Introduction

On the basis of the analysis of the 16S rRNA gene sequences, the genus Paenibacillus was proposed by Ash et al. (1993), and the description was later amended by Shida et al. (1997). At the time of writing, the genus comprises 277 species with validly published names (https://lpsn.dsmz.de/genus/paenibacillus). Strains of the genus have MK-7 as the predominant quinone and anteiso-C15:0 as the major fatty acid. Bacteria belonging to the genus Paenibacillus have been isolated from a variety of sources that were relevant to our humans, animals, plants and environment (Yoon et al. 2005; Hwang and Ghim 2017; Simon et al. 2017; Yun et al. 2017; Yang et al. 2021). Many Paenibacillus species have specific functions, such as promoting crop growth, producing antimicrobial compounds which can be used as medicine or pesticides, yielding enzymes including carboxylesterases, lipases, pectinases, cellulases, etc., which could be utilized for bioremediation, detergents, biofuel or healthcare (Grady et al. 2016). Thermotolerant enzymes are widely used in industry or academic studies due to their better stable characteristics under high-temperature processes, and hot springs are excellent sources for screening thermostable enzymes or microorganisms. In this study, a bacterial strain YIM B00624T isolated from a hot spring which represents a novel Paenibacillus species.

Materials and methods

Bacterial isolation and growth conditions

Strain YIM B00624T was isolated from the Tengchong Hamazui hot spring using T3 medium after 14 days of incubation at 45 ℃. The components of the medium include 1 g microcrystalline cellulose, 1 g casein acid hydrolyzed, 0.5 g CaCO3, 1 g FeSO4, 20 g agar, 1 L H2O, the pH is set to 6. The strain was maintained on ISP 2 agar at 4 ℃ and at 20% glycerol (w/v) at −80 ℃. Biomass for molecular and chemical research is on ISP2 without agar (pH 6.0) at 50 ℃ and 180 r.p.m. for 1 week.

Phylogenetic analysis

The extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Yang et al. (2021). Multiple alignments of the most closely related bacterial sequences were followed out using the CLUSTAL X 1.8 program (Thompson et al. 1997). Using Molecular Evolutionary Genetics Analysis 7.0 (Kumar et al. 2016), the phylogenetic tree was reconstructed by the neighbour-joining (Saitou and Nei 1987), maximum parsimony (Fitch 1971) and maximum-likelihood (Felsenstein 1981) algorithms. The stability of strain relationships was evaluated by bootstrap analysis of 1000 resamplings (Felsenstein 1985). Sequence similarity values between the most closely related strains were determined using the EzBioCloud 16S rRNA gene sequence database (https://www.ezbiocloud.net/; Yoon et al. 2017). The whole genome of strain YIM B00624T was sequenced using PacBio and Illumina Hiseq 2000 sequencers at Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China). Using SOAPdenovo software version 2.04 to assemble the sequenced reads (Li et al. 2010). The DNA G+C mol% value was obtained from the genomic sequences.

Physiology and morphology

Light microscopy (BX41, Olympus) and transmission electron microscopy (JEM-2100, JEOL) were used to observe the morphology, motility and size of cell, and the cells were grown exponentially. Gram staining was confirmed by KOH lysis test using standard Gram reaction (Cerny 1978). The temperature range of bacterial growth was determined by culture at 4, 10, 15, 20, 25, 30, 37, 40, 45, 50, 55 and 60 ℃ on ISP 2 for 1 week. The pH range for growth was determined by culture in ISP 2 without agar adjusted to pH 4.0–11.0 (at 1.0 intervals using the buffer system described by Xu et al. 2005). NaCl concentrations (0–10%, w/v, at 0.5% intervals) for growth of the strain were measured over a week. Oxidase activity was determined by dimethyl-p-phenylenediamine hydrochloride. Catalase activity was determined by titrating H2O2 solution to produce bubbles. Urease, lipase, gelatinase activities and H2S and melanin production were tested as described by Dong and Cai (2001). Substrate utilization and chemical sensitivity were determined using BIOLOG GEN III MicroPlate. The other enzyme activities and acid production of carbohydrates were tested using the API ZYM, API 20NE and API 50CH systems (bioMérieux) according to the manufacturer’s instructions.

