
==== Front
J Microbiol Biotechnol
J Microbiol Biotechnol
Journal of Microbiology and Biotechnology
1017-7825
1738-8872
The Korean Society for Microbiology and Biotechnology

39086223
10.4014/jmb.2404.04038
jmb-34-8-1636
Research article
Environmental Microbiology and Biotechnology (EMB)
Microbial Ecology and Diversity
Paenibacillus gyeongsangnamensis sp. nov., Isolated from Soil
Lee Hyosun 1
Chaudhary Dhiraj Kumar 2
Kim Dong-Uk 1*
1 Department of Biological Science, College of Science and Engineering, Sangji University, Wonju 26339, Republic of Korea
2 Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea
* Corresponding author E-mail: dukim@sangji.ac.kr
28 8 2024
17 6 2024
17 6 2024
34 8 16361641
23 4 2024
16 5 2024
31 5 2024
Copyright © 2024 by the authors. Licensee KMB
2024
https://creativecommons.org/licenses/by/4.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license
A Gram-stain-positive, aerobic, white-coloured, rod-shaped bacteria, designated as a strain dW9T, was isolated from soil. Strain dW9T was catalase-positive and oxidase-negative. Strain dW9T grew at temperature of 20–37°C and at pH of 5.0–7.0. Phylogenetic and 16S rRNA gene analysis indicated that strain dW9T belonged to the genus Paenibacillus with its closest relative being Paenibacillus filicis S4T (97.4% sequence similarity). The genome size of dW9T was 7,787,916 bp with DNA G+C content of 51.3%. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values of dW9T with its closest relatives were found to be <22.0% and <74.0%, respectively. The only respiratory quinone was MK-7, and the major fatty acids were antiso-C15:0 and iso-C16:0. Overall, the comprehensive taxonomic analysis revealed that strain dW9T met all the fundamental criteria to be classified as a novel species within the genus Paenibacillus. Accordingly, we propose the name Paenibacillus gyeongsangnamensis sp. nov., with the type strain dW9T (=KCTC 43431T =NBRC 116022T).

Paenibacillus gyeongsangnamensis sp. nov.
soil
Paenibacillaceae
taxonomy
phylogeny
==== Body
pmcIntroduction

The genus Paenibacillus was initially established by Ash et al. in 1993 for the taxonomic classification of 16S rRNA group 3 bacilli [1]. Subsequently, various species originally classified under the genus Bacillus were reassigned to the genus Paenibacillus [2, 3]. The type species of this genus is P. polymyxa. At present, Paenibacillus is categorized under the family Paenibacillaceae, which belongs to the phylum Bacillota. This genus currently includes 399 species, 304 of which are validly published with correct names (accession date: April 04, 2024; https://lpsn.dsmz.de/genus/paenibacillus). Paenibacillus species have been obtained from various sources, including soil, air, sediment, eutrophic lake, hot spring, freshwater, mountain, rhizosphere, phyllosphere, plant, seed, food, gut, insect, necrotic wound, and fecal samples [3-14]. This study characterized and determined the taxonomic status of strain dW9T in the genus Paenibacillus, which was isolated from a soil sample collected from the Republic of Korea.

Materials and Methods

Isolation of Strains

Strain dW9T was isolated from a soil sample collected from Gyeongsangnam in the Republic of Korea (35°28'48.0''N 128°13'12.0''E). The strain was isolated by the standard dilution plating technique using R2A media (MB Cell, Republic of Korea). After plating, the Petri dishes were placed in an incubator at 25°C for 7 days. Subsequently, white colonies were selected and repeatedly streaked on R2A agar. Pure colonies of strain dW9T were obtained and temporarily stored at 4°C. After the completion of taxonomic analyses, strain dW9T was preserved in glycerol stocks at −80°C and was submitted to the Korean Collection for Type Cultures and NITE Biological Resource Center.

16S rRNA Gene Sequence and Phylogenetic Analysis

Genomic DNA from strain dW9T was extracted using the HiGene Genomic DNA Prep Kit (BioFact, South Korea). PCR amplification of the 16S rRNA gene was performed using forward (27F) and reverse (1492R) primers [15]. The amplified PCR products were sequenced and analyzed as described previously [16]. The closest phylogenetically related taxa were sorted by analyzing and comparing the 16S rRNA nucleotide sequences using the EzBioCloud server [17]. Phylogenetic trees were constructed with MEGA X software [18] using the maximum likelihood (ML) [19], neighbor-joining (NJ) [20], and maximum parsimony (MP) algorithms [21]. The topologies of phylogenetic trees were estimated using the bootstrap resampling method with 1,000 replications [22]. The evolutionary distances were determined using Kimura’s two-parameter model [23].

