Neobacillus paridis sp. nov., an endophyte of Paris polyphylla Smith var. yunnanensis

A novel endophytic strain, designated YIM B02564T, was isolated from the root of Paris polyphylla Smith var. yunnanensis obtained from Yunnan Province, southwest China. By using a polyphasic approach, cells of the strain were characterized as facultative anaerobic, Gram-positive and rod-shaped. The growth conditions of the strain were found to occur at 20–55 °C (optimum, 30 °C), pH 6.0–9.0 (optimum, pH 7.0). Strain YIM B02564T can tolerate 2% NaCl concentration. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM B02564T belonged to the genus Neobacillus and the 16S rRNA gene sequence similarity values of strain YIM B02564T to the type strains of members of this genus ranged from 95.6 to 97.8%. The DNA G+C content of strain YIM B02564T calculated from the whole genome sequence was 41.6 mol%. Values of the ANI and the dDDH between strain YIM B02564T and its closely related Neobacillus species were below 77.9% and 21.5%. Strain YIM B02564T contained MK-7 as the major menaquinone, iso-C15:0 and anteiso-C15:0 as the major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid and four unidentified lipids. It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. On the basis of polyphasic analysis, strain YIM B02564T could be differentiated genotypically and phenotypically from recognized species of the genus Neobacillus. The isolate therefore represents a novel species, for which the name Neobacillus paridis is proposed. The type strain is YIM B02564T (= JCM 34668T = CGMCC 1.18655T).


Introduction
The genus Neobacillus and other five genera were proposed by dividing the former genus Bacillus based on phylogenomic and comparative genomic approaches (Patel and Gupta 2020). At the time of writing, this genus harbors 16 valid published species isolated from diverse environmental habitats, including soil, human gun and plant roots (https:// lpsn. dsmz. de/ genus/ neoba cillus). During an investigation of endophytic bacterial communities associated with traditional Chinese medicinal plants in Yunnan Province, southwest China, strain YIM B02564 T was isolated from the surfacesterilized root of Paris polyphylla Smith var. yunnanensis. Its harvested rhizomes have become the indispensable component of more than 70 popular patented medicines for treatment of carbuncle, sore throat, venomous snake bites, and Communicated by Erko Stackebrandt. traumatic pain in China (Qin et al. 2018). In this work, based on a polyphasic approach, we describe the characterizations of a novel strain YIM B02564 T isolated from this medicinal plant and report that this strain represents a novel species of the genus Neobacillus.

Bacterial isolation and maintenances
Healthy roots of Paris polyphylla var. yunnanensis were collected from Shilin County, Yunnan Province, southwest China for further sterilizing and pulverizing before distribution on R2A medium which was described by Yang et al. (2016). During 10 days' incubation at 25 °C, the colonies obtained were re-streaked on the same medium until pure colonies were obtained and stored both on R2A slants at 4 °C and in 15% (v/v) glycerol at − 80 °C for long-term preservation.

16S rRNA gene sequencing and phylogenetic analysis
Genomic DNA of strain YIM B02564 T was extracted using a genomic DNA extraction kit (Tiangen, China) whose 16S rRNA was subsequently PCR-amplified with the forward primer 27F (5′-AGA GTT TGA TCC TGG CT-3′) and reverse primer 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). Amplified products were purified and cloned into vector pClone007 (TsingKe, China). This clone was sequenced using vector primers M13F (5′-TGT AAA ACG GCC AGT-3′) and M13R (5′-CAG GAA ACA GCT ATG ACC-3′). The 16S rRNA gene sequence of strain YIM B02564 T for analysis had a size of 1514 bp and was submitted to the Gen-Bank database. 16S rRNA sequence similarities to closely related type strains were calculated using EZBioCloud server (https:// www. ezbio cloud. net/) (Yoon et al. 2017). The sequences of closely related strains retrieved from GenBank and EZBioCloud were aligned using Clustal Omega (Sievers et al. 2011) and aligned sequences were used to constructed the phylogenetic tress with neighbor-joining (NJ) (Saitou and Nei 1987) method by Mega X software (Kumar et al. 2018). Phylogenetic distances were calculated with Kimura's two-parameter model (Kimura 1980). The bootstrap values were calculated based on 1000 replications (Felsenstein 1985). The sequence of Lysinibacillus boronitolerans NBRC 103108 T was selected as outgroup.

