Archives of Microbiology (2021) 203:3997–4004 https://doi.org/10.1007/s00203-021-02389-1 ORIGINAL PAPER Paenibacillus roseus sp. nov., a ginsenoside‑transforming bacterium isolated from forest soil Shahina Akter1 · Xiaoqing Wang2 · Sun‑Young Lee2 · M. Mizanur Rahman3 · Jong‑Hyun Park1 · Muhammad Zubair Siddiqi4 · Sri Renukadevi Balusamy5 · Kihong Nam6 · Md. Shahedur Rahman7 · Md. Amdadul Huq2 Received: 30 January 2021 / Revised: 30 April 2021 / Accepted: 18 May 2021 / Published online: 25 May 2021 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021 Abstract A novel, pink-pigmented, Gram-stain-positive, aerobic, motile, rod-shaped and ginsenoside-converting bacterium, designated strain MAHUQ-46T, was isolated from soil of a forest. Strain MAHUQ-46T grew in the pH range 6.0–9.0 (optimum, 7.5), at temperatures between 10 and 37 °C (optimum, 30 °C) and at 0–3% (w/v) NaCl (optimum, 0.5%). 16S rRNA gene sequence analysis showed that strain MAHUQ-46T was closely related to Paenibacillus pinihumi S23T (97.3% similarity), followed by Paenibacillus elymi KUDC6143T (96.7%). The draft genome of strain MAHUQ-46T had a total length of 5,367,904 base pairs. A total of 4,857 genes were identified, in which 4,629 were protein-coding genes and 137 were RNA genes. The genome annotation of MAHUQ-46T showed 172 carbohydrate genes, some of them may be responsible for the biosynthesis of ginsenoside Rd from major ginsenoside Rb1. The DNA G + C content was 48.4 mol% and the major quinone was MK-7. Main fatty acids of strain MAHUQ-46T were C15: 0 anteiso, C16: 0 and C17: 0 anteiso. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyl-N-methylethanolamine, two unidentified aminophospholipids and five unidentified phospholipids. Diagnostic diamino acid of peptidoglycan was meso-diaminopimelic acid. The novel strain MAHUQ-46T was able to rapidly synthesize ginsenoside Rd from major ginsenoside Rb1. The synthesized ginsenoside was confirmed by TLC and HPLC analysis. According to the phenotypic, genetic and chemotaxonomic evidence, strain MAHUQ-46T was clearly distinguishable from validly published species of genus Paenibacillus and should, therefore, be categorized as a novel species for which the name Paenibacillus roseus sp. nov. is proposed. The type strain is MAHUQ-46T (= KACC 21242T = CGMCC 1.17353T). Keywords Paenibacillus roseus · Genome sequence · Ginsenoside Rd · Rapid synthesis Introduction Communicated by Erko Stackebrandt. Shahina Akter, and Xiaoqing Wang authors equally contributed to this work as co-first author. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and draft genome sequence of strain MAHUQ-46T are MK680121 and JAELUP000000000, respectively. * Md. Amdadul Huq amdadbge@gmail.com; amdadbge100@cau.ac.kr Extended author information available on the last page of the article The genus Paenibacillus was proposed in 1993 by Ash et al. (1993) and separated from genus Bacillus according to the comparative analysis of 16S rRNA gene sequence. Species of genus Paenibacillus have been isolated from diverse environments including soil (Huq et al. 2015; Akter and Huq 2018), food (Berge et al. 2002), air (Rivas et al. 2005), water (Baik et al. 2011), human feces (Clermont et al. 2015), roots (Zhang et al. 2016), seeds (Liu et al. 2010), leaves (Madhaiyan et al. 2017) and flowers (Siddiqi et al. 2015). Isolation and characterization of bacteria are important due to their potential applications (Farh et al. 2015; Huq et al. 2014). Ginsenoside is the pharmacological active compound of ginseng, and has been reported to contain different biological efficacies such as anti-obesity, anticancer, 13 Vol.:(0123456789) 3998 tumor-suppressing, hepatoprotective, neuroprotective effects (Siraj et al. 2015; Chae et al. 2009; Huq et al. 2016). Minor ginsenosides possess higher activity than major ginsenosides (Huq et al. 2016). Ginsenoside Rb1 is a major ginsenoside and abundant in ginseng root. Therefore, many scientists have tried to convert major