Pseudonocardia lutea sp. nov., a novel actinobacterium isolated from soil in Chad

This dataset contains the digitized treatments in Plazi based on the original journal article Gao, Yuhang, Piao, Chenyu, Wang, Han, Shi, Linlin, Guo, Xiaowei, Song, Jia, Xiang, Wensheng, Zhao, Junwei, Wang, Xiangjing (2018): Pseudonocardia lutea sp. nov., a novel actinobacterium isolated from soil in Chad. International Journal of Systematic and Evolutionary Microbiology 68 (6): 1992-1997, DOI: 10.1099/ijsem.0.002780, URL: dx.doi.org novel actinomycete, designated strain NEAU-G57 T was isolated from a soil sample collected from the bottom of a river in Chad. A polyphasic approach was employed to determine the status of strain NEAU-G57 T. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that the organism should be assigned to the genus Pseudonocardia and formed a monophyletic clade with its closest relatives Pseudonocardia yuanmoensis YIM 75926 T (98.8 %), Pseudonocardia halophobica DSM 43089 T (98.2 %) and Pseudonocardia kujensis A 4038 T (97.6 %). Moreover, morphological and chemotaxonomic properties of strain NEAU-G57 T also confirmed the affiliation of the isolate to the genus Pseudonocardia. The cell wall contained meso -diaminopimelic acid and whole-cell sugars were glucose, xylose, arabinose and galactose. The predominant menaquinone was MK-8(H4). The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylmethylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol mannosides, one unidentified glycolipid and one unidentified lipid. The major fatty acids were C 16: 0 and C 16: 1 Ɯ 7 c. The DNA G+C content was 73.3 mol%. However, the low level of DNA – DNA relatedness and some phenotypic characteristics allowed the isolate to be differentiated from its closely related species. Therefore, it is concluded that strain NEAU-G57 T can be classified as representing a novel species of the genus Pseudonocardia, for which the name Pseudonocardia lutea sp. nov. is proposed. The type strain is NEAU-G57 T (= JCM 32387 T = CGMCC 4.7397 T).The genus Pseudonocardia within the family Pseudonocardiaceae was originally described by Henssen [1], and since then the description of the genus has been emended repeatedly [2 – 5]. At the time of writing, the genus encompassed 55 recognized species (bacterio.net), most of which have been described in the last decade. Members of the genus Pseudonocardia have the following common characteristics: a type IV cell wall; aerial mycelia may be present and substrate mycelium may fragment; the spores are normally smooth and form chains by acropetal budding or septation on the substrate or aerial mycelium; MK-8(H 4) or MK-9 are their major menaquinones; and iso-branched hexadecanoic acid is the predominant fatty acid. Mycolic acids are absent and the phospholipids are type II, III or IV. The DNA G+C content ranges from 68 to 79 mol%. Novel species of the genus Pseudonocardia have been isolated from diverse environments such as active sludge soils, including those polluted by chemical compounds [5, 6], wastewater activated sludge [7], plant samples [8, 9], soils [10, 11] and deep-sea sediments [12, 13]. During our investigation on the diversity of species from a soil sample collected from the bottom of a river in Chad, a novel strain, designated NEAU-G57 T, was isolated. In this study, we performed polyphasic taxonomy on this strain and proposed that it represents a new species of the genus Pseudonocardia, for which the name Pseudonocardia lutea sp. nov. is proposed.Strain NEAU-G57 T was isolated from the bottom soil of a river in Chad (13 Ǫ 38′ N, 15 Ǫ 22′ E). The soil sample (5 g) was suspended in distilled water (2 ml) followed by ultrasonic treatment (160 W) for 3 min. After the addition of distilled water (43 ml), the soil suspension was incubated at 28 Ǫ C and 250 r.p.m. on a rotary shaker for 30 min. Subsequently, a 200 µl sample of the suspension was spread on a plate of sodium succinate–asparagine agar medium (0.2 g asparagine; 1 g sodium succinate; 0.2 g CaCl 2 ·2H 2 O; 1 mg FeSO 4 ·7H 2 O; 0.3 g KCl; 0.9 g KH 2 PO 4; 0.6 g K 2 HPO 4 ·3H 2 O; 20 g agar; 1 l distilled water; pH, 7.2) supplemented with cycloheximide (50 mg l ‒1) and nalidixic acid (20 mg l ‒1). After 20 days of aerobic incubation at 28 Ǫ C, colonies were transferred and purified on International Streptomyces Project (ISP) 3 medium [14] and maintained as glycerol suspensions (20 %, v/v) at- -80 Ǫ C. The type strains of Pseudonocardia yuanmoensis, Pseudonocardia halophobica and Pseudonocardia kujensis were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and the Japan Collection of Microorganisms (JCM), respectively, and cultured under the same conditions for comparative analysis.Morphological characteristics were observed by light microscopy (Nikon Eclipse E200) and scanning electron microscopy (Hitachi SU8010) using cultures grown on ISP 2 agar at 28 Ǫ C for 4 weeks. Spore motility was assessed by light microscopic (Nikon Eclipse E200) observation of cells suspended in phosphate buffer (pH 7.0 1 mM). Cultural characteristics were determined after 2 weeks at 28 Ǫ C using ISP 1–7, nutrient agar (NA) and Bennett’ s agar (BA) [14 – 16]. Colour determination was done with colour chips from the ISCC-NBS colour charts [17]. Hydrolysis of Tweens (20, 40 and 80) and production of urease were tested as described by Smibert and Krieg [18]. The utilization of sole carbon and nitrogen sources, decomposition of cellulose, hydrolysis of starch and aesculin, reduction of nitrate, peptonization of milk, liquefaction of gelatin, and production of H 2 S were examined as described previously [19, 20]. Growth at different temperatures (5, 10, 15, 20, 25, 28, 30, 35, 40, 42 and 45 Ǫ C) was determined on ISP 2 medium after incubation for 14 days. The pH range for growth (pH 4–12, at intervals of 1 pH units) was tested in ISP 2 medium [21] using the buffer system described by Xie et al. [22], and NaCl tolerance was determined in ISP 2 medium supplemented with 1–10 % NaCl (w/v, with an interval of 1 % w/v) at 28 Ǫ C for 14 days on a rotary shaker.Biomass for chemotaxonomic studies was prepared by growing strain NEAU-G57 T in ISP 2 medium in shake flasks at 28 Ǫ C for 7 days. Cells were harvested by centrifugation, washed with distilled water and freeze-dried. The isomers of diaminopimelic acid in the cell wall were derivatized according to McKerrow et al. [23] and analysed by a high-performance liquid chromatography (HPLC) method using an Agilent TC-C18 column (250× 4.6 mm i.d. 5 µm). The whole-cell sugars were analysed according to the procedures developed by Lechevalier and Lechevalier [24]. Phospholipids in cells were examined by two-dimensional thin-layer chromatography and identified using the method of Minnikin et al. [25]. Menaquinones were extracted from freeze-dried biomass and purified according to Collins [26] and analysed by an HPLC–UV method as described previously [27]. To determine cellular fatty acid compositions, strain NEAU-G57 T was cultivated in ISP 2 medium for 7 days in shake flasks at 28 Ǫ C. Fatty acid methyl esters were extracted from the biomass as described by Gao et al. [28] and analysed by gas chromatography–mass spectrometry using the method of Xiang et al. [29].Extraction of chromosomal DNA and PCR amplification of the 16S rRNA gene sequence were carried out by using a standard procedure [30]. The PCR product was purified and cloned into the vector pMD19-T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL). The almost full-length 16S rRNA gene sequences of strain NEAU-G57 T were obtained and aligned with multiple sequences obtained from the GenBank/ EMBL/DDBJ databases using CLUSTAL_X 1.83 software. Phylogenetic trees were generated with the neighbour-joining [31] and maximum-likelihood [32] algorithms using molecular evolutionary genetics analysis (MEGA) software version 6.06 [33]. The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 1000 replicates [34]. A distance matrix was generated using Kimura’ s two-parameter model [35]. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). Pairwise alignment analysis of 16S rRNA gene sequence similarities between strains were calculated by using the EzBioCloud [36]. Strains NEAU-G57 T and its 32 closely related species were included in the phylogenetic trees.The G+C content of strain NEAU-G57 T was determined by using the thermal denaturation (Tm) method [37] with Escherichia coli JM109 DNA used as the control. DNA– DNA relatedness tests were carried out as described by De Ley et al. [38], using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6×6 multicell changer and a temperature controller with an in situ temperature probe (Varian) under the conditions and modifications described by Huss et al. [39]. The DNA samples used for hybridization were diluted to