Paenibacillus Sinensis sp. nov., a Nitrogen-fixing Species Isolated from Plant Rhizospheres

doi:10.21203/rs.3.rs-867873/v1

Download PDF

Research Article

Paenibacillus Sinensis sp. nov., a Nitrogen-fixing Species Isolated from Plant Rhizospheres

https://doi.org/10.21203/rs.3.rs-867873/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 31 Oct, 2021

Read the published version in Antonie van Leeuwenhoek →

You are reading this latest preprint version

Two strains HN-1^T and 39 were isolated from rhizospheres of different plants grown in different regions of PR China. The two strains exhibited high nitrogenase activities and possessed almost identical 16S rRNA gene sequences. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between the two strains were 99.9 and 93.8% respectively, suggesting that they belong to one species. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strains HN-1^T and 39 are the members of the genus Paenibacillus and both strains exhibited 99.5% similarity to Paenibacillus stellifer DSM 14472^T and the both strains represented a separate lineage from all other Paenibacillus species. However, the ANI of type strain HN-1^T with P. stellifer DSM 14472^T was 90.69, which was below the recommended threshold value (< 95–96% ANI). The dDDH showed 42.1% relatedness between strain HN-1^T and P. stellifer DSM 14472^T, which was lower than the recommended threshold value (dDDH < 70%). The strain HN-1^T contain anteiso-C_15:0 as major fatty acids and MK-7 as predominant isoprenoid quinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four aminophospholipids and an unidentified glycolipid. Unlike the most closely related P. stellifer DSM 14472^T, strain HN-1^T or 39 was positive for catalase reaction. Distinct phenotypic and genomic characterisations from previously described taxa support the classification of strains HN-1^T or 39 as representatives of a novel species of the genus Paenibacillus, for which the name Paenibacillus sinensis is proposed, with type strains HN-1^T (= CGMCC 1.18902, JCM 34620), and reference strain 39 (= CGMCC 1.18879, JCM 34616), respectively.

Agricultural Engineering

Agroecology

Genomic characterisations

Nitrogen-fixing bacteria

Paenibacillus sinensis sp. nov.

Rhizosphere of plant

The genus Paenibacillus was proposed by Ash et al. (1993) and its description was emended by Shida et al. (Shida et al. 1997). Some species of the genus Bacillus were transferred to the genus Paenibacillus (Ash et al. 1993; Heyndrickx et al. 1996; Shida et al. 1997; Lee et al. 2004; Hu et al. 2010), and further descriptions of novel members increased the number of species of the genus Paenibacillus. At the time of writing, the genus comprises 256 species and four subspecies with validly published names (www.bacterio.net/paenibacillus.html). Members of the genus Paenibacillus are rod-shaped, aerobic or facultatively anaerobic, spore-forming bacteria with anteiso-C_15:0 as the major cellular fatty acid and menaquinone 7 (MK-7) as the major menaquinone, and their DNA G + C contents range from 45 to 54 mol% (Ash et al. 1993; Priest 2009).

Species of the genus Paenibacillus have been isolated from diverse environment habitats (https://lpsn.dsmz.de/genus/paenibacillus) and have diverse physiological characteristics. Presently, there are only a few species with nitrogen fixation ability in these bacteria, including Paenibacillus borealis (Elo et al. 2001), Paenibacillus brasilensis (von der Weid et al. 2002), Paenibacillus graminis (da Mota et al. 2004), Paenibacillus massiliensis (Ding et al. 2005), Paenibacillus zanthoxyli (Ma et al. 2007a), Paenibacillus sabinae (Ma et al. 2007b), Paenibacillus forsythiae (Ma and Chen 2008), Paenibacillus donghaensis (Choi et al. 2008), Paenibacillus sonchi (Hong et al. 2009), Paenibacillus riograndensis (Beneduzi et al. 2010), Paenibacillus jilunlii (Jin et al. 2011a), Paenibacillus sophorae (Jin et al. 2011b), Paenibacillus stellifer (Jin et al. 2011c), Paenibacillus triticisoli (Wang et al. 2013a), Paenibacillus polymyxa (Wang et al. 2013b), Paenibacillus beijingensis (Gao et al. 2012), Paenibacillus taohuashanense (Xie et al. 2012), Paenibacillus brassicae (Gao et al. 2013), Paenibacillus bryophyllum (Liu et al. 2018), Paenibacillus helianthi (Ambrosini et al. 2018), Paenibacillus maysiensis (Wang et al. 2018), Paenibacillus rhizophilus (Ripa et al. 2019), Paenibacillus durus (Guella et al. 2019). Most of N₂-fixating Paenibacillus species isolated from plant roots have been shown to play an important role in promoting plant growth by nitrogen fixation, phosphate solubilization, production of plant phytohormones and various enzymes (Xie et al. 2016; Grady et al. 2016; Li et al. 2019). Comparative genomic analysis revealed the conservation of nif cluster comprising 9 genes (nifB nifH nifD nifK nifE nifN nifX hesA nifV) in nitrogen-fixating Paenibacillus strains (Xie et al. 2014). In addition, some N₂-fixing Paenibacillus strains with additional nif and nif-like genes exhibited higher nitrogenase activities (Li et al. 2014; Li et al. 2021). Such traits make the study of the regulation of the multiple nif genes of N₂-fixing Paenibacillus under different environmental conditions and their adaptation to varying ecological niches interesting.

In this study, two nitrogen-fixing strains HN-1 ^T and 39 isolated from the rhizosphere of plant were charactered by a polyphasic taxonomic approach, one more presumably novel species strain belonging to the genus Paenibacillus.

Isolation and culture conditions

Strain 39 was isolated from a soil sample collected from arbor rhizosphere in Beijing of China (39°57’N, 116°17’E). 1 g soil sample was suspended in 9-mL sterile water, stirred for 30 min and heated at 80°C for 15 min. After that, 100 µl suspension was spread on nitrogen-free medium agar plates in triplicate. After incubation at 30°C for 3 days, single colonies were isolated by streaking plating. The nitrogen-free medium consisted 20 g sucrose, 0.1 g K₂HPO₄, 0.4 g KH₂PO₄, 0.2 g MgSO₄·7H₂O, 0.1 g NaCl, 0.01 g FeCl₃, and 0.002 g Na₂MoO₄ per liter water. The strain HN-1^T was previously isolated from the rhizosphere of rice and on nitrogen-free medium agar plates (Liu et al. 2019). Strains were routinely cultured in LD medium (per liter contains 2.5 g NaCl, 5 g yeast, and 10 g tryptone) at 30°C for further identification and study. The type strain of the genus Paenibacillus, P. polymyxa DSM 36^T, P. sabinae DSM 17841^T, P. stellifer DSM 14472^T, P. zanthoxyli JH29^T, P. graminis RSA19, P. triticisoli BJ-18^T and P. durus ATCC 35681^T were obtained from our bacterial collection. Bacteria were freeze-dried or frozen using a sterile glycerol solution in cryogenic tubes to preserve the samples (30% v/v), and stored at − 80℃ and − 20℃.