Whole genome sequencing and analysis
The Illumina platform was used to perform the wholegenome sequencing of strain YIM B02564 T , which was based in the China General Microbiological Culture Collection Center (CGMCC) as part of the Global Catalogue of Microorganisms (GCM) 10K project (Shi et al. 2021). The paired-end reads were de novo-assembled using SOAPdenovo 2.04 (Li et al. 2015). The DNA G+C content of strain YIM B02564 T was calculated from the whole genomic sequence. Average nucleotide identity (ANI) values, average amino acid identity (AAI) values and digital DNA-DNA hybridization (dDDH) values were respectively calculated using FastANI (Jain et al. 2018), online AAI calculator (http:// enve-omics. ce. gatech. edu/ aai/), and DSMZ Genometo-Genome Distance Calculator web server (http:// ggdc. dsmz. de/ distc alc2. php) with formula 2 (Meier-Kolthoff et al. 2013). The closely related strains with YIM B02564 T were obtained from NCBI GenBank Database for phylogenomics analysis. Functional annotation was conducted using PROKKA (Seemann 2014). OrthoFinder (Emms and Kelly 2015) was used to infer the orthologous genes that were to be aligned by Clustal Omega (Sievers et al. 2011). Gblocks was used to select conserved blocks from the concatenated alignments. Based on the conserved blocks, a maximumlikelihood tree was constructed by IQ-TREE (Nguyen et al. 2015).

Morphology and physiology and biochemical analysis
After growth for 3 days in R2A medium at 30 °C, transmission electron microscope (JEM-2100, JEOL) was used to observe the cell morphology of the strain. The presence of endospores was examined according to Schaeffer-Fulton stain method after cell growth on 1/2 TSA plate for 1 week, then observed by light microscopy (DM2000, Leica). Motility was assayed using the soft (0.4%, w/v) agar stabbing technique (tube method) for 5 days. By incubating inoculated R2A plates in a bacteria incubator at 30 °C for 7 days, the best growing condition for this new species was determined. Therefore, the species was incubated at different temperature (10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 °C) and pH values (4.0-10.0, at intervals of 1.0 pH units). The buffer systems were prepared using Na-citrate, KH 2 PO 4 /NaOH and NaHCO 3 /Na 2 CO 3 for pH 4.0-5.0, pH 6.0-8.0 and pH 9.0-10.0, respectively (Tang et al. 2010). Growth of strain YIM B02564 T at different NaCl concentration (0-5.0%, w/v, at intervals of 0.5%) was tested in R2A plates. The growth of the strain under anaerobic conditions was assessed by incubating inoculated R2A plates in anaerobic workstation (Whitley A35, Don Whitley Scientific) at 30 °C for 7 days. The Gram reaction was carried out using 3% (w/v) KOH for cell lysis (Cerny 1978). Catalase activity was examined by investigating bubble production with 3% (v/v) H 2 O 2 . Oxidase activity was detected using API oxidase reagent (bioMérieux) according to the manufacturer's instructions. API ZYM, API 20NE, API 50CH (bioMérieux, France) and Biolog GENIII microplates (BIOLOG Inc., Hayward, CA, United States) were used to view the other biochemical characters and utilization of various substrates according to the manufacturer's protocols.

Chemotaxonomic characteristics
Several standard methods were applied to analyze the chemotaxonomic characteristics of strain YIM B02564 T . Biomass for fatty acid analysis was harvested from cultures grown on TSA medium at 25 °C for 2 days. The fatty acids were extracted using a standard MIDI protocol and identified by using the Sherlock Microbial Identification System (Sherlock version 6.1; MIDI database: TSBA6) following the manufacturer's instructions (Sasser 2001). Determination of the cell-wall diaminopimelic acid was performed according to the method described by Staneck and Roberts (1974). Menaquinone and polar lipids were extracted following the method described by Minnikin et al. (1984). Menaquinone was analyzed by a reversed-phase HPLC system (Agilent Technologies 1260 Infinity) with a C18 column (25 cm × 4.6 mm, 5 μm). Identification and analysis of polar lipids were performed by a two-dimensional TLC procedure on silica gel G60 plates and then 5% molybdatophosphoric acid, 0.2% ninhydrin and molybdenum blue were used to detect the lipids, aminolipids and phospholipids, respectively.