Genomic Analysis

The genome was sequenced by the Illumina MiSeq sequencing technique, and raw sequences were assembled using Platanus-allee v. 2.2.2 [24] and SPAdes v. 3.13.0 [25] assembly tools. The quality of the genome sequence was assessed using the ContEst16S algorithm [26] and BLAST-N tool [27]. The annotation of the assembled genome sequence was performed using the Rapid Annotations using Subsystems Technology (RAST) server [28] and the Prokaryotic Genome Annotation Pipeline (PGAP) [29]. The DNA G + C content was directly determined from the genome sequence data. Biosynthetic gene clusters (BCGs) for various secondary metabolites were explored using antiSMASH 5.0 [30]. The genomic similarities between strain dW9T and reference species were calculated using the Genome-to-Genome Distance Calculator [31] and the average nucleotide identity (ANI) tool [32]. The phylogenomic tree was generated on the Type (Strain) Genome Server [33] using FastME 2.1.6.1 [34].

Morphological, Physiological, and Biochemical Analyses

Cellular morphologies of strain dW9T were analyzed by transmission electron microscopy (Talos L120C; FEI) after culturing the strain on R2A agar at 25°C for 5 days. The Gram stain reaction was determined using the Color Gram 2 Kit (bioMérieux, France). Anaerobic growth, motility, catalase, and oxidase tests were performed as described previously [16]. Endospores were examined by phase-contrast microscopy using a BX53-DIC microscope (Olympus) [35]. The temperature, pH, and NaCl ranges for growth were determined as described previously [36]. Moreover, the ability to hydrolyze cellulose, casein, DNA, starch, and Tween 80 was assessed as illustrated previously [37]. Various other biochemical, enzymatic, and carbon assimilation features were assessed using API ZYM, API 20NE, and API ID 32 GN kits (bioMérieux).

Chemotaxonomic Characterization

Cellular fatty acid compositions were assessed after growing strain dW9T and its closest reference taxa on R2A agar at 25°C for 3 days. After the late log phase of growth, the biomass of all strains was harvested and used for extracting fatty acids. The extracted fatty acids were analyzed and identified using the MIDI protocol [38]. Peptidoglycans were analyzed as described previously [39]. Quinones and polar lipids were analyzed using freeze-dried cells in accordance with previously described methods [40, 41]. Polar lipid spots on TLC plates were visualized by spraying with various reagents [42].

Results and Discussion

The length of the 16S rRNA gene nucleotide sequence of strain dW9T was 1,447 bp. Moreover, 16S rRNA gene analysis revealed that strain dW9T belonged to the genus Paenibacillus. Its closest phylogenetic neighbors were P. filicis S4T (97.4%), P. chinjuensis WN9T (97.3%), P. validus JCM 9077T (97.1%), P. mucilaginosus VKPM B-7519T (97.0%), P. puerhi SJY2T (96.8%), and P. cremeus JC52T (963.8%). The 16S rRNA gene sequence identities between strain dW9T and all other phylogenetically related taxa were below the cut-off value of <98.7% for species demarcation [43, 44]. This suggested that strain dW9T could be considered a novel species in the genus Paenibacillus. Furthermore, ML and NJ phylogenetic trees depicted that strain dW9T formed a clade with P. puerhi SJY2T (Figs. 1 and S1), whereas the MP tree revealed the formation of a clade with P. cremeus JC52T (Fig. S2).

Quality assessment confirmed that the genome sequence generated from strain dW9T was valid and contamination-free. The genome size of strain dW9T was 7,787,916 bp with a DNA G + C content of 51.3%. The genome sequence of strain dW9T was assembled in 71 contigs with an N50 value of 243,884 bp and genome coverage of 136.0× (Table S1). The annotated data obtained using RAST revealed 326 subsystem features in the genome of strain dW9T (Fig. S3). The strain also contained numerous BGCs encoding various secondary metabolites, such as linear azol(in)e-containing peptides, type III polyketide synthase, cyclic lactone autoinducer peptide, thiopeptide, phosphonate, proteusin, and terpene (Table S2). The dDDH and ANI values between strain dW9T and its closest phylogenetically related taxa ranged from 19.2% to 21.6% and 69.6% to 73.9%, respectively (Table S3). The genome relatedness values between strain dW9T and its reference species were below the threshold values [dDDH (70.0%) and ANI (95.0%)], suggesting that strain dW9T was genomically different from its closest members [45, 46]. Furthermore, the phylogenomic tree revealed that strain dW9T formed a clade with P. cremeus JC52T (Fig. S4).

