T (97.6%), while share less than 96.7% 16S rRNA gene sequence resemblance with the type specie (Luteimonas mephitis DSM 12574T) and other members of the genus. Similarly, the topology of other trees based on maximum-likelihood, neighbor-joining and maximum persimony analysis indicated that the novel isolate form a clad with Luteimonas padinae KCTC 52403T, Luteimonas terricola DSM 22344T 52403T (99.1%) and Luteimonas arsenica KCTC 42824T.

The DNA G+C contents of strain Gr-4T was 70.2 mol%. The DNA G+C content was within the range to the other describe species of the genus Luteimonas (Table 1).

Table 1 Characteristics that differentiate Luteimonas granuli sp. nov. from its closely related species of the genus Luteimonas
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Complete genome sequence analysis show a total length of 2.92 Mb. Gene prediction has also been performed and 2481 protein coding genes, 53 RNA gene, 3 rRNA and 46 tRNA genes were detected by NCBI IMG/ER (https://img.jgi.doe.gov/cgi). The genomic DNA G+C content of strain Gr-4T, directly calculated from its genome sequence, was determined to be 70.5 mol% which was in the range of the previously described species of the genus Luteimonas. Furthermore the genome features of strain GR-4T are show in Table 2. The genomic ANI values between strain GR-4T and Luteimonas padinae KCTC 52403T was 87.3%. The low ANI value supports the characterization of strain Gr-4T as a novel species of the genus Luteimonas.

Table 2 The genome features of strain Luteimonas granuli Gr-4T
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DNA–DNA relatedness value between strain Gr-4T and Luteimonas padinae KCTC 52403T and Luteimonas terricola DSM 22344T were 36.4 ± 1.3% (34.2 ± 1.8%, reciprocal) and 14.2 ± 1.7% (16.1 ± 1.9%, reciprocal), respectively. According to Wayne et al. [23], DNA–DNA relatedness values lower than 70% are considered to be the threshold value for the delineation of genospecies, so the result obtained is low enough to assign strain Gr-4T as a novel species of the genus Luteimonas.

Phenotypic and Biochemical Tests

Cells of strain Gr-4T were Gram-reaction-negative, aerobic, non-motile, non-spore-forming, and rod-shaped. Colonies of strain Gr-4T grown on R2A agar were circular, convex, opaque, and light yellow color after 24 h incubation at 30 °C. The isolate did not grow on MacConkey agar (Difco), DNase agar (Difco) and LB (Difco), whereas weakly grow on nutrient agar (Difco) and TSA (Difco) at 30 °C. Physiological characteristics of strain Gr-4T is summarized in the species description and a comparison of selective characteristics of the isolated strain and related type strains are given in Table 1.

Quinone, Polar Lipid, and Fatty Acids

The major respiratory quinone was ubiquinone Q-8. The fatty acid profile of strain Gr-4T was compared with those of the type strains of recognized Luteimonas species. The major fatty acids of strain Gr-4T were iso-C15:0 (28.6%), iso-C16:0 (15.2%), and summed feature 9 [(comprising iso-C17:1ω9c and/or C16:0 10-methyl) 13.5%], which is a typical profile of members of the genus Luteimonas [7]. However, some qualitative and quantitative variances in the fatty acids distinguished strain Gr-4T from the other recognized species of the genus Luteimonas (Table 3).

Table 3 Cellular fatty acid profiles of strain Gr-4T and phylogenetically related species of the genus Luteimonas
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On the basis of the data and observations described above, it is appropriate to conclude that strain Gr-4T should be assigned to the genus Luteimonas as the type strain, for which the name Luteimonas granuli sp. nov. is proposed.

Description of Luteimonas granuli sp. nov.

Luteimonas granuli (gra.nu'li. L. gen. n. granuli of a small grain, pertaining to a granule, from which the type strain was isolated).

Cells are Gram-reaction-negative, aerobic, non-motile and rod-shaped. The colonies grown on R2A agar plate for 2 days are light yellow color, smooth, opaque, circular with regular margins, and 1–2 mm in diameter. Growth occurs between 18–42 °C (optimum, 30 °C), pH 6–8 (optimum, pH 7) and 0–3% NaCl (optimum, without % NaCl). Does not hydrolyze casein, starch, CM-cellulose and DNA. In the API kits (API 20 NE, 32 GN, and API ZYM) system, positive for esculin hydrolysis, nitrate reduction, β-galactosidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, salicin, d-glucose, N-Acetyl-d-glucosamine, and glycogen. Q-8 is the predominant respiratory quinone and iso-C15:0, iso-C16:0 and summed feature 9 (comprising iso-C17:1ω9c and/or C16:0 10-methyl) are the major cellular fatty acids. The G+C content of the genomic DNA is 70.2 mol%.

The type strain, isolated from the granules used in the wastewater treatment lab scale reactor of in Daejeon, Republic of Korea, is Gr-4T (=KACC 16614T = JCM 18203T).