DOI: 10.15468/xzz32p
title: Dyella dinghuensis sp. nov. and Dyella choica sp. nov., isolated from forest soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Ou, Fang-hong, Gao, Zeng-hong, Chen, Mei-hong, Bi, Jie-yi, Qiu, Li-hong (2019): Dyella dinghuensis sp. nov. and Dyella choica sp. nov., isolated from forest soil. International Journal of Systematic and Evolutionary Microbiology 69 (5): 1496-1503, DOI: 10.1099/ijsem.0.003356, URL: http://dx.doi.org/10.1099/ijsem.0.003356AbstractTwo aerobic, Gram-stain-negative, non-motile, rod-shaped bacterial strains, designated as DHOA 06 T and 4 M-K27 T, were isolated from soil samples collected from the forest of Dinghushan Biosphere Reserve, Guangdong Province, PR China (112 Ǫ 31′ E 23 Ǫ 10′ N). Strains DHOA 06 T and 4 M-K27 T grew at pH 4.5–7.0 (optimum, pH 5.0–6.0) and pH 4.5–6.5 (pH 6.0), respectively. Both strains grew at 12–37 Ǫ C (optimum, 28 Ǫ C) and NaCl levels up to 1.0 % (optimum 0 %, w/v). Phylogenetic analysis based on both 16S rRNA gene sequences and the concatenated partial atpD, gyrB and lepA gene sequences showed that strains DHOA 06 T and 4 M-K27 T formed two isolated clades with members of the genus Dyella, but they each occupied a distinctive position within the genus. Strains DHOA 06 T and 4 M-K27 T showed the highest 16S rRNA gene sequence similarities to Dyellacaseinilytica DHOB09 T (98.7 %) and Dyellaacidisoli 4M-Z03 T (98.8 %), respectively. DNA–DNA hybridization values of strains DHOA 06 T /DHOB09 T and 4 M-K27 T /4M-Z03 T were 27.4±2.4 % and 38.8±1.0 %, respectively. Ubiquinone-8 was the only respiratory quinone detected in both strains. Their major fatty acids consisted of iso-C 15: 0, iso-C 16: 0 and iso-C 17: 1 Ɯ 9 c, and strain DHOA 06 T had iso-C 17: 0 in addition. Their polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid, one unidentified phospholipid and two unidentified aminolipids, and strain DHOA 06 T had phosphatidylmethylethanolamine and one unidentified lipid in addition. The DNA G+C contents of strains DHOA 06 T and 4 M-K27 T were 59.1 and 61.7 mol%, respectively. Based on the above results, we propose that strains DHOA 06 T and 4 M-K27 T represent two novel species of the genus Dyella, namely Dyelladinghuensis sp. nov. (type strain DHOA 06 T = KCTC 52129 T = NBRC 111978 T) and Dyellachoica sp. nov. (type strain 4 M-K27 T = GDMCC 1.1189 T = LMG 30267 T).The genus Dyella was first described by Xie and Yokota [1], with Dyella japonica as the type species, and the following 23 species have been proposed since then: Dyella koreensis [2], Dyella ginsengisoli [3], Dyella marensis [4], Dyella soli, Dyella terrae [5], Dyella thiooxydans [6], Dyella kyungheensis [7], Dyella jiangningensis [8], Dyella jejuensis [9], Dyella humi [10], Dyella acidisoli, Dyella flagellata, Dyella nitratireducens [11], Dyella lipolytica [12], Dyella caseinilytica, Dyella flava, Dyella mobilis [13], Dyella agri [14], Dyella halodurans [15], Dyella tabacisoli [16], Dyella solisilvae [17], Dyella monticola and Dyella psychrodurans [18]. Dyella was moved to a recently established family Rhodanobacteraceae in the order Lysobacterales [19]. Members of the genus Dyella are generally Gram-stain-negative, aerobic, yellowpigmented and rod-shaped bacteria, having Q-8 as the major respiratory lipoquinone, iso-C 15: 0, iso-C 16: 0 and iso- C 17: 1 Ɯ 9 c as the major fatty acids, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as major polar lipids, and a DNA