The cells of strain dW9T were rod shaped and flagellated (Fig. S5). Moreover, strain dW9T was motile. Catalase and nitrate reduction tests were positive, whereas the oxidase test was negative. Strain dW9T could grow at a temperature of 20–37°C and a pH of 5.0–7.0 and could tolerate 2.0% (w/v) NaCl. It could hydrolyze esculin and Tween 80. β-galactosidase and β-glucosidase activities were positive. The strain could assimilate D-glucose, L-arabinose, D-mannitol, gluconate, and D-melibiose. Other distinguishing features of strain dW9T are presented in the species protologue and provided along with those of its reference species in Table 1. All enzyme activity and assimilation data obtained using API kits are provided in Table S4.

The sole respiratory quinone in strain dW9T was menaquinone (MK)-7. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, and phosphatidylethanolamine were the predominant polar lipids (Fig. S6). One unidentified polar lipid (L) was also observed. Both respiratory quinone and polar lipid profiles of strain dW9T closely resembled those of its related reference taxa [12, 47]. The peptidoglycan was identified as meso-diaminopimelic acid (DAP). The key fatty acids in strain dW9T were antiso-C15:0 (75.6%) and iso-C16:0 (6.8%). The key fatty acid profiles aligned with those of the closest reference taxa. However, the composition of minor fatty acids differed proportionally between strain dW9T and the reference species (Table 2).

Taxonomic Conclusion

On the basis of data presented here, we proposed strain dW9T as a novel species in the genus Paenibacillus with the name Paenibacillus gyeongsangnamensis sp. nov.

Description of Paenibacillus gyeongsangnamensis sp. nov.

Paenibacillus gyeongsangnamensis sp. nov. (gyeong.sang.na.men'sis. N.L. masc. adj. gyeongsangnamensis, referring to Gyeongsangnam, the place of Republic of Korea).

Cells are aerobic, Gram-stain-positive, motile, endospore-forming, rod shaped (4.9–5.1 × 1.3–1.5 μm), and flagellated. Colonies on R2A agar are white, circular (4.6–5.4 mm in diameter), and convex. Cells grow at temperature 20–37°C (optimum, 25°C), at pH 5.0–7.0 (optimum, 7.0), and at 0–2.0% NaCl concentration (optimum without NaCl). Positive for catalase and nitrate reduction tests, and negative for oxidase activity. Hydrolyse Tween 80 and esculin, but not starch, DNA, gelatin, casein, and urea. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, β-galactosidase, α-glucosidase, β-glucosidase, α-mannosidase, and α-fucosidase. Assimilates D-glucose, L-arabinose, D-mannitol, gluconate, salicin, D-melibiose, L-histidine, 2-ketogluconate, D-ribose, inositol, D-sucrose, and glycogen. The key fatty acids are antiso-C15:0 and iso-C16:0. The sole menaquinone is MK-7, diagnostic peptidoglycan is meso-DAP, and major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, and phosphatidylethanolamine. The DNA G+C content of the type strain is 51.3%.

The type strain, dW9T (=KCTC 43431T =NBRC 116022T), was isolated from soil in Republic of Korea (GPS coordinates: 35°28'48.0"N 128°13'12.0"E).

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA sequence and genome sequence of strain dW9T are ON573456 and JAQAGZ000000000, respectively.

Supplemental Materials

Supplementary data for this paper are available on-line only at http://jmb.or.kr.

Acknowledgments

This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR202203112).

Fig. 1 Maximum likelihood tree based on 16S rRNA gene sequences of strain dW9T closest reference species.

Nodes recovered by maximum-likelihood, neighbor-joining, and maximum-parsimony trees are denoted by filled circles. The numbers at branch nodes are percentage of 1,000 bootstrap replicates (values >70% are only illustrated). NCBI GenBank accession numbers for 16S rRNA gene sequences are provided in parentheses. Geobacillus stearothermophilus IFO 12550T was used as an out-group. The scale bar indicated 0.05 substitutions per nucleotide position.

Table 1 Differentiating properties of dW9T and closely affiliated reference taxa.