NitrogenFixing Capability

To determine nitrogenase activity, strains HN-1 ^T and 39 and reference Paenibacillus strains, including P. polymyxa DSM 36^T, P. sabinae DSM 17841^T, P. stellifer DSM 14472^T, P. zanthoxyli JH29^T, P. graminis RSA19, P. triticisoli BJ-18^T and P. durus ATCC 35681^T were grown in 20 mL of LB broth medium in 50 mL flasks shaken overnight at 30°C. The cultures were collected by centrifugation, precipitations were washed three times with sterilized water and then resuspended in nitrogen-limited medium (per liter contains 10.4 g Na₂HPO₄, 3.4 g KH₂PO₄, 26 mg CaCl₂·2H₂O, 30 mg MgSO₄, 0.3 mg MnSO₄, 36 mg Ferric citrate, 7.6 mg Na₂MoO₄·2H₂O, 10 µg p-aminobenzoic acid, 5 µg biotin, 0.3 g glutamate and 4 g glucose). The nitrogenase activity was determined using the acetylene reduction assay and expressed as nmol C₂H₄ mg^− 1 protein h^− 1 as described previously (Wang et al. 2013b).

Genomic data analysis

Total DNA was extracted from strains HN-1 ^T and 39, evaluated by gel electrophoresis, and estimated using a NanoDrop 2000 (Thermo Scientific, MA, USA). The draft genome sequence was produced by using Illumina paired-end sequencing technology at the mega genomics. Assembly was conducted using SOAP de-novo v. 1.04 assembler (Li et al. 2008). Gene prediction was made using Glimmer v. 3.0 (Delcher et al. 2007). Annotation of protein-coding sequence was performed by using the Basic Local Alignment Search Tool (BLAST) against the COG, Kyoto Encyclopedia of Genes and Genomes (KEGG) databases and NCBI nr protein database.

Average nucleotide identity (ANI) were calculated in EZBioCloud [https://www.ezbiocloud.net/tools/ani] (Yoon et al. 2017a), using the algorithm published by Lee et al. (2016). Digital DNA–DNA hybridization (dDDH) values were computed at GGDC (Genome-to-Genome Distance Calculator) using GGDC 2.0 BLAST + and recommended formula 2 (Meier-Kolthoff et al. 2013).

Phylogenetic analysis

The 16S rRNA gene sequences of 39 were acquired from a PCR product using highly specific forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and universal reverse primer 1492R (5′-GGTTACCTTGTTACGACTT-3′). The DNA sequence obtained was compared to reference 16S rRNA gene sequences available in GenBank and EMBL databases obtained from the NCBI Genome Database using BLASTN software (Altschul et al. 1990) and the EzBioCloud server (https://www.ezbiocould.net) (Yoon et al. 2017b). The sequences of the gyrB gene were obtained from the genome of HN-1^T and 39 and other type strains. Multiple sequence alignments were analysed using CLUSTAL X (Thompson et al. 1997). The phylogenetic tree calculating evolutionary distance matrices was constructed by the neighbor-joining method (Saitou and Nei, 1987) using MEGA (version 7.0) (Kumar et al. 2016). Bootstrap analysis was conducted on 1000 replications (Felsenstein, 1985).

Phenotypic Characterization

Cell morphology was obtained by scanning electrical microscopy (SEM), after incubated on endospore-forming medium agar plate [yeast extract 0.07%, tryptone 0.1%, glucose 0.1%, (NH₄)₂SO₄, 0.02%, MgSO₄·7H₂O, 0.02%, K₂HPO₄, 0.1% (w/v), pH 7.2] for 72 h. The flagellation type was determined by transmission electron microscopy (TEM) after 48 h incubation of strain HN-1 on LD medium. Cell motility was evaluated in semi-solid (0.3% agar) LD medium after incubation at 30℃ for 24 h. Physiological and biochemical characteristics were determined in comparison with P. sabinae DSM 17841^T and Paenibacillus stellifer DSM 14472^T. Most physiological and biochemical tests, including activities of catalase and oxidase, nitrate reduction, hydrolysis of starch, aesculin and tween 20, production of dextrin and indole, methy red reaction, Voges-Proskauer reaction, lysozyme test and production of acid from fermentation of different substrates were performed according to Zhao et al. (2014). Temperature range for growth were determined after incubation at 4, 10, 15, 25, 28, 30, 37, 40 and 45°C on LD agar. The pH range for growth was determined in LD broth adjusted to pH 4.0–10.0 (using increments of 1.0 pH unit) by using HCl and NaOH buffers. Growth in the absence of NaCl and in the presence of 0, 0.2, 0.5, 1.0, 2.0, 3.0 and 4.0 % (w/v) NaCl was investigated by using LD broth. A spectroscopic method of monitoring turbidity at OD₆₀₀ was used to assess the growth at various temperature, pH values and NaCl concentration. The ability of strains to assimilate different substrates were tested using Biolog GEN III MicroPlate system (Biolog Microstation™, CA, USA) following the manufacturer’s instructions.

Chemotaxonomy

Strains were incubated in LD medium at 30°C for 2 days. The compositions of cellular fatty acid were analyzed according to the method described by Komagata and Suzuki (1987) using Sherlock Identification System (MIDI) (Sasser et al. 2005). Cellular menaquinones and respiratory quinones were extracted, purified, and analyzed by HPLC according to the method described by Collins (1980). Polar lipid was extracted by the method of Minnikin et al. (1979), and was identified by two-dimensional TLC as described by Collins et al. (1980).

