Introduction

The genus Paenibacillus, belonging to the phylum Bacillota, was described by Ash et al. in 1993, with the type species Paenibacillus polymyxa (Ash et al. 1993; Tindall 2000). At the time of preparing the manuscript, this genus accommodates a total of 293 species with a validly published and correct names (https://lpsn.dsmz.de/genus/paenibacillus). The members of Paenibacillus have been reported from diverse sources including rhizosphere, phyllosphere, hot spring, root nodules, freshwater wetland, necrotic wound, soil, water, food, faeces, and insects (Baik et al. 2011; Glaeser et al. 2013; Menendez et al. 2016; Kämpfer et al. 2022; Wang et al. 2022). Recently, Paenibacillus rhizolycopersici (Thin et al. 2023) and Paenibacillus sabuli (Gao et al. 2022) have been retrieved from tomato plant and sea environment. Based on polyphasic approach, this study aimed to characterize and assign the taxonomic position of strain S3N08T, which was isolated from agricultural soil and proposed the name as Paenibacillus agricola sp. nov.

Materials and methods

Isolation of strain

Strain S3N08T was isolated from an agricultural soil (rice paddy field) sample collected from Seongju-gun, Republic of Korea (GPS coordinates: 35°53′56.8′′N 128°14′04.9′′E) using the standard dilution plating technique on R2A medium (MB Cell, South Korea). After plating, all the plates were incubated at 28 °C for 7 days. A white-pigmented colonies of bacterial strain, S3N08T was obtained after repeated streaking in R2A agar. The pure colonies of strain S3N08T were then stored at -80 °C as a suspension in R2A broth with 20% (w/v) glycerol for long-term preservation. The strain S3N08T was deposited in the Korean Agricultural Culture Collection and NITE Biological Resource Center.

16S rRNA gene sequence and phylogenetic analysis

Genomic DNA from strain S3N08T was extracted using commercial DNA extraction kit (InstaGene Matrix, Bio-Rad, USA). The 16S rRNA gene was amplified using forward (27F) and reverse (1492R) primers (Frank et al. 2008). The sequencing and analysis of amplified 16S rRNA gene was performed as described previously (Chaudhary et al. 2017). The nearest phylogenetic members were concluded by evaluating 16S rRNA gene sequence in the the EzBioCloud server (Yoon et al. 2017b). The 16S rRNA gene sequences of all the phylogenetically affiliated species were downloaded from EzBioCloud database and aligned with SINA (v1.2.11) according to the SILVA seed alignment (https://www.arb-silva.de) (Pruesse et al. 2012). The generation of phylogenetic trees was accomplished by maximum–likelihood (ML) (Felsenstein 1981) and neighbour–joining (NJ) (Saitou and Nei 1987) algorithms using MEGA X software (Kumar et al. 2018). The topology of the phylogenetic trees was evaluated by the bootstrap resampling method with 1000 replicates (Felsenstein, 1985). Evolutionary distances were determined by Kimura’s two-parameter model (Kimura 1980).

Genome analysis

The genome of strain S3N08T was sequenced by Illumina MiSeq platform and assembled by SPAdes ver. 3.14.1 (Macrogen, South Korea). The quality control and contamination of the sequenced genome were investigated using ContEst16S algorithm (Lee et al. 2017). The annotation of the assembled genome sequence was performed by Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016) and Rapid Annotation Subsystem technology (RAST server) (Aziz et al. 2008). The putative secondary metabolites were detected with the program antiSMASH 5.0 (Blin et al. 2019). The whole genome similarities between strain S3N08T and the closest members (Paenibacillus periandrae PM10T, Paenibacillus vulneris CCUG 53270T, Paenibacillus rigui JCM 16352T, Paenibacillus phytorum LMG 31458T, and Paenibacillus alginolyticus DSM 5050T) were assessed by average nucleotide identity (ANI) tool using OrthoANIu algorithm (Yoon et al. 2017a) and Genome-to-Genome Distance Calculator (Meier-Kolthoff et al. 2013). The phylogenomic tree was generated on the Type (Strain) Genome Server (Meier-Kolthoff and Göker 2019) using FastME 2.1.6.1 tools (Lefort et al. 2015).

