DOI: 10.15468/k4brex
title: Nocardioides vastitatis sp. nov., isolated from Taklamakan desert soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Liu, Shao-Wei, Xue, Chun-Mei, Li, Fei-Na, Sun, Cheng-Hang (2020): Nocardioides vastitatis sp. nov., isolated from Taklamakan desert soil. International Journal of Systematic and Evolutionary Microbiology 70 (1): 77-82, DOI: 10.1099/ijsem.0.003718, URL: http://dx.doi.org/10.1099/ijsem.0.003718AbstractA Gram-stain-positive, aerobic, non-motile, rod-shaped actinobacterium, designated strain 21Sc5-5T, was isolated from a soil sample collected in the Taklamakan desert in Xinjiang Uygur Autonomous Region, PR China and investigated by using a polyphasic approach. Strain 21Sc5-5 T grew at 10–37 Ǫ C (optimum, 28–30 Ǫ C), pH 6.0–9.0 (optimum, pH 7.0) and in the presence of 0–3 % (w/v) NaCl (optimum, 0 %). Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain 21Sc5-5 T formed a distinct lineage within the genus Nocardioides and had the highest similarity to Nocardioides albidus THG-S11.7 T (97.30 %), followed by Nocardioides kongjuensis A2-4 T (97.22 %), Nocardioides nitrophenolicus NSP 41 T (97.15 %) and Nocardioides caeni MN 8 T (97.15 %). The results of chemotaxonomic analyses showed that the isolate possessed - LLdiaminopimelic acid as the diagnostic diamino acid of the peptidoglycan and MK-8(H 4) as the predominant menaquinone. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unidentified phospholipid and three unidentified lipids. The major fatty acids were iso-C and 10-methyl C. The genome length of strain 21Sc5-5 T 16: 0 18: 0 was 4.67 Mb containing 372 contigs and with a DNA G+C content of 70.4 mol%. On the basis of data from phylogenetic, phenotypic and chemotaxonomic analyses, strain 21Sc5-5 T represents a novel species of the genus Nocardioides, for which the name Nocardioides vastitatis sp. nov. is proposed. The type strain is 21Sc5-5 T (=JCM 33365 T =CGMCC 4.7608 T).The genus Nocardioides, a member of the family Nocardioidaceae within the suborder Propionibacterineae [1], was first described in 1976 by Prauser et al. with Nocardioides albus as the type species [2]. Since then, the number of described Nocardioides species has increased rapidly. At the time of writing, the genus comprises more than 100 species with validly published names (www.bacterio.net/nocardioides.html). In 2018 and 2019, eight Nocardioides species were reported, which were isolated from animal faeces [3], freshwater mud [4], karst cave [5], air-conditioning system [6], soil [7, 8] plant [9] and glacier [10]. Members of the genus Nocardioides are typically Gram-stain-positive, aerobic, motile or non-motile, and coccoid or rod-shaped. In some species, such as Nocardioides albus [2] and Nocardioides luteus [11], branched vegetative hyphae can also be observed. The chemotaxonomic markers characterizing the genus Nocardioides include LL- diaminopimelic acid as the diagnostic amino acid of in the cell-wall peptidoglycan, MK-8(H 4) as the predominant menaquinone, iso-C 16: 0 as the major fatty acid, and phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and/or phosphatidylinositol (PI) as the major polar lipids [2 – 12]. In this study, we describe a novel species of the genus Nocardioides, designated 21Sc5- 5 T, isolated from a soil sample collected in the Taklamakan desert.Strain 21Sc5-5 T was isolated from a soil sample at the rhizosphere of Inula salsoloides collected in the Taklamakan desert (38 Ǫ 41′ 40″ N 80 Ǫ 58′ 47″ E) in Xinjiang Uygur Autonomous Region, PR China. The soil sample was diluted serially in sterile distilled water, and then spread on Reasoner’ s 2A (R2A) agar (BD) supplemented with cycloheximide (45 mg l ‒1), nalidixic acid (25 mg