NitrogenFixing Capability

Since bacteria in the soil sample were cultured in nitrogen-free medium on the purpose of isolating nitrogen-fixing strain, strain 39 is possible to have nitrogen-fixing capability. Strains HN-1^T isolated from the rhizosphere of rice was detected by acetylene reduction to have nitrogen-fixing capacity (Liu et al. 2019). To confirm genes encoding nitrogenase are contained in their genome, nifH gene was amplified and sequenced. However, the sequencing result of nifH fragment both displayed double peaks, which indicated that there were multiple nifH genes in their genome. Acetylene reduction assays were performed to verify the nitrogenase activity of HN-1^T and 39. As shown in Table 1, strains HN-1^T and 39 exhibited very high nitrogenase activity compared to other nitrogen-fixing Paenibacillus species, suggesting a high efficiency of the nitrogen fixation process.

Table 1

Nitrogenase activity of strains HN-1^T and 39 in comparison with some nitrogen-fixing species of the genus *Paenibacillus*
Strain	Nitrogenase activity [nmol C₂H₄ (mg protein h)⁻¹]
P. polymyxa DSM 36^T	1355.1 ± 152.4
P. stellifer DSM 14472^T	6099.5 ± 497.3
P. zanthoxyli JH29^T	6282.4 ± 307.7
P. graminis RSA19^T	4272.9 ± 207.9
P. sabinae DSM 17841^T	7749 ± 371.8
P. durus ATCC 35681^T	5511.5 ± 260.2
P. triticisoli BJ-18^T	743.6 ± 82.9
39	7160.3 ± 584.6
HN-1^T	6937.2 ± 625.1

Molecular Characterization

The newly generated 16S rRNA gene sequence of strain 39 were compared with sequences of bacterial type strains in the Genbank nucleotide database. Phylogenetic trees were inferred using the neighbour-joining and maximum-parsimony methods in the software MEGA7. As no significant differences were found among the phylogenetic trees obtained by the different methods used, only the trees constructed by using the neighbour-joining method are shown. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains HN-1^T and 39 clustered with species of the genus Paenibacillus and formed a monophyletic cluster with P. stellifer DSM 14472^T, as the three strains formed a separate phylogenetic branch within the genus Paenibacillus with a high bootstrap resampling value of 100 % (Fig. 1). The strain 39 shares 100% 16S rRNA gene sequence identity with strain HN-1^T. The strains HN-1^T or 39 showed the highest 16S rRNA gene sequence identity to P. stellifer DSM 14472^T (99.5%), followed by P. durus ATCC 35681^T (97.4%) and P. sabinae DSM 17841^T (97.0%).

Generally, 98.7% sequence identity on the 16S rRNA gene are considered to be within the same species (Kim et al. 2014). However, several reports have been published showing that Paenibacillus species with > 99 % 16S rRNA gene sequence similarity may not belong to the same species (Kamfer et al. 2017; Kim and Cha 2018; Ghio et al. 2019; Guella et al. 2019; Velazquez et al. 2020). Thus, housekeeping genes are now routinely used to complement the 16S rRNA gene analysis for species level determination (da Mota et al. 2004; Holmes et al. 2004; Rodriguez et al. 2019). Due to the low level of discrimination based on 16S rRNA gene between closely related species, the gyrB gene (coding for the b subunit of DNA gyrase) was used as an alternative phylogenetic marker (Wang et al. 2007). The gyrB genes were retrieved from the HN-1^T and 39 genomes. The gyrB gene clearly distinguishes HN-1^T and 39 from other Paenibacillus species with only 93.04% gene sequence identity to P. stellifer DSM 14472 (Fig. S1). Based on the 95–96% gyrB gene sequence similarity as the interspecies gap (Lee et al. 2008; Liu et al. 2013), strains HN-1^T and 39 could be assigned to novel species.

Phenotypic Characteristics

Strains HN-1^T and 39 were found to be Gram-positive, facultatively anaerobic, motile and rod-shaped. Colonies of strain HN-1^T and 39 grown on LD medium after 72 h of incubation at 30℃ were usually 0.8–1.2 mm in diameter, circular, moist, milky and convex. The transmission electron micrographs of type strain HN-1^T showed the presence of peritrichous flagella on cell surface (Fig. 2a). Strain HN-1^T produced ellipsoidal spores in swollen sporangia in the terminal region of the cell by scanning electron microscope (Fig. 2b).

In order to determine physiological and biochemical characteristics of HN-1^T and 39 in comparison with P. stellifer DSM 14472^T and P. sabinae DSM 17841^T, a series of tests were carried out following the proposed minimal standards for describing new taxa of facultatively anaerobic, endospore-forming bacteria (Logan et al. 2009). The ability of strains to assimilate different substrates were tested using GEN III microplates by Biolog system (Biolog Microstation TM, CA, USA) (Kiran et al. 2017; Ripa et al. 2019).

The strains HN-1^T and 39 grew well in up to 4% NaCl (w/v), however, strain P. stellifer DSM 14472^T tolerated only 3% NaCl. The pH range for growth was 5.0–9.0 and the temperature range for growth is 15–42°C. Strains HN-1^T and 39 was determined to be negative for the Voges–Proskauer reaction, and positive for the methyl red reaction. Strains HN-1^T and 39 were positive for catalase reaction and can produce acid from rhamnose and sorbitol, which differentiated HN-1^T and 39 from the most related P. stellifer DSM 14472^T. The physiological and biochemical characteristics of HN-1^T and 39 are shown in Table 2. In Biolog analysis, significant differences were observed in the metabolization of different substrates on Biolog GEN III microplate by strains HN-1^T and 39 from its closest type strain P. stellifer DSM 14472^T (Table 3). Strain HN-1^T and P. stellifer DSM 14472^T differed in the metabolization of D-Fucose, D-Maltose, 3-Methyl glucose, D-Sorbitol, Stachyose, Citric acid, α-Keto-butyric acid, Mucic acid, Methyl pyruvate, Gelatin, Inosine, D-Glucose-6-PO₄, Pectin, Aztreonam, Fusidic acid, Nalidixic acid, Vancomycin, Lithium chloride, Sodium bromate, Sodium lactate 1%, Rifamycin sv and Troleandomycin as a sole carbon source. Compared with type strains of closely related Paenibacillus species, strain HN-1^T and 39 exhibited nearly identical phenotypic characteristics.

