DOI: 10.15468/k8azgt
title: Paenibacillus shunpengii sp. nov., isolated from farmland soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Yang, You-Jian, Zhang, Yan-Ting, Chen, Guo-Qiang, Cheng, Dan, Qiu, Ji-Guo, He, Qin, He, Jian (2018): Paenibacillus shunpengii sp. nov., isolated from farmland soil. International Journal of Systematic and Evolutionary Microbiology 68 (1): 211-216, DOI: 10.1099/ijsem.0.002484, URL: http://dx.doi.org/10.1099/ijsem.0.002484AbstractA bacterial strain designated YYJ7-1 T was isolated from farmland soil in China and characterized using a polyphasic taxonomic approach. Cells of strain YYJ7-1 T were Gram-staining-positive, aerobic or facultatively anaerobic, rod-shaped, motile and endospore-forming. Growth occurred at 18–42 Ǫ C (optimum at 35 Ǫ C), at pH 6.0–8.0 (optimum at pH 7.5) and with 0.0–4.0 % NaCl (optimum with 0.5 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Paenibacillus and showed high levels of sequence similarity with respect to Paenibacillus provencensis 4401170 T (98.6 %) and Paenibacillus urinalis 5402403 T (98.4 %), while lower 16S rRNA gene sequence similarities were observed with all other type strains (97.0 %). However, strain YYJ7-1 T showed low DNA–DNA relatedness with P. provencensis 4401170 T 48.7±4.5 % (43.6±7.1 % in a reciprocal experiment), and P. urinalis 5402403 T 38.9±5.7 % (35.6 ±6.8 %). The major cellular fatty acids (&gt;10.0 %) of strain YYJ7-1 T were anteiso-C 15: 0, iso-C 16: 0 and anteiso-C 17: 0. The polar lipid profile consisted of phospholipids, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major isoprenoid quinone was MK-7. The DNA G+C content was 39.4 mol%. Based on these results, it is concluded that strain YYJ7-1 T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus shunpengii sp. nov. is proposed, with YYJ7-1 T (= ACCC 19965 T = KCTC 33849 T) as the type strain.The genus Paenibacillus, belonging to the family Paenibacillaceae, was proposed by Ash et al. [1] and emended by Shida et al. [2]. At the time of writing, the genus comprised 213 species with validly published names (http://www.bacterio. net/paenibacillus.html), which were isolated from various sources such as marine envoironments [3], soil [4], plants [5], food and human urine [6]. Typically, members of the genus Paenibacillus are Gram-stain-positive or -negative, rod shaped, endospore-forming and aerobic, facultatively anaerobic or strictly aerobic bacteria. The DNA G+C contents range from 36.9 to 52.8 mol% [7 – 10]. The major cellular fatty acid is anteiso-C 15: 0, the major menaquinone is MK-7 [11], and the major diamino acid in peptidoglycan is meso -diaminopimelic acid [1, 2, 12]. The present study was carried out to clarify the taxonomic position of a Paenibacillus -like bacterial strain by a polyphasic taxonomic approach.During our investigations on the biodiversity of bacteria in a farmland soil (GPS location: 27 Ǫ 53′ N 120 Ǫ 36′ E) in Wenzhou city, Zhejiang Province, China, a novel bacterium, designated YYJ7-1 T, was isolated using standard dilutionplating techniques on Luria–Bertani (LB) agar. The strain was preserved at 80 Ǫ C as a suspension in 20.0 % (v/v) ‒ glycerol. Paenibacillus provencensis CCUG 53519 T (=4401170 T) and Paenibacillus urinalis CCUG 53521 T (=5402403 T) were obtained from the Culture Collection, University of Göteborg, Sweden (CCUG). The two type strains were used as reference strains and evaluated together under identical experimental conditions to those for strain YYJ 7-1 T.For 16S rRNA gene sequencing, genomic DNA was extracted and purified according to standard procedures [13]. The 16S rRNA gene was amplified by PCR using one pair of universal primers, namely 27F (5′-AGAGTTTGA TCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′), as described previously [14]. Phylogenetic analysis using a housekeeping gene was performed; the RNA polymerase b subunit (rpoB) gene sequence of YYJ7- 1 T was amplified by PCR with primers rpo B1698f (5′-AACATCGGTTTGATCAAC-3′) and rpo B2041r (5′-GTTGCA TGTTGGTACCCAT-3′) [4]. The PCR product was purified using an agarose gel