The draft genome of strain IB182493T contained 86 contigs and with a size of 7.06 Mbp. 10 rRNAs (2, 5, 3 for 5S, 16S, 23S rRNA, respectively) and 70 tRNAs were detected. The General features of the genome of strain IB182493T were listed in table S1. The mol% G + C of the genome of the isolate was 56.2 mol% and within the range of 40–59 mol% reported for the genus Paenibacillus (Priest 2015) but higher than those of P. harenae DSM 16969T (49.9 mol %) and P. alkaliterrae DSM 17040T (49.4 mol %). The distribution of the genes into clusters of orthologous groups (COGs) functional categories is presented in Fig. S1. In the phylogenomics tree (Fig. S2), strain IB182493T clustered with P. harenae DSM 16969T. The ANIb, ANIm and rthoANIu values between strain IB182493T and the closely related type strains were ranged from 69.1–78.6%, 82.9–84.7% and 71.8–80.1%, respectively, while dDDH values were 23.6–18.5% (Table S2). All of these values meet the criteria for bacterial species demarcation (Richter and Rosselló-Móra 2009; Chun et al. 2018) and support the hypothesis that IB182493T represents a novel species within the genus Paenibacillus.
The obtained 16S rRNA gene sequence of strain IB182493T was 1470 bp long and the GenBank /EMBL/ DDBJ accession number was MK249696. According to the EzTaxon-e search, strain IB182493T represented a member of the genus Paenibacillus and showed the highest 16S rRNA gene sequence similarity to P. harenae DSM 16969T (96.6%), P. alkaliterrae DSM 17040T (96.1%), P. agarexedens DSM 1327T (96.1%) and P. agaridevorans DSM 1355T (96.1%). Phylogenetic analyses showed that the isolate formed a discrete cluster with P. harenae DSM16969T and P. alkaliterrae DSM 17040 T (Fig. 1). The ME and ML trees also showed the similar results (Figs S3 and S4, available in the online version of this article). Based on 16S rRNA gene sequence similarities and phylogenetic analyses, the most closely related species, P. harenae DSM 16969T and P. alkaliterrae DSM 17040 T were used as reference strains and examined for their genomic and phenotypic characteristics in comparison with those of the new isolate. Both the two reference strains were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).
Strain IB182493T was strictly aerobic and motile by mean of peritrichous flagella. Cells are Gram-stain-negative, rod-shaped and swollen by subterminal ellipsoidal spores. Under the electron microscope, cells were approximately 0.5–0.9 µm in diameter and 2.8–3.3 µm in length (Fig. 2). Cells grow well on R2A agar, LB, TSA, MA and NA, while poor on ISP3, but could not grow on ISP2 and PDA. Growth occurred at temperatures ranging from 20–40°C (optimum 25–30°C), pH 5.0–10.0 (optimum pH 7.0–8.0) and 0–9% (w/v) NaCl (optimum 2–4%). The strain was positive for catalase and reduced nitrate to nitrite. Starch, CM-cellulose, tween 20, tween 40, tween 60 and tween 80 were not hydrolysed. In antibiotic tests, strain IB182493T was found to be sensitive to cefamezin, cefradine, cefuroxime, ceftazidime, ceftriaxone, cefoperazone, norfloxacin and polymyxin B, but resistant to all of the other antibiotics tested. In the API 20NE kit tests, strain IB182493T was found to be unable to reduce nitrate to nitrite, showed positivity for ß-glucosidase and ß-galactosidase, assimilationed of glucose, mannitol and maltose. with API ZYM kit, strain IB182493T was found to be positive for the activities of esterase (C4), acid phosphatase, naphthol-AS-BI-phosphohydrolase, ß-galactosidase and ß-glucosidase. In the API 50CH B kit, cells produce acid from arbutin, salicin, D-cellobiose, maltose, D-lactose, D-melibiose, D-saccharose sucrose, D-trehalose, inulin, D-melezitose, D-raffinose, starch amylase, D-gentiobiose, D-turanose; weakly produce acid from methyl-ß-D-xylopyranoside, D-galactosemethyl, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, esculin ferric citrate, glycogen and potassium 5-ketogluconate. The phenotypic characteristics of strain IB182493T which were used for comparison with the reference species were summarized in Table 1.
The predominant respiratory quinone of strain IB182493T was MK-7, which is also the major menaquinone in other species of the genus Paenibacillus (Priest. 2009). The diagnostic diamino acid in the cell-wall peptidoglycan of strain was meso-diaminopimelic acid. The polar lipids included phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and two unidentified phospholipids (PL1–PL2) (Fig. S5). The major cellular fatty acids (> 5%) were anteiso-C15:0 (47.1%), iso-C16:0 (23.4%), C16:0 (8.9%), and iso-C15:0 (6.1%). The novel isolate had similar relative proportions of the fatty acids to reference strains (Table S3).