G+C content of 58.2– 67.0 mol% [1 – 18]. Most Dyella species were isolated from soil and rhizosphere, where they may play an important role in soil element and material recycling, thus may be used for bioremediation of contaminated soils [3]. In this study, we described two novel species of the genus Dyella based on phenotypic, phylogenetic and chemotaxonomic analyses, in combination with DNA–DNA hybridization experiments.Strains DHOA 06 T and 4 M-K27 T were isolated from forest soil samples collected at Dinghushan Biosphere Reserve (DHSBR), Guangdong Province, PR China (112 Ǫ 31′ E 23 Ǫ 10′ N). The soil in the study sites is acidic lateritic red earth with a pH of 3.5–4.5. For the isolation, the soil sample was suspended in PBS buffer and the resulted suspensions were serially diluted with the same buffer. Aliquots of the diluted suspensions were spread on Reasoner’ s 2A (R 2A) medium [0.5 g yeast extract, 0.5 g proteose peptone, 0.5 g casamino acids, 0.5 g glucose, 0.5 g soluble starch, 0.03 g Na–pyruvate, 0.3 g K 2 HPO 4, 0.05 g MgSO 4 ·7H 2 O, 15.0 g agar, in 1 l distilled water, buffered at pH 7.0 and 4.0 (20.0 g agar was used for medium buffered at pH 4.0)] and the plates were incubated at 28 Ǫ C for 1 week. Once a single colony was obtained, it was purified by sub-culturing three times at the same conditions. Both strains DHOA 06 T and 4 M-K27 T were isolated in this way, and they were preserved in 25 % (v/v) glycerol in a refrigerator at ‒80 Ǫ C. The growth of strains DHOA 06 T and 4 M-K27 T were tested on trypticase soy agar, R 2A agar, Luria–Bertani agar and MacConkey agar (all from HKM). Growth was observed for both strains cultured on all media tested except for MacConkey agar, and the best growth was achieved on R 2A agar at 28 Ǫ C for 4 days. Strains DHOA 06 T and 4 M-K27 T together with the following related type species, D. acidisoli 4M-Z03 T, D. caseinilytica DHOB 09 T, D. nitratireducens DHG 59 T and D. flagellata 4 M-K16 T (all from this lab), were cultured on R 2A agar at 28 Ǫ C for 4 days during the course of this study, except otherwise stated.The Gram-staining was performed by the standard method [20]. The cell morphology was examined using an optical microscope (×1000 magnification; Nikon) with cells grown on both liquid and solid R 2A media. Cell motility and oxygen demand was studied by observing the development of turbidity throughout a tube of semi-solid (0.3 % agar) R 2A agar inoculated by stabbing. The range and optimal growth temperature (4, 12, 20, 28, 37 and 42 Ǫ C), pH (pH 3.0–10.0 at intervals of 0.5 pH unit) and salinity (0–5 % NaCl at intervals of 0.5 %, w/v) were assessed by monitoring the OD 600 of the bacterial density incubated under the tested conditions for 4 days, except for the temperature of 4 and 12 Ǫ C, which was determined by observing the growth of the culture on R 2A agar plates for 3 weeks. For the pH experiments, three different buffers were used: buffer with 1.0 mol l ‒1 sodium acetate/1.0 mol l ‒1 acetic acid for pH 3.0–5.5; 0.2 mol l ‒1 disodium hydrogen phosphate/0.2 mol l ‒1 sodium dihydrogen phosphate for pH 6.0–8.0, and 0.2 mol l ‒1 Tris/0.2 mol l ‒1 HCl for pH 8.5–10.0. The colonies of both strains were smooth, circular, convex and yellow, but the yellow of strain DHOA 06 T was a little darker than that of strain 4 M-K27 T, while strain DHOAO6 T colonies had greater viscosity and humidity than that of strain 4 M-K27 T. Cells of strains DHOA 06 T and 4 