l ‒1) and potassium dichromate (45 mg l ‒1). A colony of strain 21Sc5-5 T appeared after incubation at 28 Ǫ C for 8 weeks. The colony was selected carefully and then purified on International Streptomyces Project (ISP) 2 agar [13] using the serial streaking technique. The purified isolate was maintained at 4 Ǫ C on ISP 2 agar slants for short-term preservation and in aqueous glycerol suspensions (20 %, v/v) at- -80 Ǫ C for longterm preservation. To verify the evolutionary position of strain 21Sc5-5 T, four reference type strains, Nocardioides albidus CCTCC AB 2015297 T, Nocardioides kongjuensis CGMCC 4.6877 T, Nocardioides nitrophenolicus CGMCC 4.6873 T and Nocardioides caeni CGMCC 4.7043 T were obtained from the China Centre for Type Culture Collection (CCTCC) and China General Microbiological Culture Collection Centre (CGMCC).Author affiliations: 1 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China; 2 Jiamusi University, Heilongjiang 154007, PR China.*Correspondence: Cheng-Hang Sun, chenghangsun@hotmail.com or sunchenghang@imb.pumc.edu.cnKeywords: Nocardioides vastitatis; actinobacteria; taxonomy; strain 21 Sc 5-5 T.Abbreviations: AL, aminolipid; DPG, diphosphatidylglycerol; ISP, International Streptomyces Project; L, lipid; MK-8, menaquinone-8; NA, nutrient agar; PG, phosphatidylglycerol; PI, phosphatidylinositol; PL, phospholipid; R2A, Reasoner’ s 2A; TSA, tryptic soy agar.†These authors contributed equally to this work.The GenBank accession number for the 16S rRNA gene sequence of strain 21Sc5-5 T is MK787305. The draft genome sequences of strain 21Sc5-5 T, N. albidus CCTCC AB 2015297 T and N. caeni CGMCC 4.7043 T have been deposited at DDBJ/ENA/GenBank under the accession numbers SSXI00000000, VDMP00000000 and STGW00000000, respectively.One supplementary table and four supplementary figures are available with the online version of this article.Cultural characteristics of strain 21Sc5-5 T were tested on ISP 2, 3, 4, 5 and 7 agars [13], tryptic soy agar (TSA; BD), nutrient agar (NA; BD) and R2A agar. The Inter-Society Colour Council–National Bureau of Standards (ISCC–NBS) colour charts [14] were used to determine the colony colour. Cell morphology was observed by transmission electron microscopy (JEM-1400, JEOL) after cultures had been incubated on ISP 2 agar at 28 Ǫ C for 3 days. Cell motility was studied on ISP 2 swarming agar (0.4 % agar, w/v). The Gram-staining test was performed as described by Magee et al. [15]. Growth under anaerobic conditions was tested on ISP 2 agar at 28 Ǫ C for 14 days in a sealed jar containing Anaerocult A (Merck) as a reducing agent. Growth at different temperatures (4, 8, 10, 15, 20, 25, 28, 30, 37, 42 and 45 Ǫ C) and various NaCl concentrations (0, 1, 2, 3, 5, 7 and 10 %, w/v) were determined on ISP 2 agars for 14 days. The pH range for growth was measured in ISP 2 broth at various pH values (pH 4.0–12.0, at intervals of 1.0 pH unit) for 14 days. For the pH experiments, the different buffers were used as described by Xu et al. [16]. Catalase activity was determined by bubble production in 3 % (v/v) H 2 O 2. Oxidase activity was assessed by using 1 % (w/v) tetramethyl- p - phenylenediamine [17]. Hydrolysis of Tween 20, Tween 40 and Tween 80, production of H 2 S, and gelatin liquefaction were examined as described by Gonzalez et al. [18]. Hydrolysis of starch, carboxymethyl-cellulose and casein were tested using R2A agar as a basal medium, supplemented with certain concentrations of corresponding substrates as described by Kim et al. [19]. Sole carbon source utilization and acid production from carbon sources were tested by using Biolog GEN III MicroPlates and the API 50 CH (bio-Ḿerieux) system according to the manufacturer’ s