OD 260 around 1.0 using 0.1× SSC (saline sodium citrate buffer), then sheared using a JY92-II ultrasonic cell disruptor (ultrasonic time 3 s, interval time 4 s, 90 times). The DNA renaturation rates were determined in triplicate in 2× SSC at 70 Ǫ C with three replications and the DNA–DNA relatedness value was expressed as a mean value.Fig. 1. Scanning electron micrograph of strain NEAU-G57 T grown on ISP 2 agar for 4 weeks at 28 Ǫ C.Morphological observation of a 4-week-old old culture of strain NEAU-G57 T revealed that both aerial and substrate mycelia were abundant, well developed and fragmented. Chains of rod-shaped spores with smooth surfaces were produced on ISP 2 medium (Fig. 1). The strain showed good growth on ISP 2, Czapek’ s agar (CA) and BA media, moderate growth on ISP 1, ISP 3, ISP 4, ISP 6 and ISP 7 media, poor growth on ISP 5 and NA. The cultural characteristics of the strain are summarized in Table S1 (available in the online version of this article). Growth was observed at 20–45 Ǫ C (optimum, 28–37 Ǫ C), pH 6.0–8.0 (optimum, pH 7.0) and 0–5 % (w/v) NaCl (optimum, 0–3 %). Detailed physiological characteristics are presented in the species description.The cell walls of strain NEAU-G57 T contained mesodiaminopimelic acid and whole-cell sugars included glucose, arabinose, xylose and galactose. The phospholipid profile of the strain was found to consist of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylmethylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol mannosides, one unidentified glycolipid and one unidentified lipid (Fig. S1). The menaquinones of strain NEAU-G57 T were identified as MK-8(H 4) (79.0 %), MK-8(H 0) (12.1 %) and MK-8(H 2) (8.9 %), and the cellular fatty acids of strain NEAU-G57 T were C 16: 0 (59.8 %), C 16: 1 Ɯ 7 c (11.9 %), C 18: 0 (6.0 %), 10-methyl C 16: 0 (4.6 %), iso-C (4.5 %), anteiso-C 17: 0 (4.2 %), iso-C 15: 0 (4.1 %), 10-methyl C 18: 0 (3.0 %) and C 18: 1 Ɯ 5 c (1.9 %).17: 0EzBioCloud analysis of the 16S rRNA gene sequence showed that strain NEAU-G57 T should be assigned to the genus Pseudonocardia. The novel isolate exhibited the highest 16S rRNA gene sequence similarity to P. yuanmoensis YIM 75926 T (98.9 %), followed by P. halophobica DSM 43089 T (98.2 %) and P. kujensis A 4038 T (97.6 %), and the 16S rRNA gene sequence similarities between strain NEAU- G57 T and the type strains of other recognized species of the genus Pseudonocardia were less than 97.6 %. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain NEAU-G57 T formed a monophyletic clade with P. yuanmoensis YIM 75926 T, P. halophobica DSM 43089 T and P. kujensis A 4038 T (Fig. 2). This relationship was also observed in the maximum-likelihood tree (Fig. S2). DNA–DNA hybridization was employed to further clarify the relatedness between strain NEAU-G57 T and its related type strains. The DNA–DNA relatedness values between strain NEAU-G57 T and its closest strains, P. yuanmoensis YIM 75926 T, P. halophobica DSM 43089 T and P. kujensis A 4038 T, were 34.3±4.0 %, 40.8±5.8 % and 47.2 ±5.5 %, respectively, which was significantly lower than the threshold value (70 %) for the recognition of genomic species suggested by Wayne et al. [40], thus we suggest that strain NEAU-G57 T should be considered as a representative of a different genomic species to the genus Pseudonocardia.Fig. 2. Neighbour-joining tree showing the phylogenetic position of strain NEAU-G57 T and related species based on 16S rRNA gene sequences. Asterisks denote branches that were also recovered using the maximum-likelihood method. Bootstrap values>50 % (based on 1000 replications) are shown at branch points. Bar, 0.01 substitutions per nucleotide position.Besides the genotypic evidence above, strain NEAU-G57 T also had some other obvious differences from its closely related strains (Fig. S3, Table 1). Such as, the strain could be easily distinguished from its closely related species by cultural characteristics on ISP 5 and BA media (Fig. S3), hydrolysis of cellulose and starch, and patterns of carbon and nitrogen utilization (Table 1). Moreover, the isolate could grow with 6 % (w/v) NaCl. In contrast, P. yuanmoensis YIM 75926 T and P. kujensis A 4038 T could not. Therefore, it is evident from the genotypic and phenotypic data that strain NEAU-G57 T shoud be recognized as a novel species of genus Pseudonocardia, for which the name Pseudonocardia lutea sp. nov., is proposed.