M-K27 T were Gram-stainnegative, non-spore-forming and rod-shaped. Cell motility and oxygen demand test showed that growth of both strains was observed only at the top third of the inoculating line, indicating that both of them are aerobic and non-motile. Strains DHOA 06 T and 4 M-K27 T were able to grow at pH 4.5–7.0 (optimum, pH 5.0–6.0) and pH 4.5–6.5 (pH 6.0), respectively. Both strains grew at 12–37 Ǫ C (optimum, 28 Ǫ C) and NaCl concentration up to 1.0 % (0 %, w/v).Catalase activity was determined by bubble production in 3 % (v/v) H 2 O 2 and oxidase activity was determined using 1 % (w/v) tetramethyl- p -phenylenediamine (HKM). Carbon source utilization and enzyme activities were tested using API ZYM, API 20 NE and API 50 CHB/E galleries (bio- Ḿerieux) according to the manufacturer’ s instructions. Tests for the hydrolysis of casein, Tween 80 (on R 2A supplemented with 1 % Tween 80) and starch (medium with 10 g soluble starch, 10 g casamino acids, 0.3 g Na–pyruvate, 0.3 g K 2 HPO 4, 0.05 g MgSO 4 ·7H 2 O, 15.0 g agar, in 1 l distilled water, buffered at pH 7.0) were evaluated after 4 days of incubation at 28 Ǫ C. H 2 S production was determined with H 2 S test ager (HKM). Antimicrobial susceptibility of the strains were tested by using the Kirby–Bauer method [21]. The following 14 antibiotics were tested: amikacin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), netilmicin (30 µg), novobiocin (5 µg), penicillin (10 IU), polymyxin (300 U), streptomycin (10 µg), tetracycline (30 µg), tobramycin (10 µg) and vancomycin (30 µg). The results (Table S1, available in the online version of this article) were determined by measuring the zone of inhibition appeared on R 2A agar medium after incubation for 4 days at 28 Ǫ C. Both strains were catalase- and oxidase-positive, nitratereduced to nitrite-negative, and H 2 S and indole production negative; casein was hydrolysed, but gelatin, starch and Tween 80 were not. The detailed physiological and biochemical characteristics that can be used to differentiate strains DHOA 06 T and 4 M-K27 T from their phylogenetically closest relatives are listed in Table 1. Strain DHOA 06 T was resistant to chloramphenicol and penicillin G, but sensitive to amikacin, ciprofloxacin, erythromycin, gentamicin, kanamycin, netilmicin, novobiocin, polymyxin B, streptomycin, tetracycline, tobramycin and vancomycin. Strain 4 M-K27 T was resistant to chloramphenicol, ciprofloxacin, erythromycin, penicillin G, polymyxin B, tetracycline and vancomycin, but sensitive to amikacin, gentamicin, kanamycin, netilmicin, novobiocin, streptomycin and tobramycin.Genomic DNA of the strains was extracted with a commercial genomic DNA-extraction kit (GeneStar). The almost full-length 16S rRNA gene was amplified by PCR using the universal primers 27F and 1492 R [22]. The partial atpD, gyrB and lepA gene sequences of both strains were amplified and sequenced following the methodology detailed by Chen et al. [11] and confirmed with genome sequence when available. The PCR products were purified and sequenced by Ruibiotech Co. Ltd. The similarity and pairwise distance values were calculated using MegAlign software (DNASTAR). Then the newly determined gene sequences of 16S rRNA, partial atpD, gyrB and lepA used in this study were qualitychecked and submitted to GenBank (Table S2). Phylogenetic trees were reconstructed using MEGA software version 5.0 [23] with the neighbour-joining [24], maximum- parsimony [25] and