Chryseobacterium endalhagicum sp. nov., isolated from seed of leguminous plant.

A Gram-stain-negative, yellow-pigmented bacterium, designated as L7T, was isolated from seeds of Alhagi sparsifolia Shap., a leguminous plant that grows in northwest PR China. Strain L7T was found to be non-flagellated, non-spore forming rods which can grow at 10-37 °C, pH 6.0-8.5 and in 0-3 % (v/w) NaCl concentration. The 16S rRNA gene sequence analysis showed that strain L7T belongs to the genus Chryseobacterium with sequence similarities to Chryseobacterium vietnamense GIMN1.005T (98.1%), C. bernardetii NCCTC13530T (98.0%), C. vrystaatense LMG 22846T (97.9%), C. nakagawai NCTC13529T (97.7%), C. shigense DSM 17126T (97.6%) and C. rhizosphaerae RSB3-1T (97.5%). The average nucleotide identity of strain L7T to 31 reference strains were 78.6-85.6 %, lower than the species delineation threshold of 95 %. MK-6 was the only respiratory quinone of L7T and major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1  ω7c and/or C16 : 1  ω6c, isoC17 : 1  ω9c and/or C16 : 0 10-methyl. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminophospholipids, two unidentified aminolipids, three unidentified glycolipids and two unidentified lipids. The G+C content of the genome was 38.58 mol%. On the basis of polyphasic taxonomy analyses in this study, strain L7T is considered to represent a novel species in the genus Chryseobacterium, for which the name Chryseobacterium endalhagicum sp. nov. is proposed. The type strain is L7T (=MCCC 1K05687T=JCM 34506T).

Introduction

The genus belongs to the family , and was first described by Vandamme in 1994 [1]. There are 120 species of the genus that have been reported until now (https://lpsn.dsmz.de/genus/chryseobacterium), and most species could form yellow colonies on solid medium. Species of were isolated from varied environments, such as terrestrial and aquatic environments [2-5], even including organisms and food [6-8].

In this study, strain L7T was isolated from seeds of Alhagi sparsifolia Shap., a leguminous plant grown in northwest China. Comparing physiological, biochemical and genetic characteristics with reference strains GIMN1.005T, NCTC13530T, LMG 22846T, NCTC 13529T, DSM 17126T, RSB3-1T and type strain ATCC35910T, strain L7T represents a novel species in the genus .

Methods

Isolation and ecology

Strain L7T was isolated from seeds of Alhagi sparsifolia Shap. collected from Turpan basin of Xinjiang (N 45°16´, E 85°2´). We took about 1.0 g of the seeds and surface sterilized them according to the method of Yuan [9]: firstly, we rinsed the seeds with distilled water three times, and then soaked them with 0.01 % (v/w) Tween-20 for 1 min; followed by soaking with 5 % (v/w) sodium hypochlorite solution, then rinsed with sterile water three times and dried with sterile paper. We then ground the sterile seeds into an homogenate, took 0.1 ml the slurry and spread it on Tryptic Soy Agar (TSA, Qingdao Rishui Bio-techologies Co., Ltd), incubated at 30 °C for 72 h, then selected single colonies and purified the colonies to obtain a pure culture. We isolated a bacterium which formed yellow colonies on the plate, named it L7, and preserved it in Tryptic Soy Broth (TSB, Qingdao Rishui Bio-techologies Co., Ltd) with 15 % (v/v) glycerol at −80 °C. GIMN1.005T (GDMCC1.2012), RSB3-1T (KCTC22548), and ATCC35910T (GDMCC1.870) were obtained from the Korean Collection for Type Cultures (KCTC) and Gongdong Microbial Culture Collection Centre (GDMCC) as reference strains for phenotypic comparison. To characterize the features of strain L7T, the isolate and reference strains were routinely cultured on TSA at 28 °C for 24 h unless otherwise noted.