ginsenosides to highly active minor ginsenosides (Huq et al. 2016). This report describes the isolation, characterization and genome analysis of a novel bacterial species Paenibacillus roseus sp. nov., and utilization of this novel species for the rapid synthesis of ginsenoside Rd. Materials and methods Bacterial isolation Novel strain, MAHUQ-46T was isolated from soil sample of a forest located in Anseong, South Korea (37° 00′ 44″ N, 127° 23′ 17″). Soil samples were diluted serially using sterile NaCl solution (0.85%, w/v) and these dilutions were plated onto R2A agar medium (MB cell) and spread thoroughly. The plates were incubated for 3 days at 30 °C. The culture was further purified by repeated streaking onto the R2A agar until pure strains were obtained. The purified strain was cultivated and stored as aqueous glycerol suspensions (30%, v/v) at − 80 °C. Strain MAHUQ-46T has been deposited to the KACC and CGMCC. Cell growth, morphology, physiology and biochemical tests Cultural characteristics were investigated by observing the growth of the strain MAHUQ-46T at 30 °C for 5–7 days on Reasoner’s 2A (R2A) agar (Difco), nutrient agar (NA; Difco), Luria–Bertani agar (LB agar), tryptic soy agar (TSA; Bacto) and MacConkey agar. The morphology of the cells was observed and recorded by transmission electron microscopy (Model JEM1010; JEOL) after incubation on R2A agar at 30 °C for 2 days. Salt tolerance was determined in R2A broth containing 0–6% (w/v) NaCl (0.5% interval) after 7 days of incubation at 30 °C. The temperature range (4–45 °C) for growth was assessed on R2A agar for 7 days. The growth pH range (pH 4.0–11.0) was assessed in R2A broth medium for 7 days using the buffer system described by Huq (2018). Gram-staining test was conducted according to the method of Magee et al. (Magee et al. 1975). Catalase activity was checked using 3% (v/v) H2O2 through bubble production. 1% (w/v) tetramethyl-p-phenylenediamine was used to determine the oxidase activity (Huq 2018). Hydrolysis of DNA, casine, gelatin, starch, Tweens 20 and, Tweens 80 were tested according to the method of Gonzalez et al. (Gonzalez et al. 1978). The enzyme activities 13 Archives of Microbiology (2021) 203:3997–4004 and biochemical characteristics were assessed using the API ZYM kits (bioMérieux) and API 20NE kits (bioMérieux) according to the manufacturer’s instructions. The close type strains, Paenibacillus pinihumi KACC 14199T and Paenibacillus elymi KCTC 33853T were used as reference strains and were grown under the identical experimental conditions of novel strain MAHUQ-46T. Analysis of 16S rRNA gene and phylogenetic tree The 16S rRNA gene was amplified from extracted genomic DNA using bacterial universal primers 27F and 1492R (Weisburg et al. 1991). The 16S rRNA gene sequence of strain MAHUQ-46T was compared with related type species from EzBioCloud database (www.ezbiocloud.net) to determine the similarity values (Yoon et al. 2017a). Multiple sequence alignments were conducted using clustal_x software package (Thompson et al. 1997) and BioEdit software (Hall 1999). Kimura’s two-parameter model was used to determine the evolutionary distances (Kimura 1980). Phylogenetic trees were constructed using both the neighborjoining (Saitou and Nei 1987) and maximum-likelihood (Felsenstein 1981) methods with mega (version 5.0) software (Tamura et al. 2011). To evaluate the topologies of the phylogenetic trees, bootstrap analysis was carried out with 1000 replications (Felsenstein, 1985). Genome sequence analysis Draft-genome sequencing of strain MAHUQ-46T was performed on the Illumina HiSeq X Ten platform. The goodquality paired reads were assembled using SOAPdenovo v. 3.10.1 de novo assembler into a number of scaffolds. Genome annotation was completed using NCBI prokaryotic genome annotation pipeline (PGAP). The wholegenome sequence data have been submitted to DDBJ/ENA/ GenBank. The DNA G + C content of strain MAHUQ-46T was directly calculated from the draft genome