Table 2

Differential phenotypic characteristics between strain HN-1^T, 39 and closely related strains.
Characteristic	HN-1^T	39	DSM14472^T	DSM 17841^T
Growth conditions
Temperature (℃)	15–42	15–42	15–42	15–42
pH	5–9	5–9	5–9	5–9
NaCl	0–4%	0–4%	0–3%	0–3%
0.001% lysozyme	-	-	-	-
Oxidase	-	-	-	-
Catalase	+	+	-	+
Voges-Proskauer	-	-	-	+
Methyl red reaction	+	+	+	+
Nitrate reduction	-	-	-	+
Hydrolysis of:
Starch	+	+	+	-
Tween 20	-	-	-	-
Tyrosine	-	-	-	-
Esculin	+	+	+	+
Production of:	-	-	-	-
Indole
Dihydroxyacetone	-	-	-	-
Dextrin	+	+	+	-
Production of acid from:
Glucose	+	+	+	+
Fructose	+	+	+	+
Rhamnose	+	+	-	-
Glycerol	-	-	-	-
Trehalose	+	+	+	+
Arabinose	+	+	+	-
Mannitol	-	-	-	+
Xylose	+	+	+	-
Sorbitol	+	+	-	+
Maltose	+	+	+	+
Starch	+	+	+	+
Strains: 1. HN-1^T; 2. 39; 3. P. stellifer DSM 14472^T; 4, P. sabinae DSM 17841^T; +. Positive reaction; 2. negative reaction.

Table 3

Phenotypic fingerprinting of 1. strain HN-1^T; 2. strain 39; 3. *P. stellifer* DSM 14472^T; 4. *P. sabinae* DSM 17841^T by oxidation of sole substrates on Biolog GEN III MicroPlates (All data from present study generated under the same conditions).
substrate	1	2	3	4		1	2	3	4
Carbohydrates:					Esters:
n-Acetyl-β-D-mannosamine	-	-	-	-	D-Lactic acid methyl ester	-	-	-	-
n-Acetyl-D-galactosamine	-	-	-	-	Methyl pyruvate	w	w	-	+
n-Acetyl-D-glucosamine	-	-	-	-	Alcohols:
n-Acetyl neuraminic acid	-	-	-	-	Glycerol	-	-	-	-
D-Arabitol	-	-	-	-	Amides:
α-D-Glucose	+	+	+	+	Glucuronamide	w	w	w	-
α-D-Lactose	+	+	+	-	Amino acids:
D-Cellobiose	+	+	+	+	L-Alanine	-	-	-	-
D-Fructose	+	+	+	+	L-Arginine	-	-	-	-
D-Fucose	-	-	w	-	L-Aspartic acid	-	-	-	-
D-Galactose	+	+	+	+	D-Aspartic acid	-	-	-	-
D-Gentiobiose	+	+	+	+	Gelatin	w	w	-	-
β-Methyl-D-glucoside	+	+	+	+	Glycyl-l-proline	-	-	-	-
D-Maltose	+	+	+	+	L-Glutamic acid	-	-	-	-
D-Mannitol	-	-	-	+	L-Histidine	-	-	-	-
D-Mannose	+	+	w	+	L-Pyroglutamic acid	-	-	-	-
D-Melibiose	w	w	+	+	L-Serine	-	-	-	-
3-Methyl glucose	+	+	w	+	D-Serine	-	-	-	-
D-Raffinose	+	+	+	+	Nucleosides
D-Salicin	+	+	+	+	Inosine	w	w	-	-
D-Sorbitol	+	+	-	+	Phosphorylated compounds:
D-Trehalose	+	+	+	+	D-Fructose-6-PO₄	w	w	w	-
D-Turanose	+	+	+	+	D-Glucose-6-PO₄	w	w	-	-
L-Fucose	w	w	w	-	Polymers:
L-Rhamnose	w	w	w	-	Dextrin	+	+	+	+
Stachyose	+	+	w	+	Pectin	+	+	w	+
Sucrose	+	+	+	+	Tween 40	-	-	-	-
Carboxylic acid:					Antibiotics:
Acetic acid	-	-	-	-	Aztreonam	+	+	w	+
Acetoacetic acid	w	w	w	+	Fusidic acid	w	w	-	+
γ-Amino-butryric acid	-	-	-	-	Lincomycin	-	-	-	+
Bromo-succinic acid	-	-	-	-	Minocycline	-	-	-	+
Citric acid	w	w	-	-	Nalidixic acid	+	+	-	+
Formic acid	-	-	-	-	Rifamycin sv	-	-	+	+
D-Galacturonic acid	w	w	w	-	Troleandomycin	-	-	-	+
D-Gluconic acid	-	-	-	+	Vancomycin	+	+	w	+
D-Glucuronic acid	w	w	w	-	pH 5	w	w	w	w
L-Galactonic acid lactone	w	w	w	-	pH 6	+	+	+	+
α-Hydroxy-butyric acid	-	-	-	-	NaCl 4%	+	+	-	w
β-Hydroxy-D, L butyric acid	-	-	-	-	NaCl 8%	-	-	-	-
p-Hydroxy-phenylacetic acid	-	-	-	-	Guanidine HCl	-	-	-	-
α-Keto-butyric acid	w	w	-	w	Lithium chloride	+	+	w	+
α-Keto-glutaric acid	w	w	w	-	Myo-inositol	-	-	-	-
L-Lactic acid	-	-	-	-	Niaproof 4	-	-	-	-
D-Malic acid	-	-	-	-	Potassium tellurite	+	+	+	+
L-Malic acid	-	-	-	-	Sodium bromate	+	+	-	w
Mucic acid	w	w	-	-	Sodium butyrate	+	+	+	+
Propionic acid	-	-	-	-	Sodium lactate 1%	+	+	w	+
Quinic acid	-	-	-	-	Tetrazolium blue	-	-	w	w
D-Saccharic acid	-	-	-	-	Tetrazolium violet	w	w	+	w
+, Positive; −, negative; w, weak reaction

Chemotaxonomic Characteristics

In order to determine the composition of cellular fatty acid, HN-1^T and type strains P. stellifer DSM 14472^T and P. sabinae DSM 17841^T were incubated in LD medium at 30°C for 2 days. Whole cell fatty acid analysis revealed that anteiso-C_15:0, C_{14: 0}, C_{16: 0}, iso-C_{14: 0}, iso-C_{16: 0} and iso-C_{15: 0} are present as major (> 5 %) fatty acids, and anteiso-C15: 0, anteiso-C_{17: 0}, iso-C_{17: 0} and C_18:1ω9c are present as minor (< 5 but > 1 %) fatty acids (Table S1). Anteiso-C_15:0 is the predominant fatty acid of members of the genus Paenibacillus (Ash et al. 1993), consistent with strain HN-1^T (41.14%) being a member of this genus. However, in the closely related type strains P. stellifer DSM 14472^T, the fatty acid C_16:0 was found to be more abundant than anteiso-C_15:0. The major menaquinone of HN-1^T was MK-7, in conformity to genus Paenibacillus. The polar lipids detected by two-dimensional TLC are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), four aminophospholipids (APL) and unidentified glycolipid (Fig. S1), in agreement with the profile of genus Paenibacillus.