Based on the result of the gene cluster prediction, strain IB182493T contained 9 secondary metabolite gene clusters in the genome (Table S4), while 8 clusters in P. harenae DSM16969T and 8 clusters in P. alkaliterrae DSM 17040T. The genome mining revealed that this strain has the potential to produce many secondary metabolites including lasso peptide paeninodin, ectoine, basiliskamide A/B and staphylobactin (Table S4). The lasso peptide belongs to a new class of natural product with highly compact and stable structure, which has various biological activities such as anti-tumor metastases, receptor antagonist, enzyme inhibitor, and antibacterial agents (Wilson et al. 2003; Knappe et al. 2010; Iwatsuki et al.2006). As known that lasso peptides are non-pathogenic, and their great resistibility to high temperature, acidic condition, and most proteases, lasso peptides may be used as multifunctional backbones for further medical use (Knappe et al. 2011; Hegemann et al. 2014; Meyer et al. 2006). In the genome of strain IB182493T, lasso peptide paeninodin biosynthetic gene clusters with 100% similarity to that of strain P. dendritiformis C454 (Sirota-Madi A et al, 2012) were found. The lasso peptide paeninodin cluster has a gene encoding a kinase, which was represented as member of a new class of lasso peptide tailoring kinases. By employing a wide variety of peptide substrates, it was shown that the novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere (Zhu et al, 2016). In genomic data analysis, 75% similarity of ectoine gene cluster were found in the genome of strain IB182493T compared to Streptomyces anulatus (Beijerinck 1912). The isolate and the closely related species (P. harenae DSM16969T and P. alkaliterrae DSM 17040T) contain ectoine gene cluster and were isolated from the similar habitat such as sand and soil which were dry or alkaline-like extreme. This evidence suggests that the ectoine is primarily associated with extreme environments, as has been reported by Brown (1976) that ectoine is essential for extremophiles to survive in extreme environments. In addition, basiliskamide A/B biosynthetic gene cluster with 9% similarity to that of strain Brevibacillus laterosporus PE36 (Theodore et al. 2014) were found in the genome of strains IB182493T. Despite the relatively closeness, no basiliskamide A/B biosynthetic gene clusters were detected in the genome sequence of strain P. harenae DSM16969T and P. alkaliterrae DSM 17040T. In conclusion, the complete genome of strain IB182493T will help further studies regarding the biosynthesis of diverse secondary metabolites and their regulatory mechanisms.
Based on phenotypic, phylogenetic and genomics analyses, strain 182493T is considered as a typestrain of a novel species with the proposed name, Paenibacillus arenilitoris.
Description of Paenibacillus arenilitoris sp. nov.
Paenibacillus arenilitoris sp. nov. (a.re.ni.li.to′ris. L. n. arena sand; L. n. litus-oris the seashore, coast; N.L. gen. n. arenilitoris of sand of the seashore, from which the type strain was isolated).
Cells are Gram-stain-negative, strictly aerobic and motile with peritrichous flagella. Cells are rod-shaped with 0.5–0.9×2.8–3.3 µm in size. subterminal ellipsoidal endospores are observed in swollen sporangia. Colonies are non-pigmented, white-cream, punctiform circular and smooth, with 0.5–1.5 mm in diameter on MA medium after 48 h. Growth occurs at 20–40°C (optimum 25–30°C), pH 5.0–10.0 (optimum pH 7.0–8.0) and 0–9% (w/v) NaCl (optimum 2–4%). Cells were Catalase-positive and reduced nitrate to nitrite, but negative for urease and oxidase. Starch, CM-cellulose, tween 20, tween 40 and tween 80 are not hydrolysed.
The predominant isoprenoid quinone is MK-7 and the cell-wall peptidoglycan contains meso-diaminopimelic acid. The major cellular fatty acids are anteiso-C15:0 and iso-C16:0. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and two unidentified phospholipids. The mol% G + C content of the DNA is 56.2 mol%. The genomics analyses predicted 9 secondary metabolite gene clusters and revealed that this strain has the potential to produce many multifunctional active ingredients including lasso peptide paeninodin, ectoine, basiliskamide A/B and staphylobactin.
The type strain IB182493T (= MCCC 1K04626T = JCM 34215T), was isolated from seashore sand of South China Sea in China. The GenBank/ENBL/DDBJ accession numbers of 16S rRNA gene and the draft genome sequences of the type strain IB182493T are MK249696 and JACXIY000000000, respectively.