sequences (Chun et al. 2018). The average nucleotide identity (ANI) was calculated as described previously (Yoon et al. 2017b). While, the digital DNA–DNA hybridization (dDDH) value was determined using the genome-to-genome distance calculator (http://ggdc.dsmz.de/ggdc.php) according to MeierKolthoff et al. (2013). Cellular fatty acid, respiratory quinone, polar lipid and peptidoglycan analysis Cell biomass of strain MAHUQ-46 T and two reference strains for analysis of the cellular fatty acid were collected by cultivation on R2A agar medium at 30 °C, for 48 h. The cellular cell fatty acids were saponified, methylated and extracted using a standard protocol as described by Sasser Archives of Microbiology (2021) 203:3997–4004 3999 (1990), and analyzed by gas chromatograph GC (HewlettPackard 5890 Series II) and identified through the rtsba 6.00 database of the Microbial Identification System (Sasser 1990). Respiratory quinones of strain MAHUQ-46T were extracted and purified according to Collins et al. (1977), then analyzed and confirmed by HPLC (Guo et al. 2015). Polar lipids of strain MAHUQ-46T were extracted from dry cells (100 mg) as described by Minnikin et al. (1984). The extracted polar lipids were analyzed by two-dimensional TLC according to the previous description (Akter and Huq 2020). The diamino acid of the peptidoglycan was extracted and determined by TLC (Cellulose; Merck) as described by Komagata and Suzuki (1987). MAHUQ-46T belonged to the genus Paenibacillus and had moderately high similarity to Paenibacillus pinihumi S23T (97.3%) and Paenibacillus elymi KUDC6143T (96.7%). The relationship between isolated strain MAHUQ-46T and recognized Paenibacillus species is also shown in the constructed phylogenetic trees. The neighbor-joining and maximumlikelihood trees placed strain MAHUQ-46T within cluster for Paenibacillus species, as shown in Fig. 1 and Supplementary Fig. S2. The 16S rRNA gene sequence analysis revealed that strain MAHUQ-46T could be clearly separated from validly published species of the genus Paenibacillus and should be assigned as a novel species of this genus (Wayne et al. 1987; Stackebrandt and Goebel 1994). Biotransformation of ginsenoside Rb1 Genome sequence analysis The novel strain MAHUQ-46T was grown in R2A broth medium at 30 °C for 48 h and cells were harvested by centrifuging at 9,000 rpm for 15 min at 4 °C. Then, the harvested cells were dissolved in sodium phosphate buffer (20 mM, pH 7.0) and lysed by short pulse sonication. Then, the supernatant was collected by centrifugation and used as crude enzyme. This is the modification of Huq et al. (2016) protocol. The bioconversion of ginsenoside Rb1 was performed in screw-capped tubes. 3 ml of crude enzyme and 3 ml ginsenoside Rb1 (2.0 mg/ml) were mixed and incubated in a shaking incubator (160 rpm) at 30 °C. Every 1-h interval, 0.5 ml sample was withdrawn and analyzed by both TLC and HPLC (Huq et al. 2014). The draft genome of strain MAHUQ-46T was 5,367,904 bp long in size with a G + C content of 48.4 mol%. It comprised of 118 contigs. Among 4,857 predicted genes, 4,629 were protein-coding genes and 137 were encoding RNAs (116 tRNA genes, four 5S rRNA genes, seven 16S rRNA genes and six 23S rRNA genes). The common features of genome sequence of strain MAHUQ-46T are given in Supplementary Table S2. Taxonomic and functional research of microorganisms has increasingly relied upon genomebased data and methods (Shi et al. 2021). Distribution of genes in the genome of strain MAHUQ-46T was investigated using RAST server (Overbeek et al. 2014), via the RASTtk pipeline (Brettin et al. 2015). It was found that 207 of the genes were involved with protein metabolism, 241 genes were associated with the metabolism of amino acids and derivatives, 172 genes were linked with carbohydrate metabolism and 102 genes were involved with metabolism of vitamins, cofactors and pigments (Supplementary Fig. S3). The closest type strain P. pinihumi DSM 23905T contains a 