Genome sequence and similarity analysis

Genome sequencing was performed to evaluate the genomic relatedness of the strains HN-1^T and 39 to its closely related recognized species in the genus Paenibacillus. Genomes of strains HN-1^T and 39 were approximately 6.32 and 6.45 Mbp, respectively. The DNA G + C content of the strains HN-1^T and 39 were 53.36 and 52.99 mol%, respectively, which is within the range 39–59 mol% reported for the members of genus Paenibacillus (Im et al. 2017). The total number of protein coding genes in HN-1^T and 39 were 5631 and 5782, respectively. While, the related strain P. stellifer DSM 14472^T had a complete genome of 5.65Mb, comprising 4958 protein coding genes with a DNA G + C content of 55.6 mol% (Genbank: CP009286). More detailed genomic information of strains HN-1^T and 39 were shown in Supplementary Table S2 and high-quality draft genomes of strains HN-1^T and 39 were deposited in GenBank under accession numbers JAHCMB000000000 and JAHBAZ000000000, respectively.

In this study, digital DNA–DNA hybridization (dDDH) values were calculated using GGDC. The dDDH value of genomes for strain HN-1^T and strain 39 was 99.8%. The level of DNA–DNA relatedness was determined to be 42.1% by strain HN-1^T with P. stellifer DSM 14472^T, which is lower than the threshold value of 70 % for species delineation (Chun et al. 2018).

The average nucleotide identity (ANI) is widely used to define bacterial species (Konstantinidis and Tiedje 2005; Varghese et al. 2015; Chun et al. 2018; Ciufo et al. 2018). The ANI between the whole-genome sequence of HN-1^T and that of the most closely related species P. stellifer DSM 14472^T was calculated on ezbiocloud (Yoon et al., 2017a). The ANI value of genomes for HN-1^T and 39 was 99.93%, but the ANI values of strain HN-1^T were less than 91% with the related species P. stellifer DSM 14472^T, P. sabinae DSM 17841^TT and P. durus 35681^T (Table 4), lower than the threshold of 95–96% for differentiating bacterial species (Richter and Rossello´-Mo´ra 2009), suggesting that the new isolate HN-1^T represents a distinctive species.

Table 4

Pairwise genome comparisons between the strains HN-1^T and 39, and their close relatives
Query Genome	Reference genome	ANI (%)	dDDH (%)
HN-1^T	39	99.93	99.8
HN-1^T	P. stellifer DSM 14472^T (CP009286)	90.69	42.1
HN-1^T	P. sabinae DSM 17841^T (CP004078)	76.96	22.0
HN-1^T	P. durus ATCC 35681^T (CP011114)	76.80	22.0
39	P. stellifer DSM 14472^T (CP009286)	90.83	42.2
39	P. sabinae DSM 17841^T (CP004078)	77.01	22.2
39	P. durus ATCC 35681^T (CP011114)	76.78	22.2

The nitrogen fixation genes of strains HN -1^T and 39 were extracted by using Prokka software from the genome sequences (Seemann 2014). The genome of strains HN-1^T and 39 contain a compact nif cluster comprising ten genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, orf1, hesA and nifV encoding Mo-nitrogenase, which is unique features of the Paenibacillus nitrogen fixation system. In addition to the nif cluster, the two strains have anfHDGK encoding Fe-nitrogenase and linked to additional copies of nifBENX genes, while the closely related species P. stellifer DSM 14472^T contains anfHDGK preceding additional nifV gene. Beyond the nif and anf cluster, there are multiple nifHDK-like genes located at different sites in their genomes. The organization of nif, anf and nif-like genes in type strain HN-1^T and the closely related species P. stellifer DSM 14472^T was shown in Fig. S3. Previous studies showed that 3 nifH genes of P. sabinae DSM 17841^T are functional by complementing K. oxytoca ΔnifH mutant (Hong et al. 2012). Thus, the high nitrogenase activity exhibited by these strains may be due to their additional nif genes.

Paeniacillus durus ATCC 35681^T can fix nitrogen even in the presence of nitrate due to the absence of nitrate reductase (Seldin et al. 1984). Whole genome sequence analysis strains HN -1^T and 39 revealed that nitrate reductase gene cluster narIJHG were not detected, which suggested these two strains can also fix nitrogen in the nitrate-enriched medium. The draft genome of strains HN -1^T and 39 harbor two sets of NAD(P)H-nitrite reductases (nirBD) which are involved in the reduction of nitrite to ammonium in both assimilatory and dissimilatory reduction processes. Additional searches for genes associated with nitric oxide (nirS or nirK) and nitrous oxide reduction (norBC) were performed, but these genes were not detected in their genomes. Therefore, strains HN-1^T and 39 may possess dissimilatory nitrate reduction to ammonium pathway, but lack denitrification pathway.

The phenotypic, phylogenetic and genomic data of strains HN-1^T and 39 showed that they are different from all other closely related species of genus Paenibacillus. Therefore, we conclude that strain HN-1^T or 39 should be recognised as a novel species of the genus Paenibacillus, for which the name Paenibacillus sinensis sp. nov. is proposed.

Description of Paenibacillus sinensis sp. nov.

Paenibacillus sinensis (sin. en’sis. L.gen. n. sinensis of China, where the type strain HN-1^T was isolated).