instructions. Other biochemical characteristics and enzyme activities were tested by using the API 20NE and API ZYM Kits (bioḾerieux).Cells of strain 21Sc5-5 T were Gram-stain-positive, aerobic, non-spore-forming and non-flagellated rods (approximately 0.4–0.7×1.1–2.3 µm; Fig. S1, available in the online version of this article). Colonies grown on ISP 2 agar plates were circular, smooth, opaque and creamy white in colour after incubation for 5 days at 28 Ǫ C. No aerial mycelia formed. Growth occurred at temperatures ranging from 10 to 37 Ǫ C (optimum, 28–30 Ǫ C), at pH 6.0–9.0 (optimum, pH 7.0) and in the presence of NaCl concentrations of up to 3.0 % (w/v; optimum, 0 %). The other detailed physiological and biochemical characteristics of strain 21Sc5-5 T are given in the species description. The differences between strain 21Sc5-5 T and four reference strains are summarized in Table 1.Extraction of genomic DNA and PCR amplification of the 16S rRNA gene from strain 21Sc5-5 T were performed as described by Li et al. [20]. The purified PCR products were cloned using the pEASY-T1 Cloning kit (TransGen Biotech) according to the manufacturer’ s instructions, and sequenced by using an ABI PRISM 3730XL DNA Analyzer. The 16S rRNA gene sequence similarity values between strain 21Sc5- 5 T and the related species were obtained using the Nucleotide Similarity Search program in the EzBioCloud server (www.ezbiocloud.net/identify) [21]. Multiple alignments were made using the CLUSTAL_W tool in MEGA version 7.0 [22]. Phylogenetic trees were reconstructed using the neighbour-joining [23], maximum-parsimony [24] and maximum-likelihood [25] methods with MEGA version 7.0. Evolutionary distances were calculated by using Kimura’ s two-parameter model [26]. The topologies of the phylogenetic trees were evaluated by using the bootstrap method of Felsenstein [27] with 1000 repeats.The nearly full-length 16S rRNA gene sequence (1480 bp) of strain 21Sc5-5 T was obtained to determine its phylogenetic position. Comparative analyses of 16S rRNA gene sequences showed that strain 21Sc5-5 T was phylogenetically most related to the genus Nocardioides. The highest degrees of sequence similarity of strain 21Sc5-5 T were found to N. albidus THG-S11.7 T (97.30 %), N. kongjuensis A2-4 T (97.22 %), N. nitrophenolicus NSP 41 T (97.15 %) and N. caeni MN 8 T (97.15 %), respectively. Lower sequence similarities (&lt;97.0 %) were found to all other recognized species of the genus Nocardioides. The phylogenetic tree reconstructed based on the neighbour-joining algorithm showed that strain 21Sc5-5 T was within the radiation of the genus Nocardioides and formed a distinct phyletic line (Fig. 1). The overall topology of the neighbour-joining tree was similar to that of the maximum-likelihood and maximum-parsimony trees (Figs S2 and S3).For the comparison of genome relatedness, the whole genomes of strain 21Sc5-5 T, N. albidus CCTCC AB 2015297 T and N. caeni CGMCC 4.7043 T were obtained by extraction of total genomic DNA following the method of Marmur [28]. Genome sequencing were performed using the Illumina HiSeq X Ten platform with paired-end reads of 150 bp by the Oebiotech Company (Beijing, PR China). Sequenced reads were quality-filtered with software SOAPnuke [29] and assembled de novo with SPAdes (version 3.11.1) [30]. Contigs with length greater than 500 bp were annotated by the NCBI prokaryotic genome annotation pipeline. The genomic DNA G+C contents were calculated directly from the draft genome sequences. Draft genomes of N. kongjuensis CGMCC 4.6877 T and N. nitrophenolicus CGMCC 4.6873 T were directly retrieved from NCBI (www. ncbi.nlm.nih.gov/). The average nucleotide identity (ANI) values between strain 21Sc5-5 