Morphological, physiological, and biochemical analysis

For morphological studies, colony properties were determined by observing the colonies of strain S3N08T grown on R2A agar for 7 days at 28 °C. Cellular structure and flagella was visualized using transmission electron microscopy (TEM; Talos L120C; FEI). Gram reaction was performed using Color Gram 2 kit (bioMérieux). Endospore formation was observed by phase-contrast microscopy using BX53-DIC microscope (Olympus) after staining with 0.5% (w/v) malachite green (Oktari et al. 2017). Motility was evaluated in sulphide indole motility medium (SIM; Oxoid). Catalase and oxidase tests were conducted using ID Color Catalase and Oxidase Reagents (bioMérieux), respectively. The anaerobic growth was analysed by cultivating strain S3N08T on R2A agar for 15 days at 28 °C in an anaerobic jar with an anaerobe atmosphere generation bag. The growth at various temperatures (4, 10, 15, 20, 25, 28, 35, 37, 40, and 45 °C) was assessed on R2A agar after 10 days incubation. NaCl tolerance was investigated in R2A broth formulated with different NaCl content (0–1.5%, w/v, at 0.5% intervals). Growth at various pH 0.5–10.0 (at intervals of 0.5 pH unit) was studied in R2A broth. The pH of the medium was adjusted prior autoclaving using suitable buffers (Breznak and Costilow 2007). Casein, DNA, and starch hydrolysis tests were conducted as described (Smibert 1994). Other various biochemical studies were executed using commercial kits API ZYM, API 20NE, and API 20E (bioMérieux).

Chemotaxonomic characterization

The cellular fatty acids profile was determined after cultivating target and reference strains on R2A agar at 28 °C. Fatty acids were extracted from biomass of all strains harvested at late log phase. The extraction, analysis, and identification of fatty acids were accomplished by MIDI protocol (Sasser 1990). The peptidoglycan was analyzed as described previously (Staneck and Roberts 1974). Both polar lipids and quinones were analysed using freeze-dried cells (Collins and Jones 1981; Minnikin et al. 1984).

Results and discussion

The nearly full-length of 16S rRNA gene of strain S3N08T was 1481 bp (NCBI nucleotide accession number MH159222). The 16S rRNA gene sequence similarity data showed that the strain S3N08T was affiliated to the genus Paenibacillus, with phylogenetically closest members being P. periandrae PM10T (95.6%), P. vulneris CCUG 53270T (94.5%), P. rigui JCM 16352T (94.1%), P. phytorum LMG 31458T (93.1%), and P. alginolyticus DSM 5050T (92.3%). The 16S rRNA gene sequence similarities of strain S3N08T with all closest species were below the cut-off values of < 98.7% used for species delineation (Stackebrandt 2006; Yarza et al. 2008). Furthermore, the phylogenetic trees (ML and NJ) found that strain S3N08T formed a clade with P. periandrae PM10T with high bootstrap values (Figs. 1 and S1). Overall, the 16S rRNA gene sequence and phylogenetic results supported the assignment of strain S3N08T as a novel species in the genus Paenibacillus.

Fig. 1
figure 1

Maximum likelihood tree constructed using 16S rRNA gene sequences of strain S3N08T and closely affiliated taxa. Filled circles represent nodes recovered by both phylogenetic trees (maximum-likelihood and neighbor-joining). The numbers at the nodes indicate the percentage of 1,000 bootstrap replicates (values > 70% are only illustrated). NCBI nucleotide accession numbers are illustrated in parentheses. Geobacillus stearothermophilus IFO 12550T was used as an out-group. The scale bar represents 0.05 substitutions per nucleotide position

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The quality control and contamination test of genome sequence data assured that the generated genome sequence was valid to the strain S3N08T. The genome size of strain S3N08T was 8,375,108 bp. The entire genome sequence was assembled in 76 contigs with N50 value of 319,977 bp and genome coverage of 122.6x (Table S1). The annotation of genome executed in the RAST program revealed 316 subsystem features (Fig. S2). The annotated genome data showed various functional genes and proteins related to synthesis of plant growth promoting factors (Table S2). The genome of S3N08T comprised tryptophan synthase enzymes for auxin biosynthesis (GenBank accession numbers: NHN28712.1 and NHN28713.1) (Kriechbaumer and Glawischnig 2005), ammonium transporter for ammonia assimilation (GenBank accession numbers: NHN29230.1 and NHN31246.1), and iron-siderophore protein for iron acquisition (GenBank accession numbers: NHN30782.1 and NHN31881.1). These functional proteins of strain S3N08T help to enhance the plant growth activities (Grady et al. 2016). The biosynthetic gene clusters (BGCs) assessment revealed several genes in the genome of strain S3N08T that are responsible for putative secondary metabolites (terpene, cyclic-lactone-autoinducer, and type III polyketide synthase) (Table S3).