maximum-likelihood [26] algorithms. During the phylogenetic analysis, evolutionary distances were calculated using the Kimura two-parameter model [27], and bootstrap values were calculated based on 1000 replications [28]. Strain DHOA 06 T showed high 16S rRNA gene sequence similarities to D. caseinilytica DHOB 09 T (98.7 %), D. flava DHOC 52 T (98.6 %), D. acidisoli 4M-Z03 T (98.5 %), D. choica 4 M-K27 T (98.3 %) and D. nitratireduens DHG 59 T (98.2 %); while strain 4 M-K27 T had high sequence similarities to D. acidisoli 4M-Z03 T (98.8 %), D. flagellata 4 M-K16 T (98.4 %) and D. nitratireduens DHG 59 T (98.3 %). Evolutionary trees based on 16S rRNA gene sequences (Fig. 1) showed that strain DHOA 06 T formed a highly supported clade with D. caseinilytica DHOB 09 T, and strain 4 M-K27 T formed a clade with D. flagellata 4 M-K16 T, D. acidisoli 4M-Z03 T, D. humi DHG 40 T and D. nitratireducens DHG 59 T. The maximum-likelihood phylogenetic tree (Fig. 2) based on the concatenated partial atpD, gyrB and lepA gene sequences confirmed that strains DHOA 06 T and 4 M-K27 T belong to the genus Dyella, but differ from any described species of the genus. The genomes of strains DHOA 06 T and 4 M-K27 T obtained in this study were approximately 4.22 and 5.49 Mbp in size, with contig nos. of 15 and 68, N50 of 1437205 and 3 00 329 bp, and genome coverage of 181× and 145×, respectively, which satisfied the criteria of genomic data for the taxonomy of prokaryotes [29]. The genomes of strains DHOA 06 T and 4 M-K27 T harbour 3749 and 4742 predicted mRNA, 48 and 46 tRNA, and 7 and 3 rRNA genes, respectively. The genome size, numbers of predicted protein, tRAN and rRNA genes etc. are comparable with those reported for other Dyella species. The DNA G+C contents of strains DHOA 06 T and 4 M- K27 T were 59.1 and 61.7 mol% based on total genome calculations, within the range of the genus Dyella.Characteristic 1 2 3 4 5 6 Motility ‒ ‒ ‒ ‒ ‒ + pH optimum 4.5–7.0 4.5–6.5 4.0–7.0 5.5–9.0 4.0–7.5 4.0–7.0 NaCl tolerance (%) 0–1.0 0–1.0 0–0.5 0–4.0 0–3.0 0–1.5 Enzymic activities: Oi -Mannosidase + + + + + ‒ β -Glucosidase + + ‒ + + + Naphthol-AS + ‒ + + + + -BI-phosphohydrolase Trypsin W ‒ ‒ ‒ ‒ ‒ Biochemical characteristics: Aesculin hydrolysis + ‒ ‒ + + + Arginine dihydrolase + + ‒ + + ‒ Glucose fermentation ‒ ‒ + ‒ ‒ + Nitrate reduction ‒ ‒ ‒ + + ‒ Urease + + ‒ + + ‒ Utilization of: Aesculin ferric citrate ‒ ‒ + + + + Amygdalin + ‒ ‒ ‒ ‒ ‒ Arbutin + ‒ ‒ ‒ ‒ ‒ D- Arabinose ‒ + ‒ ‒ ‒ + Cellobiose + ‒ ‒ ‒ + ‒ D- Fructose + ‒ + + + + D- Fucose ‒ + ‒ ‒ ‒ ‒ D- Galactose + + ‒ + + ‒ D- Gentiobiose ‒ ‒ ‒ ‒ + ‒ D- Lyxose ‒ + ‒ ‒ + ‒ Melibiose ‒ ‒ ‒ + + ‒ Raffinose ‒ ‒ ‒ ‒ + + Sucrose ‒ + ‒ ‒ + ‒ Trehalose ‒ + ‒ + + ‒ Turanose ‒ + ‒ ‒ ‒ ‒ D- Xylose + ‒ + + + ‒ Glycogen ‒ + + ‒ ‒ ‒ Inositol ‒ ‒ + ‒ ‒ ‒ L- Arabinose ‒ ‒ ‒ ‒ + ‒ L- Fucose ‒ ‒ + ‒ ‒ + Methyl Oi- D- glucopyranoside + ‒ ‒ ‒ ‒ ‒ Xylitol ‒ + ‒ ‒ ‒ ‒ DNA G+C content (mol%) 59.1 61.7 58.2 59.4 58.8 59.0Fig. 1. A 16S rRNA gene sequence-based maximum-likelihood tree showing phylogenetic relationships of strains DHOA06 T and 4 M- K27 T. Filled circles at nodes indicate generic branches that were also recovered using neighbour-joining and maximum-parsimony algorithms. Xanthomonas campestris ATCC 33913 T was used as an outgroup. Bootstrap values are shown in percentages of 1000 replicates; only bootstrap values Ȅ50 % are indicated. Bar, 0.01 substitutions per nucleotide position.Fig. 2. A