Molecular phylogenetic analysis
The complete 16S rRNA gene sequence (1514 bp) was obtained and submitted to GenBank under the accession number MW911620. The 16S rRNA gene sequence similarity values showed that strain YIM B02564 T had the highest similarity to Neobacillus fumarioli (97.8%), followed by Neobacillus mesonae (97.4%), Neobacillus soli (97.4%), Neobacillus endophyticus (97.3%) and Neobacillus drentensis (97.3%). The values between YIM B02564 T and Neobacillus species were less than the threshold for recognizing a novel species (98.7-99%) (Stackebrandt and Ebers 2006;Kim et al. 2014). The NJ phylogenetic tree based on 16S rRNA gene sequences also showed that strain YIM B02564 T was closely related to members of the genus Neobacillus and formed a cluster with N. fumarioli and N. endophyticus (Fig. 1). In conclusion, the phylogenetic analyses based on 16S rRNA gene sequences clearly suggested that the isolate can be considered a species of the genus Neobacillus.
The draft genome of strain YIM B02564 T had a total length of 4,629,313 bp with N50 length of 179,987 bp. 12 rRNA genes, 98 tRNA genes and 4449 protein-coding genes were predicted in the annotated result of strain YIM B02564 T . The G+C content of strain YIM B02564 T calculated from the genome was 41.6 mol%. To confirm the phylogenetic relationship of strain YIM B02564 T , a maximumlikelihood (ML) phylogenomic tree was constructed on the basis of 882 orthologous genes. YIM B02564 T formed a branch with N. mesonae FJAT-13985 T , and formed a broader distinct cluster which also contained N. fumarioli NBRC 102428 T and N. endophyticus BRMEA1 T (Fig. 2). The dDDH estimate values for YIM B02564 T were 21.5% with

Lysinibacillus boronitolerans NBRC 103108 T (AB681946)
Neobacillus thermocopriae SgZ-7 T (JX113681) .8% with N. endophyticus BRMEA1 T , which were clearly lower than the recommended cut-off value (70%) (Wayne et al. 1987). Additionally, the ANI values between YIM B02564 T and its closely related species N. mesonae, N. fumarioli and N. endophyticus were 77.9%, 76.1% and 75.6%, respectively. AAI values ranged from 63.4 to 77.8% between YIM B02564 T and other species in Neobacillus (Table S1). ANI (Richter and Rosselló-Móra 2009) and AAI (Konstantinidis and Tiedje 2005) values were also significantly lower than the threshold of 95-96% for describing prokaryote species. Detailed information for ANI, AAI and dDDH values are given in Table S1. The eleven specific conserved signature indels (CSIs) in protein sequences of strain YIM B02564 T were identical with the description of genus Neobacillus (Patel and Gupta 2020). Based on the analysis above, strain YIM B02564 T can represent a novel species of the genus Neobacillus.

Phenotypic and chemotaxonomic characteristics
Strain YIM B02564 T was found to grow on NA, TSA and R2A medium. Colonies grown on TSA medium ware found to be circular, cream-colored and smooth after 3 days of cultivation. Cells of strain YIM B02564 T was facultative anaerobic, Gram-positive, oxidase-negative, catalase-positive, rod-shaped (0.4-0.7 µm wide and 2.0-5.0 µm long) and motile with peritrichous flagella (Fig. S1). Ellipsoidal spores were formed at the subterminal position in cells. The optimum growth condition of strain YIM B02564 T occurred at 30 °C, pH 7.0 and the peak tolerance to NaCl concentration was 2%. The other results of physiological and biochemical analyses are summarized in the species description and supplementary Table S2, and the properties comparison of strain YIM B02564 T with other related species are listed in Table 1.

Description of Neobacillus paridis sp. nov.
Neobacillus paridis (pa'ri.dis. L. gen. n. paridis of Paris, a plant genus, from which the type strain was isolated).
The GenBank accession numbers for the 16S rRNA gene sequence of Neobacillus paridis YIM B02564 T is MW911620. The whole genome sequences have been deposited at Gen-Bank and GCM Type Strains Genome Database under accession JAESWB000000000 and GCM60020047, respectively.