Cells are Gram-positive, facultative anaerobic, rod-shaped (0.4–0.5 µm×2.0–3.2 µm) and motile by means of peritrichous flagella. In slightly swollen sporangia, an ellipsoidal spore is formed and located in terminal position of cells. Colonies on LD medium are circular, convex, cream white, with diameter 1.0–2.0 mm. Nitrogen fixation positive and multiple nifH genes are present. The growth temperature is 15–42°C, optimal at 30°C. The growth pH range is 5.0–9.0, optimal at pH 7.0. NaCl concentration of 0–4% (w/v) is tolerable for growth, optimal at 0–0.2%. Positive tests for catalase, methyl red test, starch and aesculin hydrolysis, but negative for oxidase, Voges–Proskauer reaction, nitrate reduction. The various substrates are assimilated examined using Biolog GEN III microplates: dextrin, D-maltose, D-trehalose, D-cellobiose, D-gentiobiose, sucrose, D-turanose, stachyose, D-raffinose, α-D-lactose, D-melibiose, β-Methyl-D-glucoside, D-salicin, α-D-glucose, D-mannose, D-fructose, D-galactose, 3-methyl glucose, 1% sodium lactate, D-serine, D-sorbitol and pectin were utilized. Strains are resistant to inhibitory chemicals: aztreonam, nalidixic acid, vancomycin, lithium chloride, potassium tellurite, sodium bromate, sodium butyrate, sodium lactate 1% and sensitive to troleandomycin, lincomycin, guanidine HCl, niaproof 4, tetrazolium blue. The major menaquinone is MK-7. The predominant fatty acid is anteiso-C_15:0. The major polar lipids are DPG, PE, and PG. The DNA G + C contents for strains HN-1^T and 39 are 53.36 and 52.99 mol%, respectively.

The type strain, HN-1^T (= CGMCC 1.18902, JCM 34620), was isolated from the rhizosphere soil of rice in Hunan P. R. China. The GenBank (EMBL) accession number for the 16S rRNA gene sequence of strain HN-1^T is MF967304 and the GenBank accession number for the draft genome sequence is JAHCMB000000000.

Funding

This work was supported by the the National Key Research and Development Program of China (No. 2019YFA0904700) and the National Natural Science Foundation of China (No. 32000048).

Conflict of interest

The authors declare no conflict of interest.

Availability of data and material

The GenBank accession numbers for 16S rRNA gene sequences of strains HN-1^T and 39 are MF967304 and MZ153121, repectively. The draft genome sequences of strains HN-1^T and 39 have been deposited at NCBI under the accession no.JAHCMB000000000 and JAHBAZ000000000.