6,760,575 bp long genome (number of contig 45) with 48.5 mol% GC, 6,062 CDSs, 64 tRNA and 14 rRNA genes (https://www.ezbiocloud.net/genome/explore?puid= 20755). The genomic ANI (average nucleotide identity) values between strain MAHUQ-46T and P. pinihumi S23T were 82.2%, well below (≥ 95–96%) to suggest a novel species. The dDDH value based on the draft genomes between strain MAHUQ-46T and P. pinihumi S23T was 26.0% which was also far below the threshold value (70%) for species delineation (Supplementary Table S3). Results and discussion Cell growth, morphology, physiology and biochemical characteristics Cells of strain MAHUQ-46T were Gram stain positive, aerobic, rod shaped (0.6–1.2 × 1.4–2.6 µm) and motile with flagella (Supplementary Fig. S1). Strain MAHUQ-46T formed pink colonies when grown on R2A agar plates after incubation for 48 h at 30 °C. The detailed physiological, morphological and biochemical characteristics of strain MAHUQ46T and most closely related type strains are given in the species description and in Table 1. The negative properties of strain MAHUQ-46T carried out by commercial test kits (API 20NE and API ZYM) are shown in Supplementary Table S1. Cellular fatty acid, respiratory quinone, polar lipid and peptidoglycan analysis 16S rRNA gene sequence and phylogenetic analysis Almost complete length of 16S rRNA gene sequence for strain MAHUQ-46 T was 1,485 bp. 16S rRNA gene sequences analysis revealed that the novel strain Strain MAHUQ-46T contained C15: 0 anteiso (48.6%), C16: 0 (12.1%) and C17: 0 anteiso (11.5%), as the dominant fatty acids. Strain MAHUQ-46 T also contained considerable amount of C15: 0 iso (8.2%) and C16: 0 iso (9.7%). As for the 13 4000 Archives of Microbiology (2021) 203:3997–4004 Table 1 The biochemical and physiological characteristics of strain MAHUQ-46T and the reference strains of genus Paenibacillus Characteristics 1 2 3 Isolation source Forest soil Rhizosphere of the pine tree Cell size (um) Colony color Aerobic/facultative anaerobic Catalase Oxidase 4-Nitrophenyl-BD-galactopyranoside Growth temperature (°C) Growth pH NaCl tolerance (%) Hydrolysis of Gelatin (API 20 NE) Tween 80 Enzyme activity (API ZYM) Esterase (C4) Alkaline phosphatase Lipase (C14) Valine arylamidase Cystine arylamidase Trypsin α-chymotrypsin Naphthol-AS-BI-phosphohydrolase a-galactosidase β-glucosidase β-galactosidase Assimilation of (API 20 NE) d-glucose l-arabinose d-mannose Gluconate DNA G + C content (mol%) 0.8–1.2 X 1.4–2.6 Pink Strictly aerobic W+ − − 10–37 6.0–9.0 0–3 1.6–3.5 X 0.6–0.8a Cream Strictly aerobic − + + 15-37a 5.5–9.0a 0-3a Rhizosphere of Elymus tsukushiensis 0.5–0.6 X 2.0–2.7b Creamy white Facultative anaerobic − + − 25-45b 6.0–12.0b 0–4.0b − W+ − − + − + + W w w w − w − + − − + − − − − − − + − + + − − − + − + + − − − + + − + 48.4 − − − − 49.5a + + + − 50.3b Strains: 1, P. roseus MAHUQ-46T; 2, P. pinihumi KACC 14199T and P. elymi KCTC 33853T All data were obtained in this study, except a and b that were taken from Kim et al. (2009) and Hwang et al. (2018), respectively. All strains are rod shaped and motile. All strains are positive for esterase lipase (C8), hydrolysis of starch, esculin, assimilation of D-maltose and D-mannitol. All strains are negative for nitrate reduction, indole production, arginine dihydrolase, acid phosphatase, leucine arylamidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, a-glucosidase, and a-fucosidase, hydrolysis of casein and urea, and assimilation of malic acid, N-acetyl-glucosamine, phenylacetic acid, adipic acid, trisodium citrate and capric acid. + , positive; −, negative; W + , weakly positive major fatty acids, strain MAHUQ-46T was similar to close reference strains P. pinihumi KACC 14199T and P. elymi KCTC 33853T, except for some small amounts of fatty acids. Although, the major fatty acid profiles were similar with close type strains but there were significant quantitative differences. The cellular fatty acid profiles of strain MAHUQ46T and two close