16S rRNA gene phylogeny

The genomic DNA of strain L7T was extracted using TIANamp Bacteria DNA Kit (TIANGEN Biotech Co., Ltd.) according to the protocol. Using the genomic DNA of L7T as a template the 16S rDNA was amplified with Taq PCR Master Mix (Sangon Biotech Co., Ltd.) and universal bacterial primers 27F/1492R [10]. The PCR product was purified using TIANgel Mini Purification Kit (TIANGEN Biotech Co., Ltd.), and sequencing was done by Sangon Sequencing (Sangon Biotech Co., Ltd.). The 16S rRNA gene sequence analysis of strain L7T and closely related strains from the EZ BioCloud (https://www.ezbiocloud.net/) [11] were aligned using clustal W programme [12]. Phylogenetic trees were reconstructed according to the neighbour-joining and maximum-likelihood methods [13, 14] using mega X software [15], bootstrap analyses were performed using 1000 replications.

Genome features

The whole-genome of strain L7T was sequenced and assembled by Majorbio Whole Genome Analysis Service (Majorbio Co., Ltd.) using the Illumina Hiseq ×10 platform and SOAPdenovo2 [16], respectively. The assembled genome was annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [17]. To confirm the taxonomic status of strain L7T, average nucleotide identity (ANI) and digital DDH (dDDH) values were analysed using the ANI online tool of Chunlab (http://www.ezbiocloud.net/ezgenome/ani) [18] and Genome-to-Genome Distance Calculator version 2.1 (http://ggdc.dsmz.de/ggdc.php#) [19], respectively. The genome sequences of reference strains were downloaded from GenBank database.

Morphological and physiological analysis

The morphology of strain L7 T was observed by transmission electron microscopy (JEM-1230; JEOL), and the production of flexirubin-type pigments was tested using 20 % (w/v) KOH [20]. Gram-staining was performed using a Gram-staining kit (Solarbio). The range of growth temperatures was tested in TSB over the range of 5–50 °C (4, 10, 15, 20, 25, 28, 30, 35, 37, 40, 45 and 50). The pH range for growth was tested from pH 5.0 to pH 10.0 (in 0.5 pH unit intervals) at 28 °C. The pH was adjusted using MES (pH 4.0–6.0), PIPES (pH 7.0–8.0), HEPES (pH 8.0–9.0), and Gly-NaOH (pH 9.0–10.0) [21]. The catalase and oxidase activities were tested using 3 % (w/v) H2O2 and 1 % (w/v) dimethylaniline [22], respectively. The biochemical properties of strain L7T were determined using the API 20NE kit (bioMérieux), GNШ microplate (Biolog) and API ZYM kit (bioMérieux) according to protocols.

Chemotaxonomic analysis

Cellular fatty acids were analysed using biomass of strain L7T and reference strains which were obtained by culturing in nutrient broth at 28 °C. The fatty acid methyl esters were prepared, separated and identified according to the instructions of the Microbial Identification System (MIDI) [23]. The respiratory quinone content of strain L7T was analysed by following the method of Lee [5]. Polar lipids were extracted and analysed by two-dimensional TLC (silica gel plates, layer thickness 0.2 mm; Merck) according to the method modified by Indu [24]. Relevant data of strains NCTC13530T, LMG 22846T, NCTC 13529T, and DSM 17126 T were quoted from database and literature.

Results and discussion

Phylogenetic characteristics

Analysis of the 16S rRNA gene sequence of strain L7T (accession no. MT810475) revealed that L7T belonged to the genus , the most closely related strain was GIMN1.005T, with 98.1 % 16S rRNA gene sequence similarity, followed by NCCTC13530T (98.0%), LMG 22846T (97.9%), NCTC13529T (97.7%), DSM 17126T (97.6%), and RSB3-1T (97.5%) (Table 1).

Table 1.

16S rRNA similarity and genomic differences between strain L7T and reference type strains

