Paenibacillus sinensis sp. nov., a nitrogen-fixing species isolated from plant rhizospheres

Two strains HN-1T 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 99.8%, respectively, suggesting that they belong to one species. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strains HN-1T and 39 are the members of the genus Paenibacillus and both strains exhibited 99.5% similarity to Paenibacillus stellifer DSM 14472T and the both strains represented a separate lineage from all other Paenibacillus species. However, the ANI of type strain HN-1T with P. stellifer DSM 14472T was 90.69, which was below the recommended threshold value (< 95–96% ANI). The dDDH showed 42.1% relatedness between strain HN-1T and P. stellifer DSM 14472T, which was lower than the recommended threshold value (dDDH < 70%). The strain HN-1T contain anteiso-C15:0 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 14472T, strain HN-1T or 39 was positive for catalase reaction. Distinct phenotypic and genomic characterisations from previously described taxa support the classification of strains HN-1T or 39 as representatives of a novel species of the genus Paenibacillus, for which the name Paenibacillus sinensis is proposed, with type strains HN-1T (=CGMCC 1.18902, JCM 34,620), and reference strain 39 (=CGMCC 1.18879, JCM 34,616), respectively.


Introduction
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).
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.

Materials and methods
Isolation of the bacterial strains 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 lL 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 K 2 HPO 4 , 0.4 g KH 2 PO 4 , 0.2 g MgSO 4 Á7H 2 O, 0.1 g NaCl, 0.01 g FeCl 3 , and 0.002 g Na 2 MoO 4 per liter water. The strain HN-1 T was previously isolated from the rhizosphere of rice on nitrogen-free medium agar plates . 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. azotofixans 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°C and -20°C.
azotofixans 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 Na 2 HPO 4 , 3.4 g KH 2 PO 4 , 26 mg CaCl 2 Á2H 2 O, 30 mg MgSO 4 , 0.3 mg MnSO 4 , 36 mg Ferric citrate, 7.6 mg Na 2 MoO 4 Á2H 2-O, 10 lg p-aminobenzoic acid, 5 lg biotin, 0.3 g glutamate and 4 g glucose). The nitrogenase activity was determined using the acetylene reduction assay and expressed as nmol C 2 H 4 mg -1 protein h -1 as described previously (Wang et al. 2013b).

Genome sequencing and analysis
The whole genomic DNA of the strains HN-1 T and 39 was extracted using the TIANamp Bacteria DNA Kit, 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.
The DNA sequence obtained was compared to reference 16S rRNA gene sequences available in the Genbank 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 maximum likelihood method (Felsenstein 1981) using MEGA (version 7.0) (Kumar et al. 2016). Bootstrap analysis was conducted on 1000 replications (Felsenstein 1985).

Phenotypic characterization
Colony shape and size of strains were observed after 72 h of incubation on LD medium at 30°C. For endospore staining, the strains grown on LD agar for 2 days at 30°C, following 7 days at 4°C was stained using schaeffer-fulton method (Mormak et al. 1985) and visualized by light microscopy. Cell morphology was also obtained by scanning electrical microscopy (SEM), after incubated on endospore-forming medium agar plate [yeast extract 0.07%, tryptone 0.1%, glucose 0.1%, (NH 4 ) 2 SO 4 , 0.02%, MgSO 4 Á7H 2 O, 0.02%, K 2 HPO 4 , 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°C 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 OD 600 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 TM , CA, USA) following the manufacturer's instructions.

Chemotaxonomic characterization
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).

Result and discussion
Bacterial isolation and acetylene-reduction assay The two strains were isolated from rhizospheres of different plants grown in different regions of PR China. The designated type strain HN-1 T was previously isolated from rhizosphere soil of rice collected from Xiangtan City, Hunan Province ); Strain 39 was isolated from rhizosphere soil of arbor collected from Haidian District of Beijing. Since bacteria in the soil sample were cultured in nitrogenfree medium on the purpose of isolating nitrogenfixing strain, strain 39 is possible to have nitrogenfixing capability. Strains HN-1 T isolated from the rhizosphere of rice was detected by acetylene reduction to have nitrogen-fixing capacity . 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.
Phylogenetic analysis of 16S rRNA gene and gyrB gene The almost-complete 16S rRNA gene sequence of strain 39 was obtained and used for initial BLAST searches of the GenBank database. Comparisons of 16S rRNA gene sequences revealed that strain 39 was shown to belong to the genus Paenibacillus and share 99.9% 16S rRNA gene sequence identity with strain HN-1 T . These two strains showed highest 16S rRNA gene similarity to P. stellifer DSM 14472 T (99.5%), followed by P. azotofixans ATCC 35681 T (97.1%) and P. sabinae DSM 17841 T (97.0%). According to EzBiocloud database, high level of similarities included 99.5% (P. stellifer DSM 14472 T ), 97.1% (P. azotofixans ATCC 35681 T ). Others were below 97%: 96.9% (P. bryophyllum L201 T ), 96.7% (P. albidus Q4-3 T ), 96.7% (P. apii 7124 T ), etc. Phylogenetic trees were inferred using the maximum-likelihood (ML) methods in the software MEGA7. 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).
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 T (Fig. S1). Based on the 95-96% gyrB gene sequence similarity as the interspecies gap Liu et al. 2013), strains HN-1 T and 39 could be assigned to novel species.
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 Mb, respectively.
The DNA G ? C content of the strains HN-1 T and 39 were 53.36 and 52.99%, respectively. 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 5.66 Mb, comprising 5007 protein coding genes with a DNA G ? C content of 53.5%. An overview of the genome sequences of strains HN-1 T and 39 and other genome sequences from related species was given in Table 2. The high-quality draft genomes of  (Chun et al. 2018;Richter and Rossello-Mora 2009), suggesting that the new isolate HN-1 T represents a distinctive species.

Analysis of nitrogen fixation and nitrogen metabolism genes
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. S2. Previous studies showed that 3 nifH genes of P. sabinae DSM 17841 T are functional by complementing K. oxytoca DnifH mutant (Hong et al. 2012). Thus, the high nitrogenase activity exhibited by these strains may be due to their additional nif genes. Paeniacillus azotofixans 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.

Phenotypic characteristics
Strains HN-1 T and 39 were found to be Gram-positive, facultatively anaerobic, motile and rod-shaped. Colonies grown on LD medium after 72 h of incubation at 30°C were usually 0.8-1.2 mm in diameter, circular, moist, milky and convex (Fig. S3a). Endospores were stained with malachite green and observed under light microscope (Fig. S3b). 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 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 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). 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, a-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. Strain HN-1 T and 39 exhibited nearly  Table 3 shows the phenotypic properties that distinguishes the novel strains HN-1 T and 39 from the other Paenibacillus species.
In summary, the phylogenetic, genomic, phenotypic and chemotaxonomic 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.
The type strain, HN-1 T (= CGMCC 1.18902, JCM 34,620), 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. 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, respectively. The draft genome sequences of strains HN-1 T and 39 have been deposited at NCBI under the accession no. JAHCMB000000000 and JAHBAZ000000000.

Declaration
Conflict of interest The authors declare no conflict of interest.