Ethics approval

Not applicable.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215; 403-410
Ambrosini A, Sant'Anna FH, Heinzmann J, Fernandes GD, Bach E et al (2018) Paenibacillus helianthi sp. nov., a nitrogen fixing species isolated from the rhizosphere of Helianthus annuus L. Antonie Van Leeuwenhoek 111(12):2463-2471
Ash C, Priest FG, Collins MD (1993) Molecular identification of rRNA group 3 bacilli (ash, farrow, wallbanks and collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie Van Leeuwenhoek 64:253–260
Beneduzi A, Costa PB, Parma M, Melo IS, Bodanese-Zanettini MH et al (2010) Paenibacillus riograndensis sp nov., a nitrogen-fixing species isolated from the rhizosphere of Triticum aestivum. Int J Syst Evol Microbiol 60:128-133
Choi JH, Im WT, Yoo JS, Lee SM, Moon DS et al (2008) Paenibacillus donghaensis sp nov., a xylan-degrading and nitrogen-fixing bacterium isolated from East Sea sediment. J Microbiol Biotechnol 18(2):189-193
Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, Rooney AP, Yi H, Xu XW, De Meyer S et al (2018) Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 68: 461-466
Ciufo S, Kannan S, Sharma S, Badretdin A, Clark K, Turner S, Brover S, Schoch CL, Kimchi A, DiCuccio M (2018) Using average nucleotide identity to improve taxonomic assignments in prokaryotic genomes at the NCBI. Int J Syst Evol Microbiol 68:2386–2392
Collins MD, Goodfellow M, Minnikin DE (1980) Fatty acid, isoprenoid quinone and polar lipid composition in the classification of Curtobacterium and related taxa. J Gen Microbiol 118: 29-37
da Mota FF, Gomes EA, Paiva E, Rosado AS, Seldin L (2004) Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene. Lett Appl Microbiol 39: 34-40
Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679
Ding Y, Wang J, Liu Y, Chen S (2005) Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region. J Appl Microbiol 99(5):1271-1281
Elo S, Suominen I, Kampfer P, Juhanoja J, Salkinoja-Salonen M et al (2001) Paenibacillus borealis sp, nov., a nitrogen-fixing species isolated from spruce forest humus in Finland. Int J Syst Evol Microbiol 51:535-545
Felsenstein J (1985). Confidence -limits on phylogenies-an approach using the bootstrap. Evolution 39: 783-791
Gao M, Xie LQ, Wang YX, Chen J, Xu J et al (2012) Paenibacillus beijingensis sp nov., a novel nitrogen-fixing species isolated from jujube garden soil. Antonie Van Leeuwenhoek 102(4):689-694
Gao M, Yang H, Zhao J, Liu J, Sun YH et al (2013) Paenibacillus brassicae sp nov., isolated from cabbage rhizosphere in Beijing, China. Antonie Van Leeuwenhoek 103(3):647-653
Ghio S, Sauka DH, Ferrari AE, Piccini RE, Ontanon OM, Campos D (2019) Paenibacillus xylanivorans sp. nov., a xylan-degrading bacterium isolated from decaying forest soil. Int J Syst Evol Microbiol 69: 3818-3823
Grady EN, MacDonald J, Liu L, Richman A, Yuan ZC (2016) Current knowledge and perspectives of Paenibacillus: a review. Microb Cell Fact 15: 203
Guella F, Porto RZ, Sant'Anna FH, Ambrosini A, Passaglia LMP（2019）Genomic metrics analyses indicate that Paenibacillus azotofixans is not a later synonym of Paenibacillus durus. Int J Syst Evol Microbiol 69(9):2870-2876
Heyndrickx M, Vandemeulebroecke K, Scheldeman P, Kersters K, DeVos P, Logan NA, Aziz AM, Ali N, Berkeley RCW (1996) A polyphasic reassessment of the genus Paenibacillus, reclassification of Bacillus lautus (Nakamura 1984) as Paenibacillus lautus comb nov and of Bacillus peoriae (Montefusco et al 1993) as Paenibacillus peoriae comb nov, and emended descriptions of P. lautus and of P. peoriae. Int J Syst Bacteriol 46: 988-1003
Holmes DE, Nevin KP, Lovley DR (2004) Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov. Int J Syst Evol Microbiol 54: 1591-1599
Hong YY, Ma YC, Zhou YG, Gao F, Liu HC et al (2009) Paenibacillus sonchi sp nov., a nitrogen-fixing species isolated from the rhizosphere of Sonchus oleraceus. Int J Syst Evol Microbiol 59:2656-2661
Hong Y., Ma YC, Wu LX, Maki M, Qin WS, Chen SF (2012) Characterization and analysis of nifH genes from Paenibacillus sabinae T27. Microbiol Res 16: 596-601
Hu XF, Li SX, Wu JG, Wang JF, Fang QL, Chen JS (2010) Transfer of Bacillus mucilaginosus and Bacillus edaphicus to the genus Paenibacillus as Paenibacillus mucilaginosus comb. nov and Paenibacillus edaphicus comb. nov. Int J Syst Evol Microbiol 60: 8-14
Im WT, Yi KJ, Lee SS, Moon HI, Jeon CO, Kim DW, Kim SK (2017) Paenibacillus konkukensis sp nov., isolated from animal feed. Int J Syst Evol Microbiol 67: 2343-2348
Jin HJ, Zhou YG, Liu HC, Chen SF (2011a) Paenibacillus jilunlii sp nov., a nitrogen-fixing species isolated from the rhizosphere of Begonia semperflorens. Int J Syst Evol Microbiol 61:1350-1355
Jin HJ, Lv J, Chen SF (2011b). Paenibacillus sophorae sp. nov., a nitrogen-fixing species isolated from the rhizosphere of Sophora japonica. Int J Syst Evol Microbiol 61:767-771
Jin HJ, Tu R, Xu F, Chen SF (2011c) Identification of nitrogen-fixing Paenibacillus from different plant rhizospheres and a novel nifH gene detected in the P. stellifer. Microbiology 80(1):117-124
Kamfer P, Busse HJ, McInroy JA, Hu CH, Kloepper JW, Glaeser SP (2017) Paenibacillus rhizoplanae sp nov., isolated from the rhizosphere of Zea mays. Int J Syst Evol Microbiol 67: 1058-1063
Kim M, Oh HS, Park SC, Chun J (2014) Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64: 346-351
Kim YS, Cha CJ (2018) Paenibacillus translucens sp nov., isolated from tidal flat sediment. Int J Syst Evol Microbiol 68: 936-941
Kiran S, Swarnkar MK, Mayilraj S, Tewari R, Gulati A (2017) Paenibacillus ihbetae sp nov., a cold-adapted antimicrobial producing bacterium isolated from high altitude Suraj Tal Lake in the Indian trans-Himalayas. Syst Appl Microbiol 40: 430-439
Konstantinidis KT, Tiedje JM (2005) Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci USA 102:2567–2572
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870-1874
Lee F., Tien CJ, Tai CJ, Wang LT, Liu YC, Chern LL (2008) Paenibacillus taichungensis sp nov., from soil in Taiwan. Int J Syst Evol Microbiol 58: 2640-2645
Lee I, Kim YO, Park SC, Chun J (2016) OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66: 1100-1103
Lee JS, Pyun YR, Bae KS (2004) Transfer of Bacillus ehimensis and Bacillus chitinolyticus to the genus Paenibacillus with emended descriptions of Paenibacillus ehimensis comb. nov and Paenibacillus chitinolyticus comb. nov. Int J Syst Evol Microbiol 54: 929-933
Li Q, Zhang HW, Zhang LQ, Chen SF (2021) Functional analysis of multiple nifB genes of Paenibacillus strains in synthesis of Mo-, Fe- and V-nitrogenases. Microb Cell Fact 20(1):139-139
Li RQ, Li YR, Kristiansen K, Wang J (2008) SOAP: short oligonucleotide alignment program. Bioinformatics 24: 713-714
Liu XM, Li Q, Li YB, Guan GH, Chen SF (2019) Paenibacillus strains with nitrogen fixation and multiple beneficial properties for promoting plant growth. PeerJ 7: e744
Liu LH, Yuan T, Yang F, Liu ZW, Yang MY et al (2018) Paenibacillus bryophyllum sp. nov., a nitrogen-fixing species isolated from Bryophyllum pinnatum. Antonie Van Leeuwenhoek 111(12):2267-2273
Li XX, Deng ZP, Liu Z., Yan YL, Wang TS, Xie JB, Lin M, Cheng Q, Chen SF (2014). The genome of Paenibacillus sabinae T27 provides insight into evolution, organization and functional elucidation of nif and nif-like genes. BMC Genomics 15:723
Li YB, Li YL, Zhang HW, Wang MY, Chen SF (2019) Diazotrophic Paenibacillus beijingensis BJ-18 provides nitrogen for plant and promotes plant growth, nitrogen uptake and metabolism. Front Microbiol 10:1119.
Liu Y, Lai QL, Dong CM, Sun FQ, Wang LP, Li GY, Shao ZZ (2013) Phylogenetic diversity of the Bacillus pumilus group and the marine ecotype revealed by multilocus sequence analysis. PLoS One 8: e80097
Logan NA, Berge O, Bishop AH, Busse HJ, De Vos P, Fritze D, Heyndrickx M, Kampfer P, Rabinovitch L, Salkinoja-Salonen MS et al (2009) Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol 59: 2114-2121
Ma YC, Zhang J, Chen SF (2007a). Paenibacillus zanthoxyli sp nov, a novel nitrogen-fixing species isolated from the rhizosphere of Zanthoxylum simulans. Int J Syst Evol Microbiol 57:873-877
Ma YC, Xia ZQ, Liu XM, Chen SF (2007b) Paenibacillus sabinae sp nov., a nitrogen-fixing species isolated from the rhizosphere soils of shrubs. Int J Syst Evol Microbiol 57: 6-11
Ma YC, Chen SF (2008) Paenibacillus forsythiae sp nov., a nitrogen-fixing species isolated from rhizosphere soil of Forsythia mira. Int J Syst Evol Microbiol 58:319-323
Meier-Kolthoff JP, Auch AF, Klenk HP, Goker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14: 60
Minnikin DE, Collins MD, Goodfellow M (1979) Fatty acid and polar lipid composition in the classification of Cellulomonas, Oerskovia and related taxa. J Appl Bacteriol 47: 87-95
Priest FG (2009) Genus I, Paenibacillus. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds) The firmicutes, Bergey’s manual of systematic bacteriology, vol 2, 2nd edn. Springer, New York, pp 269–296
Richter M, Rossello-Mora R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A 106: 19126-19131
Ripa FA, Tong S, Cao WD, Wang ET, Wang TY, Liu HC, Gao JL, Sun JG (2019) Paenibacillus rhizophilus sp. nov., a nitrogen-fixing bacterium isolated from the rhizosphere of wheat (Triticum aestivum L.). Int J Syst Evol Microbiol 69: 3689-3695
Rodriguez M, Reina JC, Bejar V, Llamas I (2019) Paenibacillus lutrae sp. nov., a chitinolytic species Isolated from a river otter in castril natural park, Granada, Spain. Microorganisms 7:637
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425
Sasser M, Kunitsky C, Jackoway G, Ezzell JW, Teska JD, Harper B, Parker S, Barden D, Blair H, Breezee J et al (2005) Identification of Bacillus anthracis from culture using gas chromatographic analysis of fatty acid methyl esters. J AOAC Int 88: 178-181
Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30: 2068-2069.
Seldin L, Vanelsas JD, Penido EGC (1984) Bacillus azotofixans sp. nov. a nitrogen-fixing species from Brazilian soils and grass Roots. Int J Sys Bacteriol 34(4):451-456. 10
Shida O, Takagi H, Kadowaki K, Nakamura LK, Komagata K (1997) Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int J of Syst Bacteriol 47: 289-298
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882
Varghese NJ, Mukherjee S, Ivanova N, Konstantinidis KT, Mavrommatis K et al (2015) Microbial species delineation using whole genome sequences. Nucleic Acids Res 43:6761–6771
Velazquez LF, Rajbanshi S, Guan SH, Hinchee M, Welsh A (2020) Paenibacillus ottowii sp. nov. isolated from a fermentation system processing bovine manure. Int J Syst Evol Microbiol, 70(3):1463-1469
von der Weid I, Duarte GF, van Elsas JD, Seldin L (2002) Paenibacillus brasilensis sp nov., a novel nitrogen-fixing species isolated from the maize rhizosphere in Brazil. Int J Syst Evol Microbiol 52:2147-2153
Wang LT, Lee FL, Tai CJ, Kasai H (2007) Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group. Int J Syst Evol Microbiol 57: 1846-1850
Wang LY, Li J, Li QX, Chen SF (2013a) Paenibacillus beijingensis sp nov., a nitrogen-fixing species isolated from wheat rhizosphere soil. Antonie Van Leeuwenhoek 104(5):675-683
Wang L., Zhang LH, Liu ZZ, Zhao DH, Liu X M, Zhang B, Xie JB, Hong YY, Li PF, Chen S.F et al. (2013b). A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli. PLoS Genet 9: e1003865
Wang TS, Xie JY, Wang LY, Chen SF (2018) Paenibacillus maysiensis sp nov., a nitrogen-fixing species isolated from the rhizosphere soil of maize. Curr Microbiol 75(10):1267-1273
Xie JB, Zhang LH, Zhou YG, Liu HC, Chen SF (2012) Paenibacillus taohuashanense sp nov., a nitrogen-fixing species isolated from rhizosphere soil of the root of Caragana kansuensis Pojark. Antonie Van Leeuwenhoek 102(4):735-741
Xie JB, Du ZL, Bai LQ, Tian CF, Zhang YZ, Xie JY, Wang TS, Liu XM, Chen X, Cheng Q et al (2014) Comparative genomic analysis of N₂-fixing and non-N₂-fixing Paenibacillus spp.: organization, evolution and expression of the nitrogen fixation genes. PLoS Genet 10: e1004231
Xie JB, Shi HW, Du ZL, Wang TS, Liu XM et al. Comparative genomic and functional analysis reveal conservation of plant growth promoting traits in Paenibacillus polymyxa and its closely related species. Sci Rep 2016; 6:21329.
Yoon SH, Ha SM, Lim J, Kwon S, Chun J (2017a) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110: 1281-1286
Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017b) Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67: 1613-1617
Zhao B, Lin H, He SJ (2014) Microbiology experiment, 2nd edn. Science Press, Beijing