Strain	16S rRNA similarity (%)	ANI (%)	DDH (%)
C. vietnamense GIMN1.005T	98.1	79.5	23.6
C. bernardetii NCTC 13530T	98.0	79.3	23.3
C. vrystaatense LMG 22846T	97.9	83.4	27.3
C. nakagawai NCTC 13529T	97.7	79.0	23.1
C. shigense DSM 17126T	97.6	83.7	27.6
C. rhizosphaerae RSB3-1T	97.5	79.5	23.5
C. angstadtii KMT	97.5	83.8	27.6
C. carnipullorum DSM 25581T	97.5	83.8	27.4
C. aurantiacum F30T	97.5	79.0	23.1
C. oleae DSM 25575T	97.4	84.8	29.3
C. candidae JC507T	97.4	79.8	23.6
C. culicis DSM 23031T	97.4	79.2	23.2
C. jejuense DSM 19299T	97.4	79.1	23.1
C. pennipullorum 7_F195T	97.3	78.8	22.9
C. kwangjuense KJ1R5T	97.2	85.6	30.6
C. luteum DSM 18605T	97.2	84.1	28.5
C. indologenes NBRC 14944T	97.2	79.0	22.9
C. lactis NCTC 11390T	97.0	79.2	23.1
C. arthrosphaerae CC-VM-7T	96.9	79.3	23.5
C. ureilyticum DSM 18017T	96.9	79.3	23.1
C. viscerum 687B-08T	96.8	79.4	23.3
C. oranimense DSM 19055T	96.8	84.1	28.1
C. gleum ATCC 35910T	96.8	79.5	23.4
C. cucumeris GSE06T	96.7	79.5	23.3
C. aureum 17S1E7T	96.7	79.3	23.5
C. artocarpi UTM-3T	96.7	79.2	22.9
C. oncorhynchi 701B-08T	96.6	79.2	23.1
C. sediminis IMT-174T	96.6	79.6	23.4
C. gallinarum DSM 27622T	96.5	79.2	23.0
C. contaminans DSM 27621T	96.2	79.2	23.0
C. daecheongense DSM 15235T	96.2	78.6	22.1

GIMN1.005T

NCTC 13530T

LMG 22846T

NCTC 13529T

DSM 17126T

RSB3-1T

KMT

DSM 25581T

F30T

DSM 25575T

JC507T

DSM 23031T

DSM 19299T

7_F195T

KJ1R5T

DSM 18605T

NBRC 14944T

NCTC 11390T

CC-VM-7T

DSM 18017T

687B-08T

DSM 19055T

ATCC 35910T

GSE06T

17S1E7T

UTM-3T

701B-08T

IMT-174T

DSM 27622T

DSM 27621T

DSM 15235T

Phylogenetic trees of representative members in the genus were reconstructed. In Fig. 1, strain L7T was separated from the most similar sequences, forming a distinct lineage within the genus and the neighbour-joining phylogenetic tree shows a similar structure (Fig. S1) considering the 16S rRNA gene sequence similarities below the established thresholds [25], it was concluded strain L7T as a potential novel species of the genus .

Fig. 1.

Maximum-likelihood tree based on 16S rRNA gene sequences available in the GenBank database (accession numbers in parentheses), showing the positions of strain L7T and related type strains belonging to the genus . Bootstrap values (expressed as the percentage of 1000 replications) are shown at the branch points; only values greater than 50 % are shown. Filled circles indicate nodes that were also recovered in the tree with neighbour-joining algorithm. LQY-7T (GQ988780) was used as outgroup. Bar, 0.02 nucleotide substitutions per nucleotide position.

Genomic features

The assembled draft genome sequence of strain L7T was 4931506 bp, and was composed of ten contigs. The NCBI PGAP revealed three copies of the 5S, 16S, 23S rRNA gene, respectively, and 83 RNA genes (nine rRNA, 71 tRNA and three ncRNA). Among a total of 4371 predicted genes, 4288 genes were identified as protein-coding sequences. The genomic information of strain L7T and 31 reference species are shown in Table S1. The ANI values between strain L7T and the 31 type strains were 78.6–85.6 %. DNA–DNA hybridization between strain L7T and the 31 type strains were <31 % (Table 1). The low ANI and DDH values indicated that strain L7T represents a novel species in the genus .

Strain L7T was shown to be rod-shaped (2–3 µm×0.5–0. 6 µm) and non-flagellated, Gram-stain-negative, non-spore forming, produced flexirubin-type pigments (Fig. S2). The temperature range for growth was 10–37 °C (optimum 28 °C). The pH range for growth was 6.0–8.5 (optimum 7.0). The NaCl range for growth was 0–3 % (optimum 1 %). Activities of catalase and oxidase were positive. Carbon substrate utilization was positive for: dextrin, trehalose, gentiobiose, α-d-glucose, d-fructose, d-mannitol, glycerol, d-glucose-6-phosphate, d-fructose-6-phosphate, gelatin, glycyl-l-proline, l-arginine, l-aspartic acid, l-glutamic acid, l-serine, d-glucuronic acid, d-saccharic acid, methyl pyruvate, d-lactic acid methyl ester, l-lactic acid, citric acid, α-keto-glutaric acid, Tween 40, acetoacetic acid, acetic acid. Enzyme activity (API ZYM) was positive for: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase (Table S2). The main differential phenotypic characteristics of strain L7T and the reference strains are given in Table 2.