Characteristic	1	2	3	4	5	6	7	
Growth temperature (°C)	20–37	15–37	18–45	15–42	15–45	15–37	15–40	
Highest salt tolerance (%, w/v)	2.0	3.0	2.0	1.0	2.0	4.0	0.0	
pH range	5.0–7.0	5.5–9.0	6.5–8.0	5.0–8.5	6.0–8.5	5.0–8.0	6.0–7.0	
Catalase/oxidase	+/-	+/+	+/+	+/-	+/-	-/+	+/-	
Motility	+	+	+	+	-	+	+	
Nitrate reduction	+	-	-	+	−	+	-	
Hydrolysis of								
Starch	-	+	+	+	-	+	+	
Casein	-	-	+	-	-	-	+	
Tween 80	+	+	+	-	-	-	+	
Esculin	+	+	-	+	-	-	+	
Enzyme activity								
Alkaline phosphatase	+	+	-	-	-	-	-	
Leucine arylamidase	+	+	+	+	-	+	+	
Valine arylamidase	-	-	-	-	-	+	+	
Cystine arylamidase	-	-	-	-	-	+	+	
Acid phosphatase	+	+	-	-	-	+	-	
β-Galactosidase	+	+	-	-	+	-	-	
α-Glucosidase	+	+	-	-	-	-	-	
β-Glucosidase	+	+	-	+	-	-	+	
N–Acetyl-β-glucosaminidase	-	+	-	-	-	-	-	
α-Mannosidase	+	-	-	-	-	-	-	
α-Fucosidase	+	-	-	-	-	-	-	
Assimilation from (API 20NE and ID 32 GN test)								
D-Glucose	+	+	-	+	-	+	+	
L-Arabinose	+	-	-	-	-	+	+	
D-Mannose	-	+	+	w	-	-	+	
D-Mannitol	+	+	-	+	-	-	+	
N-Acetyl-D-glucosamine	-	+	-	-	-	-	-	
D-Maltose	-	+	+	+	-	+	+	
Gluconate	+	+	-	+	-	-	-	
Salicin	+	+	-	-	-	-	+	
D-Melibiose	+	+	-	+	-	-	+	
L-Fucose	-	-	-	-	-	-	+	
Propionate	-	-	-	+	-	-	-	
L-Histidine	+	-	-	-	-	-	-	
2-Ketogluconate	+	-	-	-	-	-	-	
4-Hydroxy-benzoate	-	-	-	+	-	-	-	
D-Ribose	+	-	-	+	-	-	-	
Inositol	+	+	-	+	-	-	+	
D-Sucrose	+	+	-	+	-	-	+	
Acetate	-	+	-	+	-	-	-	
Glycogen	w	+	-	+	-	-	-	
DNA G + C content	51.3%	53.5%	(53.0 mol%)	52.2%	(53.7 mol%)	53.1%	50.75	
Strains: 1, dW9T; 2, P. filicis KACC 14197T; 3, P. chinjuensis KACC 12279T [48]; 4, P. validus KACC 14477T ; 5, P. mucilaginosus KACC 13999T [49]; 6, P. puerhi KCTC 43242T ; 7, P. cremeus KACC 21221T. All data were generated in this study except the data provided in parentheses which were obtained from literatures. +, positive; w, weakly positive; -, negative.

Table 2 Cellular fatty acid profiles (% of totals) of dW9T and phylogenetically related reference species.

Fatty acids	1	2	3	4	5	6	7	
Saturated								
C10:0	–	1.0	1.6	1.3	1.0	–	1.0	
C12:0	tr	1.0	1.6	1.3	1.1	1.0	tr	
C14:0	1.1	1.4	2.1	1.6	1.6	1.4	2.2	
C16:0	2.8	5.5	11.1	7.5	6.2	10.8	5.6	
Unsaturated								
C16:1 ω7c alcohol	1.1	–	–	1.7	tr	1.5	–	
C16:1 ω11c	1.5	–	–	2.7	5.2	1.0	–	
Branched saturated								
iso-C14:0	4.5	6.3	1.6	3.7	2.2	3.7	3.9	
iso-C15:0	2.6	2.6	2.3	4.6	2.7	2.5	4.1	
iso-C16:0	6.8	14.1	3.6	10.3	3.9	9.8	12.1	
iso-C17:0	–	1.0	–	1.7	–	1.1	1.6	
anteiso-C13:0	tr	–	–	–	–	–	–	
anteiso-C15:0	75.6	61.8	62.9	56.8	60.6	58.4	60.8	
anteiso-C17:0	3.2	4.4	3.9	6.0	3.5	3.5	5.1	
Hydroxy								
C12:0 3OH	–	–	2.8	–	1.1	1.3	–	
C13:0 2OH	–	–	5.8	–	2.8	1.6	–	
Strains: 1, dW9T; 2, P. filicis KACC 14197T; 3, P. chinjuensis KACC 12279T; 4, P. validus KACC 14477T; 5, P. mucilaginosus KACC 13999T; 6, P. puerhi KCTC 43242T ; 7, P. cremeus KACC 21221T . TR, trace amount (<1.0%); –, not detected.

Abbreviations

KACC: Korean Agricultural Culture Collection; KCTC: Korean Collection for Type Cultures; NBRC: NITE Biological Resource Center; ANI: Average nucleotide identity; dDDH: Digital DNA-DNA hybridization.

Author Contributions

HL and DK designed the study. HL and DKC contributed the experimental work, data analysis, and original draft preparation. HL and DKC reviewed and finalized the manuscript. DK supervised the project.

Data Availability

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA sequence and genome sequence of strain dW9T are ON573456 and JAQAGZ000000000, respectively.

Conflict of Interest

The authors have no financial conflicts of interest to declare.
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