reference strains are shown in Table 2. The predominant isoprenoid quinone of strain MAHUQ-46T was menaquinone-7 (MK-7) which is one of the common characteristics of genus Paenibacillus (Ash et al. 1993; Huq et al. 2015; Kim et al. 2009; Hwang et al. 2018). The polar 13 lipids identified in strain MAHUQ-46T were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyl-N-methylethanolamine, two unidentified aminophospholipids and five unidentified phospholipids. (Supplementary Fig. S4). The predominant polar lipids of strain MAHUQ-46T were similar to those of the most closely related type strains (Hwang et al. 2018). The diagnostic diamino acid in the peptidoglycan cell wall of strain MAHUQ-46T was meso-diaminopimelic acid. This is a common feature among members of the genus Paenibacillus (Kim et al. 2009; Hwang et al. 2018). Archives of Microbiology (2021) 203:3997–4004 4001 Paenibacillus pinihumi JCM 16419T (BBDI01000085) Paenibacillus elymi KUDC6143T (KX858537) Paenibacillus roseus MAHUQ-46T (MK680121) Paenibacillus abyssi SCSIO N0306T (KC978082) Paenibacillus wooponensis WPCB018T (EU939687) Paenibacillus xanthanilyticus AS7T (KT429627) 80 99 Paenibacillus oenotherae DT7-4T (KF900218) 99 Paenibacillus harenae B519T (AY839867) 95 Paenibacillus alkaliterrae KSL-134T (AY960748) Paenibacillus paeoniae M4BSY-1T (MH714913) Paenibacillus castaneae Ch-32T (EU099594) Paenibacillus endophyticus PECAE04T (KC447384) 100 Paenibacillus glycanilyticusDS-1T (AB042938) 97 100 Paenibacillus darwinianus BrT (KK082214) Paenibacillus tarimensis SA-7-6T (EF125184) Paenibacillus translucens CJ11T (MF619925) Paenibacillus lacus Agd-32T (LN812815) Sphingomonas chungangi MAH-6T (KY964284) 0.02 Fig. 1 The neighbor-joining (NJ) tree based on 16S rRNA gene sequence analysis showing phylogenetic relationships of strain MAHUQ-46T and members of genus Paenibacillus, values less than 70% were not shown Table 2 Fatty acid profiles of strain MAHUQ-46T and the reference strains of genus Paenibacillus Fatty acid 1 2 3 C11:0 anteiso C12:0 iso C13:0 anteiso C14:0 iso C14:0 C15:0 iso C15:0 anteiso C16:0 iso C16:0 C16:1 w11c C17:0 iso C17:0 anteiso ND ND ND 1.2 1.0 8.2 48.6 9.7 12.1 ND 6.8 11.5 ND ND Tr 3.2 1.3 12.9 52.6 14.8 7.4 ND 3.5 4.1 2.2 1.5 1.9 2.7 2.8 7.6 43.8 12.1 16.2 1.7 2.4 4.4 Strains: 1, P. roseus MAHUQ46T; 2, P. pinihumi KACC 14199T and P. elymi KCTC 33853T All data were obtained in this study Biotransformation of ginsenoside Rb1 Ginsenoside Rd was biosynthesized from major ginsenoside Rb1 through hydrolysis of a glucose molecule at C-20 position of the ginsenoside aglycone. From TLC analysis, it was found that ginsenoside Rb1 was completely hydrolyzed and transformed into ginsenoside Rd within 3 h of incubation (Supplementary Fig. S5). Supplementary Fig. S5 shows that there was no more conversion of ginsenoside Rd until 24 h which indicates that this ginsenoside Rd is the stable product. The biosynthesis of ginsenoside Rd by crude enzyme of the novel strain MAHUQ-46T was also confirmed by HPLC analysis (Supplementary Fig. S6). Fig. S6A shows the peaks of standard ginsenosides including ginsenosides Rb1 (retention time of 20.68 min) and Rd (retention time of 22.59 min). Fig. S6B shows the control of ginsenoside Rb1 which was used for biosynthesis of ginsenoside Rd. From Fig. S6C, it was found that the peak for ginsenoside Rb1 was completely disappeared (100%) within 3 h of incubation and a new peak appeared. The appeared new peak had retention time same with that of ginsenoside Rd. Ginsenoside Rb1 was selected as target material because Rb1 is a major ginsenoside and abundant in ginseng root. Crude enzyme of P. 13 4002 roseus MAHUQ-46T are able to rapid and specific synthesis of ginsenoside Rd from Ginsenoside Rb1. The genome annotation of strain MAHUQ-46T showed 172 carbohydrate genes, some of them may be responsible for the biosynthesis of ginsenoside Rd from major ginsenoside Rb1. Conclusions Therefore, on the basis of phenotypic, genotypic and