Table 2.

Differential characteristics of the novel strain L7T with its closest phylogenetic relatives. Strains: 1, L7T; 2, GIMN1.005T; 3, NCTC13530T; 4, LMG 22846T; 5, NCTC 13529T; 6, DSM 17126T; 7, RSB3-1T; 8, ATCC35910T. +, Positive; -, negative; w, weakly positive; ND, no data

Characteristics	1	2	3*	4*	5*	6*	7	8
Source	leguminous plant	forest soil	human sputum	raw chicken	human kidney	bovine milk	rhizosphere	human vagina
Growth temperature (℃)	10–37	5–37†	18–37‡	4-32§	nd	5-30†††	10-37¶	18–40**
NaCl tolerance (%)	3	2†	nd	3§	3††	nd	4¶	7**
pH range for growth	6.0–8.5	6.0-9.0†	nd	nd	5.0–10.0††	5.0-8.0†††	5.0-10.0¶	6.0-10.0**
Indole production	−	−	nd	+§	-††	+†††	−	+
UREase	−	+	+‡	+§	nd	-†††	+	+
d-Glucose	w	w	nd	+§	+††	+†††	w	+
l-Arabinose	−	−	nd	nd	-††	-†††	w	+
d-Mannose	−	w	nd	+§	nd	nd	w	+
Maltose	−	w	nd	nd	nd	-†††	w	+
d-Mannitol	+	w	nd	+§	w††	-†††	+	−
d-Galacturonic acid	w	+	nd	nd	nd	nd	−	−
d-Glucuronic acid	+	+	nd	-/+‡‡	nd	-‡‡	w	−
Polar lipids§§	PE, 3 APL, 2AL, PL, 3 GL, 2L	nd	PE, 5AL, 4L¶¶	7L, 4AL‡‡	nd	6L, 4AL, PE‡‡	nd	PE, 7AL, 4 L**
DNA G+C content (mol%)***	38.58	36.07	36.32	37.06	35.40	37.46	36.28	36.81

*Data from literature.

†Data from Li et al. [26].

‡Data from Holmes et al. [27].

§Data from Beer et al. [28].

¶Data from Cho et al. [29].

**Data from Holmes et al. [30].

††Data from Lee et al. [31].

‡‡Data from Carmen Montero-Calasanz et al. [32].

§§PE, Phosphatidylethanolamine; AL, unidentified aminolipid; APL, unidentified aminophospholipid; GL, unidentified glycolipid; L, unidentified lipid.

¶¶Data from Kim et al. [33].

***The DNA G+C contents of the strains were calculated in this study using the genome sequence.

†††Data from Shimomura et al. [34].

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+†††

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-†††

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+§

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+

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-†††

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-†††

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Major fatty acids (>10 %) of strain L7T were iso-C15 : 0 (23.56 %), iso-C17 : 0 3-OH (22.9 %), C16 : 1  ω7c and/or C16 : 1  ω6c (13.7 %), isoC17 : 1  ω9c and/or 10-methyl C16 : 0 (11.9 %). The fatty acid composition of strain L7T was significantly different from those of GIMN1.005T, NCTC13530 T, LMG22846T, NCTC13529T, DSM17126 T, RSB3-1T, and ATCC35910T (Table 3). Menaquinone-6 (MK-6) was found as the only respiratory quinone of strain L7T. The major polar lipids of strain L7T were phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminophospholipids, two unidentified aminolipids, three unidentified glycolipids and two unidentified lipids (Fig. 2, Table 2).

Table 3.