maximum-likelihood tree based on the concatenated sequences of the housekeeping genes atpD, gyrB and lepA, showing the relationship of strains DHOA06 T and 4 M-K27 T to other species in the genus Dyella. Filled circles at nodes indicate generic branches that were also recovered using neighbour-joining and maximum-parsimony algorithms. Bootstrap values are shown in percentages of 1000 replicates; only bootstrap values Ȅ50 % are indicated. Xanthomonas campestris ATCC 33913 T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.Fatty acid methyl esters of strains DHOA 06 T, 4 M-K27 T and the related type strains were obtained from 40 mg cells grown on R 2A agar for 4 days at 28 Ǫ C [30]. The mixtures of fatty acid methyl esters were separated by GC and determined using Sherlock Microbial Identification System (MIDI, Microbial ID). Respiratory quinones were extracted and identified by HPLC according to the method described previously [31]. Polar lipids were extracted and then examined by two-dimensional TLC [32]. The major fatty acids of strain DHOA 06 T were iso-C 17: 1 Ɯ 9 c (24.8 %), iso-C 15: 0 (20.6 %), iso-C 16: 0 (16.4 %) and iso-C 17: 0 (11.5 %), while those of strain 4 M-K27 T were iso-C 15: 0 (19.1 %), iso-C 17: 1 Ɯ 9 c (16.9 %) and iso-C 16: 0 (12.5 %). The only respiratory quinone of both strains was ubiquinone-8. Their major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid, one unidentified phospholipid and two unidentified aminolipids, and strain DHOA 06 T had phosphatidylmethylethanolamine and one unidentified lipid in addition. (Fig. S1). The composition of fatty acids and the profiles of polar lipids of strains DHOA 06 T and 4 M-K27 T are all in line with the typical characteristics of the genus Dyella, but differ between themselves and from the other described species of the genus (Table 2, Fig. S1).The average nucleotide identity (ANI) values were calculated using the server-based software at www.ezbiocloud. net/tools/ani [33]. Digital DNA–DNA hybridization (dDDH) values were also determined online (http://ggdc. dsmz.de/distcalc2.php) using the Genome-to-Genome Distance Calculator as described by Meier-Kolthoff et al. [34]. To determine genomic relatedness between strains DHOA 06 T and 4 M-K27 T with other closely related phylogenetic members (without genome sequences), the fluorometric micro-well method [35, 36] was used for DNA– DNA hybridizations with eight replications for each hybridization reaction. The optimum hybridization temperature was 45 Ǫ C. The highest and lowest results obtained for each sample were deleted and the means of the remaining six results were converted to percentage DNA–DNA relatedness values [37]. The ANI and dDDH values between strains DHOA 06 T and 4 M-K27 T and its closely related members (with genome sequences) of the genus Dyella were in the range of 74.0–80.1 % and 20.0–23.9 % (Table S3), respectively, and DNA–DNA hybridization (DDH) rate between strain pairs DHOA 06 T / DHOB09 T and 4 M-K27 T / 4M-Z03 T was 27.4±2.4 % and 38.8±1.0 % (Table S4), respectively, which were all far below the cut-off points (95 % for ANI and 70 % for dDDH or DDH) for the delineation of genomic species [38].Based on phenotypic, phylogenetic and chemotaxonomic analyses, we propose that strains DHOA 06 T and 4 M-K27 T represent two novel members of the genus Dyella, namely Dyella dinghuensis sp. nov. (= KCTC 52129 T = NBRC 111978 T) and Dyella choica sp. nov. (= GDMCC 1.1189 T = LMG 30267 T).