T and four reference strains were calculated using ChunLab’ s online ANI calculator (www.ezbiocloud.net/tools/ani) [21]. The estimated DNA– DNA hybridization (DDH) values were calculated using the Genome-to-Genome Distance Calculator (GGDC 2.1, http://ggdc.dsmz.de/distcalc2.php) [31] and Formula 2 was used as recommended for the calculation of DDH for incomplete genomes.Table 1. Comparison of the characteristics of strain 21Sc5-5 T and its closest phylogenetic neighboursStrains: 1, 21Sc5-5 T; 2, Nocardioides albidus CCTCC AB 2015297 T; 3, Nocardioides kongjuensis CGMCC 4.6877 T; 4, Nocardioides nitrophenolicus CGMCC 4.6873 T; 5, Nocardioides caeni CGMCC 4.7043 T. All data shown were obtained in this study unless indicated otherwise. The genomic DNA G+C contents were calculated directly from their draft genome sequences.All strains were positive for Gram-staining, catalase activity and hydrolysis of Tween 20, but negative for oxidase, arginine dihydrolase, urease, nitrate reduction, indole production, glucose fermentation, hydrolysis of starch, casein, carboxymethyl-cellulose and production of H2S. In API 20NE strips, all strains were negative for assimilation of caprate and phenylacetate. In API ZYM strips, all strains were positive for alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, acid phosphatase, a- glucosidase and b- glucosidase, but negative for lipase, a- chymotrypsin, a- galac- tosidase, b- glucuronidase, N -acetyl-b- D- glucosaminidase, a- mannosidase and a- fucosidase. +, Positive; ‒, negative; W, weakly positive; ND, not determined.Characteristic * 1 2 3 4 5 Isolation source Soil Soil a Soil b Wastewater c Wastewater d Cell morphology Rods Cocci a Rods, cocci b Rods, cocci c Rods, cocci d Motile ‒ a ‒ b ‒ + c d ‒ Colony colour Creamy white White Yellowish white Yellowish white White Temperature range for growth (optimum; Ǫ C) 10–37 (28–30) 20–35(28) a 10–40(30–37) b 15–40 (30) c 10–37(28–30) d pH range for growth (optimum) 6–9 (7) 6.0–7.5(7) a 5.5–9.0 (7–8) b 6–10 (8) c 6.0–9.5 (6.5–7.5) d NaCl range for growth (%, w/v; optimum) 0–3 (0) 0.5–3.0(2) a 0–5(0) b () c ND ND 0–1(0–0.5) d Hydrolysis of: Skimmed milk ‒ + + + + Aesculin ‒ W + W ‒ Gelatin ‒ + ‒ ‒ + Tween 40 + + + W W Tween 80 ‒ ‒ + ‒ ‒ Enzyme assays (API ZYM): Valine arylamidase ‒ W W W W Cystine arylamidase ‒ + ‒ + + Trypsin ‒ + + + ‒ Naphthol-AS-BI-phosphohydrolase + ‒ + + + b- Galactosidase + + ‒ W ‒ Assimilation of (API 20NE): D- Glucose ‒ + + + ‒ L- Arabinose ‒ ‒ + ‒ + D- Mannose ‒ ‒ ‒ + ‒ D- Mannitol + ‒ + + ‒ N -Acetyl-D- glucosamine ‒ + + + ‒ Maltose ‒ W + + ‒ Potassium gluconate ‒ + + + + Adipate ‒ ‒ + + ‒ Malate + ‒ + + ‒ Citrate ‒ ‒ ‒ + ‒ DNA G+C content (mol%) 70.4 71.9 71.4 68.2 71.3*a, Data from Singh et al. [39]; b, data from Yoon et al. [40]; c, data from Yoon et al. [41]; d, Data from Yoon et al. [42].The assembled draft genome of strain 21Sc5-5 T contained 372 contigs, with a total length of 4.67 Mbp, 4557 predicted genes, 9 rRNA operons and 45 tRNA operons. The ANI values between strain 21Sc5-5 T and N. albidus CCTCC AB 2015297 T, N. kongjuensis CGMCC 4.6877 T, N. nitrophenolicus CGMCC 4.6873 T and N. caeni CGMCC 4.7043 T were 79.8, 78.5, 83.9 and 79.1 %, respectively, which were much lower than the cut-off value of 95–96 % [32]. The digital DDH values between strain 21Sc5-5 T and N. albidus CCTCC AB 2015297 T, N. kongjuensis CGMCC 4.6877 T, N. nitrophenolicus CGMCC 4.6873 T and N. caeni CGMCC 4.7043 T were 22.7, 21.9, 29.0 and 21.7 %, respectively, which were much lower than the cut-off point of 70 % for the delineation of a novel species [33]. The genomic DNA G+C content of strain 21Sc5-5 T was calculated to be 70.4 mol%, which lies within the range of DNA G+C for the genus