Cellular fatty acid contents of strain L7T and type strains of phylogenetically related species

Strains: 1, L7T; 2, GIMN1.005T; 3, NCTC13530T; 4, LMG 22846T; 5, NCTC 13529T; 6, DSM 17126T; 7, RSB3-1T; 8, ATCC35910T. Values are percentage of total fatty acids; fatty acids amounting to <1 % of the total fatty acids in all strains listed are omitted. -, Not detected; TR, trace amount (<1 %)

Fatty acid	1	2	3*	4†	5‡	6§	7	8
iso-C15 : 0	23.6	28.2	29.7	46.8	35.3	41.7	29.7	24.0
iso-C15 : 03-OH	2.9	2.8	2.9	3.2	3.0	2.4	3.2	3.2
iso-C16 : 0	4.0	3.0	–	tr	–	tr	3.0	3.1
C16 : 0	2.6	4.1	4.0	1.3	2.0	1.0	–	2.3
iso-C16 : 03-OH	3.7	1.5	1.1	TR	–	nd	2.4	2.4
iso-C17 : 0 3-OH	22.9	26.3	17.8	12.9	–	16.3	24.4	26.0
Summed feature 3¶	13.7	10.1	14.1	9.4	12.9	9.9	9.6	14.8
Summed feature 8¶	–	–	1.4	–	–	nd	–	–
Summed feature 9¶	11.9	9.8	16.8	14.4	21.3	nd	12.1	10.9

*Data from Kim et al. [33].

†Data from https://www.ccug.se/strain?id=50970

‡Data from https://www.ccug.se/strain?id=60563

§Data from Montero-Calasanz et al. [32].

¶Summed features are fatty acids that can not be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 3 contains C16 : 1  ω7c and/or C16 : 1  ω6c; summed feature 8 contained C18 : 1  ω7c and/or C18 : 1  ω6c; summed feature 9 contains isoC17 : 1  ω9c and/or C16 : 0 10-methyl.

Fig. 2.

Two-dimensional TLC of polar lipids photographs from strain L7T. A, total lipids of strain L7T; B, glycolipids of strain L7T; C, aminophospholipids and phosphatidylethanolamine of strain L7T; D, phospholipids of strain L7T. AL, unidentified aminolipid; APL, unidentified aminophospholipid; GL, unidentified glycolipid; L, unidentified lipid; PL, phospholipid; PE, phosphatidylethanolamine.

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The phenotypic and chemotaxonomic characteristics of the isolate, summarized in Tables 1–3, in additional to the 16S rRNA gene phylogenetic analyses, suggest that strain L7T represents a novel species of genus , for which the name Chryseobacterium endalhagicum sp. nov. is proposed.

Description of Chryseobacterium endalhagicum sp. nov.

Chryseobacterium endalhagicum (end. al. ha’ gi. cum. Gr. pref. endo- within; N.L. fem. n. Alhagi, a botanical genus name; N.L. neut. adj. endalhagicum, living inside Alhagi).

Cells are Gram-stain-negative, rod-shaped, 0.5–0.6 µm wide, 2.0–3.0 µm long, non-motile. Colonies on TSA are slimy, round, have a smooth margin, and bright yellow in colour, produce flexirubin-type pigments. Growth occurs at 10–37 °C, with optimum growth at 28 °C. pH range for growth is 6.0–8.5, with optimum growth at pH 7.0. Tolerates up to 3 % NaCl (w/v), optimum growth occurs at 1 %. Catalase and oxidase activities are positive. Carbon substrate utilized were dextrin, trehalose, gentiobiose, α-d-glucose, d-fructose, d-mannitol, glycerol, d-glucose-6-phosphate, d-fructose-6-phosphate, gelatin, glycyl-l-proline, l-arginine, l-aspartic acid, l-glutamic acid, l-serine, d-glucuronic acid, d-saccharic acid, methyl pyruvate, d-lactic acid methyl ester, l-lactic acid, citric acid, α-keto-glutaric acid, Tween 40, acetoacetic acid, acetic acid. Enzyme activity (API ZYM) positive for: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase. MK-6 is the only quinone. Phosphatidylethanolamine, an unidentified phospholipid, two unidentified aminolipids, three unidentified aminophospholipids, three unidentified glycolipids and two unidentified lipids are the polar lipids. Major fatty acids (>10 %) are iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1  ω7c and/or C16 : 1  ω6c, isoC17 : 1  ω9c and/or 10-methyl C16 : 0. The genomic DNA G+C content is 38.58 mol%.

The type strain is L7T (=MCCC 1K05687T=JCM 34506T), which was isolated from seeds of Alhagi sparsifolia Shap., a leguminous plant. The GenBank accession number for the 16S rRNA gene sequence and the draft genome sequence accession numbers of Chryseobacterium endalhagicum L7T were MT810475 and JAELVM000000000, respectively.

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