chemotaxonomic characteristics and the phylogenetic analysis, strain MAHUQ-46T clearly considered as a novel species of the genus Paenibacillus, for which the name Paenibacillus roseus sp. nov. is proposed. It is also concluded that the novel species Paenibacillus roseus MAHUQ-46T could be useful for the biologically rapid synthesis of ginsenoside Rd. Description of Paenibacillus roseus sp. nov. Paenibacillus roseus (ro’se.us. L. masc. adj. roseus rosy; referring to the color of the colonies). Cells are aerobic, Gram stain positive, rod shaped (0.6–1.2 × 1.4–2.6 µm) and motile with flagella. Colonies are circular (diameter, 0.5–1.2 mm), smooth and pink in color on R2A agar after 48 h of incubation at 30 °C. The growth temperature range is 10–37 °C (optimum, 30 °C). The growth pH range is 6.0–9.0 (optimum, pH 7.5) and the NaCl tolerance range is 0–3% (w/v) (optimum, 0%). Growth was observed on R2A agar, TSA, LB agar and NA, but no growth occurred on MacConkey agar. Positive for catalase but negative for oxidase. Cells are able to hydrolysis of starch, esculin and Tween 80, but unable to hydrolysis of casein (skimmed milk), DNA, l-tyrosine, gelatin, urea and Tween 20. Negative for indole production, glucose fermentation and nitrate reduction. In API ZYM strips, positive for alkaline phosphatase, leucine arylamidase, esterase lipase, esterase and β-glucosidase; weakly positive for naphtholAS-BI-phosphohydrolase, cystine arylamidase, valine arylamidase, lipase and trypsin. In API 20NE strips, d-glucose, d-maltose, l-arabinose and gluconate are utilized as sole carbon source. The predominant menaquinone is MK-7. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylN-methylethanolamine, two unidentified aminophospholipids and five unidentified phospholipids. Diagnostic diamino acid of peptidoglycan was meso-diaminopimelic acid. The DNA G + C content was 48.4 mol% and the major quinone was MK-7. Main fatty acids of strain MAHUQ-46T were C15: 0 anteiso, C16: 0 and C17: 0 anteiso. T h e t y p e st ra i n i s M A H U Q - 4 6 T ( = K AC C 21242T = CGMCC 1.17353T), isolated from the soil sample of a forest located in Anseong, South Korea. 13 Archives of Microbiology (2021) 203:3997–4004 Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00203-021-02389-1. Acknowledgements This study was performed with the support of the National Research Foundation (NRF) of Korea grant (Project no. NRF2018R1C1B5041386, Grant Recipient: Md. Amdadul Huq) funded by Korean government, Republic of Korea. I would like to give special thanks to CGM 10K project for analyzing the draft genome sequence of strain MAHUQ-46T (GCM60012307). Declarations Conflict of interest The author declares that there are no conflicts of interest. References Akter S, Huq MA (2018) Biological synthesis of ginsenoside Rd using Paenibacillus horti sp. Nov. Isolated from vegetable garden. Curr MicrobiOl 75:1566–1573 Akter S, Huq MA (2020) Sphingomonas chungangi sp. nov., a bacterium isolated from garden soil sample. Int J Syst Evol Microbiol 70:4151–4157 Ash C, PriestCollins FGMD (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. 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Amdadul Huq2 1 Department of Food Science and Biotechnology, Gachon University, Seongnam 461-701, Republic of Korea 5 Department of Food Science and Technology, Sejong University, Gwangjin-gu, Seoul 143-747, Republic of Korea 2 Department of Food and Nutrition, Chung-Ang University, Anseong, Gyeonggi-do 17546, Republic of Korea 6 3 Department of Biotechnology and Genetic Engineering, Faculty of Biological Science, Islamic University, Kushtia 7003, Bangladesh Department of Horticultural Life Science, Hankyong National University, Anseong, Gyeonggi-do 17579, Republic of Korea 7 Department of Genetic Engineering and Biotechnology, Jashore University of Science and Technology, Jashore 7408, Bangladesh 4 Department of Biotechnology, Hankyong National University, Anseong, Gyeonggi-do 17579, Republic of Korea 13