DNA extraction kit (TaKaRa) according to the manufacturer’ s instructions and was ligated to pMD19- T vector and sequenced by an automatic sequencer (3730; Applied Biosystems). The gene sequence was compared with the available DNA sequences from EzBio-Cloud [15] to examine the phylogenetic position of strain YYJ7-1 T. Alignment of sequences was performed by the CLUSTAL X program [16], and phylogenetic trees were reconstructed by the neighbour-joining [17], maximumlikelihood [18] and minimum-evolution methods [19] with Kimura’ s two-parameter calculation model in MEGA version 7.0 [20]. Bootstrap analysis was used to evaluate the tree topology by performing 1000 replications [18].Author affiliations: 1 Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China; 2 Suzhou Entry-Exit Inspection and Quarantine Bureau, Suzhou, Jiangsu 215021, PR China; 3 Laboratory Centre of Life Science, College of Life Science, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.*Correspondence: Qin He, qhe@njau.edu.cn; Jian He, hejian@njau.edu.cnKeywords: Paenibacillus shunpengii sp. nov.; farmland soil.†These authors contributed equally to this work.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YYJ7-1 T is KX900571.Five supplementary figures are available with the online version of this article.The length of the 16S rRNA gene sequence determined for strain YYJ7-1 T was 1512 bp. According to sequence similarity calculations, strain YYJ7-1 T belonged to the genus Paenibacillus of the family Paenibacillaceae. Strain YYJ7-1 T was most closely related to P. provencensis 4401170 T (98.6 % similarity) and P. urinalis 5402403 T (98.4 % similarity), and shared less than 97.0 % similarity with other type strains. In the phylogenetic tree reconstructed by using the neighbourjoining algorithm, strain YYJ7-1 T formed a subcluster with P. provencensis 4401170 T and P. urinalis 5402403 T with a robust bootstrap support (Fig. 1). This affiliation was also supported by the phylogenetic trees reconstructed by the maximum-likelihood (Fig. S1, available in the online version of this article) and minimum-evolution methods (Fig. S2). To further validate the phylogenetic affinity of the isolates, this study also analysed the rpoB sequence, which has been shown to be more discriminative than the 16S rRNA gene in distinguishing members of the genus Paenibacillus [21]. The rpoB gene fragment (371 bp) of strain YYJ7-1 T acquired shared 84.2 % sequence identity with that of Paenibacillus campinasensis KCTC 0364BP T, 83.1 % identity with Paenibacillus susongensis M327 T, Paenibacillus glucanolyticus DSM 5162 T, Paenibacillus borealis KK19 T and Paenibacillus curdlanolyticus DSM 10247 T, and less than 82.3 % identity with other members of the genus Paenibacillus. These data were below the 85.0 % cut-off value suggested for the genomic distinction of species based on rpoB gene sequence analysis [22]. The neighbour-joining (Fig. 2) and minimum-evolution (Fig. S3) trees based on rpoB gene sequences confirmed that strain YYJ7-1 T belonged to the genus Paenibacillus and formed a subclade with P. susongensis M327 T (KJ934388.1). The results based on rpoB gene analysis indicated that strain YYJ7-1 T represents a novel species of the genus Paenibacillus.Gram staining was performed according to the method described by Zhang et al. [10]. Cell morphology was observed during the exponential growth phase using transmission electron microscopy (H-7650; Hitachi). Endospore formation was determined using malachite green, as previously described by Schaeffer and Fulton [23]. Cell motility was tested by the hanging drop method [24]. Cells were Gram-stain-positive, aerobic or facultatively anaerobic, rod-shaped, motile, endospore forming, 0.4–0.6 µm in width and 0.8–1.2 µm in length (Fig. S4).Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing relationships between strain YYJ7-1 T and closely related species. The tree was rooted using the sequence of Cohnella xylanilytica MX15-2 T as the outgroup. Numbers at nodes represent bootstrap values (based on 1000 replicates). Filled circles indicate that the corresponding branches were also recovered using the maximum-likelihood and minimum-evolution algorithms. Bar, 0.01 substitutions per nucleotide position. GenBank accession numbers are shown in parentheses.Fig. 2. Neighbour-joining phylogenetic tree based on the rpoB gene sequences showing relationships between strain YYJ7-1 T and closely related species. The tree was rooted using the sequence of Brevibacillus brevis NBRC 100599 as the outgroup. Numbers at nodes represent bootstrap values (based on 1000 replicates). Bar, 0.05 substitutions per nucleotide position. GenBank accession numbers are shown in parentheses.Growth at various temperatures (16, 18, 20, 25, 30, 37, 40, 42, 45 and 50 Ǫ C), various salt concentrations [0–5.0 % (w/v) NaCl (using an increment of 0.5 %)] and various pH values (pH 4.0–10.0, using increments of 0.5 pH unit) was tested in LB broth with incubation for up to 2 days. For determination of salt tolerance, the NaCl concentration was adjusted in salt-free LB medium, and for determination of pH range, the medium was adjusted prior to sterilization using appropriate biological buffers [25]: citrate/Na 2 HPO 4 buffer, pH range 4.0–5.5; phosphate buffer, pH range 6.0–8.0; Tris buffer, pH range 8.5–10.0. Verification of the pH values after autoclaving revealed only minor changes. The strain grew in the presence of 0.0–4.0 % (w/v) NaCl (optimally at 0.5 %), at 18–42 Ǫ C (optimally at 35 Ǫ C) and at pH 6.0–8.0 (optimally at pH 7.5). Oxidase activity was tested by using oxidase reagent (bioḾerieux) according to the manufacturer’ s instructions. Catalase activity was tested as described by McCarthy et al. [26]. Catalase activity was positive but oxidase activity was negative. Substrate utilization, acid production and other physiological and biochemical tests were determined using API 20E, API 20NE, API 50 CH and API ZYM kits (bioḾerieux) following the manufacturer’ s protocols.Antibiotic sensitivity was assessed by measuring an inhibition zone of&gt;30.0 mm after 2 days of incubation with discs that contained the following antibiotics (µg per disc): erythromycin (10), penicillin (100), vancomycin (30), streptomycin (10), tetracycline (30), kanamycin (30), gentamicin (10), chloramphenicol (30), neomycin (30), lincomycin (2), rifampin (10), novobiocin (30), bacitracin (40), polymyxin B (300), or nalidixic acid (30). Cells were resistant to lincomycin (2), rifampin (10), novobiocin (30), nalidixic acid (30), bacitracin (40) and polymyxin B (300). The detailed antibiotic sensitivity and morphological, physiological and biochemical characteristics of strain YYJ7-1 T are summarized in the species description and in Table 1.To further clarify the taxonomic relationship of strain YYJ7-1 T with its closest phylogenetic neighbours, DNA– DNA hybridization was performed according to the instructions of the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche), using digoxin-labelled DNA probes and nitrocellulose [27]. Hybridization was performed with three replications, and the reciprocal experiments were examined. The level of DNA–DNA relatedness of strain YYJ7-1 T with P. provencensis CCUG 53519 T and P. urinalis CCUG 53521 T was 48.7±4.5 % (43.6±7.1 % in a reciprocal experiment) and 38.9±5.7 % (35.6±6.8 %), respectively. These values were below the 70.0 % cut-off point recommended for the assignment of strains to the same species [28], warranting that strain YYJ7-1 T represents a separate genomic species.Table 1. Characteristics of strain YYJ7-1 T and the type strains of closely related species of the genus PaenibacillusStrains: 1, YYJ7-1 T; 2, P. urinalis CCUG 53521 T; 3, P. provencensis CCUG 53519 T. All data were from this study. All strains are negative for reduction of nitrates to nitrites and assimilation of phenylacetic acid. +, Positive or resistant; ‒, negative.Characteristic 1 2 3 Isolation source Soil Human Human Gram stain + + + Growth temperature/ optimum (Ǫ C) 18–42/35 20–42/37 19–42/37 Growth pH range/optimum 6.0–8.0/ 5.5–8.0/ 6.5–8.0/ NaCl tolerance (%, w/v) 7.5 0–4 7.5 0–5 7.5 0–5 Lincomycin + ‒ ‒ Rifampicin + ‒ + Novobiocin + ‒ + Chloramphenicol ‒ ‒ + Neomycin ‒ – + Bacitracin + + ‒ Polymyxin B + + ‒ Acid production from: L- Aarabinose + ‒ + D- Xylose + ‒ + D- Galactose ‒ + + D- Fructose + + ‒ D- Mannitol + ‒ + N -Acetylglucosamine + ‒ + Arbutin + ‒ + Sucrose ‒ ‒ + Glycogen ‒ + ‒ Potassium gluconate + ‒ ‒ Enzyme activities (after 4 h) b- Galactosidase ‒ + + b- Glucosidase ‒ ‒ + Assimilation of: D- Glucose ‒ ‒ + L- Arabinose + ‒ + D- Mannose + ‒ ‒ D- Mannitol + ‒ + N -Acetylglucosamine + ‒ + Maltose + ‒ + Potassium gluconate + ‒ + Capric acid ‒ ‒ ‒Table 2. Cellular fatty acid profiles of strain YYJ7-1T and the type strains of related species of the genus Paenibacillus Strains: 1, YYJ7-1T; 2, P. urinalis CCUG 53521T; 3, P. provencensis CCUG 53519T. All data were obtained in this study. Values are percentages of total fatty acids; major fatty acids are shown in bold type; –, not detected.Fatty acid 1 2 3 C12:0 0.5 – – C14:0 0.5 – – iso-C14: 0 3.0 3.5 3.2 iso-C15: 0 3.4 4.3 3.2 anteiso-C15: 0 45.2 48.4 54.4 C15:0 – 7.2 3.4 iso-C16: 0 15.8 15.1 10.6 C16:1 Ɯ 11 c 0.6 0.9 3.5 C16:0 6.7 9.2 6.6 iso-C17: 0 3.4 2.6 2.5 C17:0 1.6 0.5 – anteiso-C17: 0 11.6 7.6 10.5 C18:0 1.8 – 0.6 C18:1 Ɯ 9 c 0.7 – –The G+C content (mol%) of genomic DNA was determined by reversed-phase HPLC according to the method of Mesbah et al. [29] with Escherichia coli K-12 used as a standard. The DNA G+C content of strain YYJ7-1 T was 39.4 mol%, a value within the range reported for strains belonging to the genus Paenibacillus (36.9– 52.8 mol%) [7 – 10].For chemotaxonomic determination, cells of strain YYJ7-1 T and its related strains were cultured in LB broth at 35 Ǫ C, harvested at the exponential growth phase, washed with distilled water and freeze-dried. The fatty acid profiles were determined according to the manufacturer’ s instructions for the Sherlock Microbial Identification System (MIDI) [30]. The fatty acid methyl esters were obtained from cells by saponification, methylation and extraction and separated in a gas chromatograph (6890 N; Agilent). Peaks were integrated automatically, and fatty acid names and percentages were determined using the MIDI Sherlock MIS system (Library: TSBA6; version, 6.0B). The cellular fatty acid profiles of strain YYJ7-1 T and the reference strains are shown in Table 2.The major fatty acids (&gt;10.0 %) of strain YYJ7-1 T were anteiso-C 15: 0 (45.2 %), iso-C 16: 0 (15.8 %) and anteiso-C 17: 0 (11.6 %). Previous reports also showed that anteiso-C 15: 0, iso-C 16: 0 and anteiso-C 17: 0 were the major fatty acids in members of the genus Paenibacillus [6, 9, 10]. Other fatty acids detected were C 16: 0 (6.7 %), iso-C 17: 0 (3.4 %), iso-C 15: 0 (3.4 %), C 18: 1 Ɯ 9 c (0.7 %), C 17: 0 (1.6), C 16: 1 Ɯ 11 c (0.6 %), C 14: 0 (0.5 %), iso-C 14: 0 (3.0 %), C 18: 0 (1.8 %) and C 12: 0 (0.5 %). However, there were some qualitative and quantitative differences in the proportions of some components between strains. Polar lipid determination was carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany), as described by Minnikin [31]. Like most members of the genus Paenibacillus, the polar lipid profile of strain YYJ7-1 T also included diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. In addition, it contained four other unidentified phospholipids (Fig. S5). Quinone was extracted according to the methods described by Collins et al. [32]. The HPLC apparatus used for analysis of quinones was as follows: a reverse-phase C18 column (100× 2.1 mm; particle size 1.8 µm; Agilent) was used, the mobile phase was methanol/acetonitrile/H 2 O (48: 2: 50, by vol.), the flow rate was 0.4 ml min ‒1 and the injection volume was 20 µl. The major respiratory quinone of the strain was ubiquinone MK-7, which is in consistent with the results reported by Wang et al. [11] for other members of the genus Paenibacillus.Therefore, on the basis of phylogenetic analysis, DNA– DNA hybridization, phenotypic differences and chemotaxonomic data, strain YYJ7-1 T represents a novel species within the genus Paenibacillus, for which the name Paenibacillus shunpengii sp. nov. is proposed.