Chemotaxonomic characteristics

Amino acids in cell walls and sugars in whole cell hydrolysates were analyzed according to the procedures described by Hasegawa et al. (1983); Lechevalier and Lechevalier (1970) and Tang et al. (2009). The extraction of polar lipids was determined by two-dimensional TLC and identified using previously described procedures (Collins and Jones 1980; Minnikin et al. 1979). Menaquinones were extracted according to Collins et al. (1977) and separated by HPLC (Tamaoka et al. 1983). The Sherlock Microbial Identification System (MIDI) was used to extract, methylate and analyze cellular fatty acids according to the manufacturer’s instructions. For fatty acid analysis, strain YIM B00624T was cultured at 45 ℃ on tryptic soy agar (Difco) for 4 days. The Microbial Identification software package (Sherlock Version 6.1; MIDI databaseTSBA6) was used to analyze fatty acid methyl esters (Sasser 1990).

Results and discussion

Phylogenetic analysis

An almost complete 16S rRNA gene sequence (1552 bp) of strain YIM B00624T was obtained. According to the comparative analysis of gene sequences, strain YIM B00624T was most similar to Paenibacillus filicis S4T (95.9%). These values were below the 98.7% cutoff point recommended for recognition of genomic species which need not to calculate overall genome related index (OGRI) (Stackebrandt and Ebers 2006; Kim et al. 2014). Based on the 16S rRNA gene sequences, the phylogenetic tree of strain YIM B00624T was constructed by clustering neighbour-joining algorithms, maximum-parsimony algorithms and maximum-likelihood algorithms at values (Fig. 1; Fig. S1, S2). The phylogeomic tree also indicated that strain YIM B00624T formed a stable phylogenetic lineage within the genus Paenibacillus (Supplementary Fig. S3). The whole genome of strain YIM B00624T contained 2 contigs, with a total length of 8,795,693 bp and the N50 length of 8,770,504 bp. Some other genome characteristics of strain YIM B00624T and other reference genomes are summarized in Table S1. Based on the preliminary analysis of the genome sequence of YIM B00624T, the peripheral pathways for metabolism of aromatic compounds such as biphenyl and benzoate using acetaldehyde dehydrogenase (EC 1.2.1.10), 4-hydroxy-2-oxovalerate aldolase (EC 4.1.3.39), etc., were found. Esterases including carboxylesterases and lipases are usually used as biocatalysts in a variety of industrial processes, including biochemical, food, pharmaceutical, and biological purposes (Nakamura et al. 2018). Three putative acetyl xylan esterases are acquired in the genome sequence of YIM B00624T and detailed characterizations will be proceeded in the future work. The DNA G+C content of strain YIM B00624T was 53.4 mol%, which comes from the genomic sequences. Analysis of these data showed that strain YIM B00624T was a new species of the genus Paenibacillus.

Fig. 1
figure 1

Neighbour-joining tree based on 16S rRNA gene sequences showing the relationships between strain YIM B00624T and other member of the genus Paenibacillus. Bootstrap values (> 50%) based on 1000 replicates are indicated at the branch nodes. Lactobacillus delbrueckii subsp. sunkii YIT 11221T was used as an outgroup. Bar, 0.01 changes per nucleotide position

Full size image

Physiology and morphology

Strain YIM B00624T was Gram-positive, aerobic and motile. The shape of the cells was rod-shaped with flagella and the endospores located terminally within a swollen sporangium (Fig. S4). Colonies were creamy yellow, flat and circular. Strain YIM B00624T was positive for catalase, oxidase, nitrate reduction, urease and H2S production, but negative for gelatin liquefaction. Tweens 20, 40, 60 and 80 are hydrolyzed. Other physiological characteristics of the strain are summarized in Table 1, Table S2 and in the species description.

Table 1 Differential characteristics of strain YIM B00624T and related Paenibacillus species
Full size table

Chemotaxonomic characteristics

The predominant menaquinone of strain YIM B00624T was MK-7. Cell wall amino acids of the strain contained meso-diaminopimelic acid as the diamino acid. Galactose (34.4%), glucose (23.8%), mannose (11.3%) and rhamnose (10.7%) as the major sugars, while ribose (7.9%), xylose (2.2%), arabinose (0.5%), as minor whole-cell sugar. The polar lipids of strain YIM B00624T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and four unidentified glycolipids (Fig. S5). The strain was found to contain anteiso-C15:0 (49.2%), anteiso-C17:0 (15.9%) and C16:0 (11.3%) as major fatty acids, iso-C16:0 (9.4%), iso-C15:0 (5.3%), iso-C17:0 (5.0%), C17:0 (2.2%) as minor fatty acids (Table S3).