Fig.S1.tif
Fig. S1 Neighbor-joining phylogenetic tree based on gyrB gene sequences showing the position of strains HN-1T and 39 among species of the genus Paenibacillus. Bootstrap analyses were performed with 1000 cycles. Only bootstrap values >50 % are shown at the branch points. Bar 0.01 substitutions per nucleotide positions.
Fig.S2.tif
Fig. S2 Two-dimensional TLC plate of polar lipids extracted from HN-1. The plate was sprayed with 10% (v/v) molybdophosphoric aicd to show all polar lipids present. PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PL, unidentified phosphoglycolipid; APL, aminophospholipid; GL, unidentified glycolipid.
Fig.S3.tif
Fig. S3 Organization of nif, anf and nif-like genes in type strain HN-1T and P. stellifer DSM 14472T.
TableS1.docx
TableS2.docx

Download PDF

Journal Publication

published 31 Oct, 2021

Read the published version in Antonie van Leeuwenhoek →

Reviews received at journal
15 Sep, 2021
Reviewers invited by journal
15 Sep, 2021
Editor assigned by journal
14 Sep, 2021
First submitted to journal
14 Sep, 2021

You are reading this latest preprint version

Paenibacillus Sinensis sp. nov., a Nitrogen-fixing Species Isolated from Plant Rhizospheres

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Isolation and culture conditions

NitrogenFixing Capability

Genomic data analysis

Phylogenetic analysis

Phenotypic Characterization

Chemotaxonomy

Result And Discussion

NitrogenFixing Capability

Molecular Characterization

Phenotypic Characteristics

Chemotaxonomic Characteristics

Genome sequence and similarity analysis

Description of Paenibacillus sinensis sp. nov.

Declarations

Funding

Conflict of interest

Availability of data and material

Ethics approval

References

Supplementary Files

Status:

Journal Publication

Version 1