Nocardioides (64.9–74.0 mol%) [8] and showed differences with the four closely related species (Table 1). Combining all the genomic analyses, our results indicated that strain 21Sc5-5 T should represent a novel species of the genus Nocardioides.Fig. 1. Neighbour-joining phylogenic tree on the basis of 16S rRNA gene sequences showing the phylogenic position of strain 21Sc5- 5 T and related species. Luedemannella helvata 3-9(24) T was used as an outgroup. Numbers at nodes refer to bootstrap values (based on 1000 replicates; only values&gt;50 % are shown). Bar, 1 nt substitution per 1000 nt.Biomass used for chemotaxonomic investigations of polar lipids and menaquinones was obtained from cultures grown in ISP 2 broth at 28 Ǫ C at 180 r.p.m. for 5 days. Polar lipids were extracted and analysed by two-dimensional TLC as described by Minnikin et al. [34]. The solvent systems of the first and second dimension were chloroform/methanol/ water (64: 27: 5, by vol.) and chloroform/methanol/acetic acid/water (80: 15: 12: 4, by vol.), respectively. Menaquinones were isolated and purified according to the method of Collins et al. [35], and identified by HPLC coupled with a single quadrupole mass spectrometer (LCMS-2020, Shimadzu) as described by Guo et al. [36]. The diagnostic diamino acids in whole-cell hydrolysates were identified by TLC as described by Schleifer and Kandler [37]. For analyses of whole-cell fatty acids, cell mass of strain 21Sc5-5 T and the four reference strains were harvested from NA plates at 28 Ǫ C for 4--7 days, when the bacterial communities reached the late-exponential stage of growth [38]. The fatty acid methyl esters were prepared according to the standard protocol described by Sasser [38], analysed by using gas chromatograph (Agilent 7890B) coupled with the Sherlock Microbial Identifications System (version 6.2; MIDI), and identified by using the RTSBA 6 database.The predominant menaquinone of strain 21Sc5-5 T was MK-8(H 4), and LL- diaminopimelic acid was detected as a diagnostic diamino acid in the cell-wall peptidoglycan. These results were in accordance with those reported for the recognized species of the genus Nocardioides [2 – 12]. The polar lipids of strain 21Sc5-5 T comprised diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI), an unidentified phospholipid (PL) and three unidentified lipids (L1–L3; Fig. S4). This polar lipid pattern was quite similar to those of the most closely related strains N. albidus and N. kongjuensis, which contained DPG, PG, PI and PL in common, however, the presence/absence of some unidentified lipids or an aminolipid (in N. albidus) could be used distinguish strain 21Sc5-5 T from its two closest relatives. The major cellular fatty acids (&gt;10 % of the total) detected in strain 21Sc5-5 T were iso-C 16: 0 (33.1 %) and 10-methyl C 18: 0 (TBSA, 13.8 %), with minor components (&gt;5 % of the total) of C 16: 0 (6.7 %), iso-C 16: 1 (5.8 %), iso-C 16: 0 2-OH (5.8 %), C 18: 0 (5.4 %) and 10-methyl C 17: 0 (5.2 %), which was similar to the other Nocardioides strains but different in proportion (Table S1).The results of the phylogenetic and chemotaxonomic analyses supported that strain 21Sc5-5 T should be assigned to the genus Nocardioides. However, on the basis of the 16S rRNA gene similarities, levels of genomic DNA–DNA relatedness and ANI values, fatty acid compositions, polar lipid profiles as well as several phenotypic and biochemical characteristics (Table 1), strain 21Sc5-5 T can be readily differentiated from the phylogenetically closest relatives of the genus Nocardioides, suggesting that strain 21Sc5-5 T represents a novel species of the genus Nocardioides, for which the name Nocardioides vastitatis sp. nov. is proposed.