Some chemotaxonomic characteristics, for example, major fatty acids and major menaquinone of strain YIM B00624T are consistent with other members of the genus Paenibacillus. On the basis of the phylogenetic and chemotaxonomic data described here, strain YIM B00624T should belong to the genus Paenibacillus. However, some distinct characteristics were easily distinguished from other strains of the genus Paenibacillus (Table 1). In summary, strain YIM B00624T is considered a novel species of the genus Paenibacillus, for which the name Paenibacillus hamazuiensis sp. nov. is proposed.

Description of Paenibacillus hamazuiensis sp. nov

Paenibacillus hamazuiensis (ha.ma.zui.en’sis. N.L. masc. adj. hamazuiensis pertaining to Hamazui Hot Spring).

A gram-positive, aerobic and motile bacterium. Colonies are creamy yellow, flat and circular. Cells are rods (width of 0.2–1.2 μm and length of 2.0–4.8 μm) and the endospores located terminally within a swollen sporangium. Growth occurs at 25–50 °C (with an optimum at 45 °C), at pH 6.0–8.0 (with an optimum pH range of 6.0) and NaCl tolerance range for growth is 0.0–2.5% (w/v) NaCl. The following substrates are utilized for growth (Biolog GENIII systems): dextrin, d-cellobiose, gentiobiose, sucrose, d-turanose, stachyose, α-d-lactose, d-melibiose, β-methyl-d-glucoside, d-salicin, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, N-acetyl-d-galactosamine, N-acetylneuraminic acid, α-d-glucose, d-mannose, d-fructose, d-galactose, 3-methyl glucose, l-fucose, l-rhamnose, inosine, d-sorbitol, d-mannitol, myo-inositol, glycerol, gelatin, glycyl-l-proline, l-alanine, l-arginine, l-aspartic acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, l-serine, pectin, d-galacturonic acid, l-galactonic acid, d-gluconic acid, d-glucuronic acid, glucuronamide, mucic acid, quinic acid, d-saccharic acid, d-lactic acid methyl ester, l-lactic acid, citric acid, α-keto-glutaric acid, d-malic acid, l-malic acid, bromo-succinic acid, tween 40, γ-amino-butryric acid, α-hydroxy-butyric acid, β-hydroxy-d, l butyric acid, acetoacetic acid, propionic acid, acetic acid, formic acid. Nitrate reduction, arginine dihydrate enzyme, urease, hydrolysis of aesculin, ρ-nitro-β-d-methylgalactosidase and the assimilation of glucose, arabinose, mannose, mannitol, maltose, malic acid were positive in tests using API 20NE. The results from API 50CH tests showed that acids were produced from l-arabinose, d-xylose, methyl-β-d-xylopyranoside, d-galactose, d-glucose, fructose, d-mannose, methyl-α-d-glucopyranoside, arbutin, esculin ferric citrate, salicin, d-celiobiose, d-maltose, d-l actose, d-melibiose, sucrose, d-trehalose, d-raffinose, starch, glycogen, gentiobiose, d-turanose and d-tagatose. In the API ZYM system, alkaline phosphatase (weak), esterase (C4), esterase lipase (C8), acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase (weak) and β-glucosidase activities are present. The diagnostic diamino acid of peptidoglycan is meso-diaminopimelic acid. Galactose, glucose, mannose and rhamnose are the major whole-cell sugars. The polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and four unidentified glycolipids. The predominant menaquinone is MK-7. Major fatty acids are anteiso-C15:0, anteiso-C17:0 and C16:0. The DNA G+C content of the strain is 53.4 mol%.

The type strain, YIM B00624T (= CGMCC 1.19245T = KCTC 43365T), was isolated from a Hamazui hot spring in Tengchong, Yunnan province, south-west China.

The GenBank accession numbers for the 16S rRNA gene sequence and genomic sequence are ON256646 and GCA_023276405.1, respectively.