The DNA G + C content analyzed from genome data was 45.1%. The ANI and digital DNA–DNA hybridization (dDDH) values between strain S3N08T and reference strains were in the ranges of 69.9–76.1% and 19.1–26.9%, respectively (Table S4). These genome relatedness data were below than the threshold values of ANI (95.0%) and dDDH (70.0%), indicating that the strain S3N08T differs genomically from closest species of the genus Paenibacillus (Wayne et al. 1987; Richter and Rosselló-Móra 2009). The phylogenomic tree generated with genome sequence data assured the affiliation of strain S3N08T with the genus Paenibacillus, forming clade with P. periandrae PM10T, P. phytorum LMG 31458T, and P. alginolyticus DSM 5050T, but generating separate lineage (Fig. S3).

Cells of strain S3N08T were Gram-stain-positive, and rod shaped with peritrichous flagella (Fig. S4). Catalase test was negative for S3N08T and P. periandrae LMG 28691T and positive for P. vulneris DSM 27954T and P. rigui KCTC 13282T. Oxidase test was positive for strain S3N08T and all reference strains except P. alginolyticus KACC 11445T. Strain S3N08T was able to grow at 10 °C and can tolerate 1.0% NaCl. Nitrate reduction was negative for S3N08T, P. periandrae LMG 28691T, P. vulneris DSM 27954T and P. alginolyticus KACC 11445T, but positive for P. rigui KCTC 13282T and P. phytorum LMG 31458T. Alkaline phosphatase and acid phosphatase were positive for strain S3N08T, whereas most of the other enzymatic and assimilation tests were negative. Leucine arylamidase, D-glucose, L-arabinose, and malic acid were negative for strain S3N08T, but were positive for all reference strains. Several other distinguishing phenotypic features of strain S3N08T are presented in the species description and illustrated along with closest reference strains in Table 1. The detail enzymatic and assimilation properties obtained from API commercial test kits are provided in Table S5.

Table 1 Differentiating features of strain S3N08T and phylogenetically closely related taxa
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The only menaquinone detected in strain S3N08T was menaquinone MK-7, which was consistent with other species of the genus Paenibacillus (Baik et al. 2011; Kämpfer et al. 2022). Phosphatidylmonomethylethanolamine (PME), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) were major polar lipids found in strain S3N08T. Additionally one unidentified phospholipid (PL1) was observed as minor polar lipid (Fig. S5). The polar lipids profiles were identical with other species of the genus Paenibacillus (Glaeser et al. 2013; Menendez et al. 2016). The predominant fatty acids of strain S3N08T were antiso-C15:0 (54.9%), C16:0 (13.2%), and iso-C15:0 (11.2%). The major fatty acids contents of strain S3N08T were similar with closely related reference strains. However, some proportional differences were observed with minor fatty acids composition between strain S3N08T and reference strains. Strain S3N08T reported C17:0 cyclo (1.2%) which was absent from reference strains (Table 2).

Table 2 Cellular fatty acid profiles (% of totals) of S3N08T and phylogenetically related references
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Taxonomic conclusion

In overall, the polyphasic taxonomic data provided in this study confirmed that the strain S3N08T represents a novel species in the genus Paenibacillus for which the name Paenibacillus agricola sp. nov. is proposed.

Description of Paenibacillus agricola sp. nov.

Paenibacillus agricola sp. nov. [a.gri′co.la. L. masc. n. ager field, L. suff. cola (from L. n. incola) a dweller, inhabitant, L. masc. n. agricola field dwelling].

Cells (3.3–4.5 × 0.7–0.9 µm) are Gram-stain-positive, aerobic, rod-shaped, and motile with flagella. Colonies on R2A agar are white, convex, circular, smooth, and translucent with 1.0–1.5 mm in diameter. Cells grow at temperature 10–40 °C (optimum, 25–28 °C), at pH 6.5–8.0 (optimum, 6.5), and at 0–1.0% NaCl concentration (optimum without NaCl). Endospores are formed in a sub-terminal position. Negative for catalase and positive for oxidase tests. Aesculin is hydrolysed, but gelatine, urea, casein, DNA, and starch are not hydrolysed. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase, naphthol-AS-BI-phosphohydrolase, and β-galactosidase. The cells assimilate d-maltose and produce acetoin. The only menaquinone is MK-7; principal polar lipids are PME, PG, and PE; and main cellular fatty acids are antiso-C15:0, C16:0, and iso-C15:0. The DNA G + C content of the type strain is 45.1%.

The type strain, S3N08T (= KACC 19666 T = NBRC 113430 T), was isolated from an agricultural soil in Seongju-gun, Republic of Korea.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA sequence and whole genome shotgun sequence of strain S3N08T are MH159222 and JAAOIW000000000, respectively.