Introduction

On the basis of the analysis of the 16S rRNA gene sequences of group 3 bacilli, the genus Paenibacillus was proposed by Ash et al. (1993) and then emended by Shida et al. (1997). At the time of writing, the names of more than 255 species of the genus Paenibacillus have been validly published (https://www.bacterio.net/paenibacillus.html). Some species had been reported to have the capacity of fixing nitrogen, such as P. polymyxa, P. taohuashanense, P. azotofixans, P. sabinae, P. sonchi, P. forsythiae and P. sophorae (Grau and Wilson 1962; Xie et al. 2012; Seldin et al. 1984; Ma et al. 2007; Hong et al. 2009; Ma and Chen 2008; Jin et al. 2011). Some Paenibacillus strains play a significant role in agriculture and industry (Seldin 2011). Previous studies have reported that microorganisms found in extreme environments can produce a variety of natural compounds and their specific mechanisms for adapting to extreme environments (Tang et al. 2002). In order to explore more multifunctional, special mechanisms and valuable microbes, we targeted halophilic microorganisms in hypersaline ecosystems. In the investigation of microbial resources of salt environment in Xinjiang province, one novel strain YIM B00363T was isolated from the hypersaline sediment of Wuzunbulake salt lake. In this study, we report the novel strain YIM B00363T belonging to the genus Paenibacillus by means of a polyphasic taxonomic study.

Methods and materials

Bacterial isolation and cultivation

Strain YIM B00363T was isolated from saline soil samples collected from Wuzunbulake salt lake, Turpan city, Xinjiang province, by the spread plate method on Nitrogen Fixing agar (NFA) medium (0.2 g KH2PO4, 0.2 g MgSO4, 0.2 g NaCl, 5.1 g CaCO3, 10 g mannitol, 15 g agar, 1L H2O and pH 7.0). The plates were then incubated at 37 °C for 7 days. Strain YIM B00363T was one of the isolates that appeared on the NFA plates under aerobic condition. Single colonies were purified by transferring them onto tryptic soy agar (TSA) medium plates. It was maintained on TSA at 4 °C and as a glycerol suspension (20%, v/v) at − 80 °C.

Phylogenetic and genotypic analysis

Genomic DNA for PCR amplification was prepared using TIANamp Bacteria DNA Kit (TIANGEN BIOTECH (BEIJING) CO., LTD), according to the manufacturer’s instructions. The 16S rRNA gene sequence of YIM B00363T was amplified using the universal bacterial primers PA (5′-CAGAGTTTGATCCTGGCT-3′) and PB (5′-AGGAGGTGATCCAGCCGCA-3′), synthesized by Sangon Biotech Co. Ltd (Shanghai, PR China). The amplification programme was used as described previously (Ueda et al. 2013). The PCR products were cloned into pEASY®-T5 Zero Cloning Vector (Kit, TransGen Biolech). The sequence was determined by the Tsingke Company (Beijing, PR China). Sequence similarities to the related type strains were calculated by using the EzBiocloud (https://www.ezbiocloud.net/) (Yoon et al. 2017). Multiple alignments with sequences of the most closely relatives were carried out using the CLUSTAL X 1.8 program (Thompson et al. 1997). Phylogenetic trees were reconstructed by the neighbour-joining (Saitou and Nei 1987), maximum parsimony (Fitch 1971) and maximum likelihood (Felsenstein 1981) tree-making algorithms using the software packages MEGA version 7.0 (Kumar et al. 2016). The stability of relationships was assessed by performing bootstrap analyses with 1000 resamplings (Felsenstein 1985).

The genome of YIM B00363T was sequenced using a PacBio + Illumina Hiseq at Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China). The sequenced reads were assembled using SOAPdenovo software version 2.04 (https://soap.genomics.org.cn/soapdenovo.html). The DNA G + C mol% value was obtained from the genomic sequences.

Phenotypic, physiological and biochemical characteristics

Cell morphology was examined by light microscopy (BX41; Olympus) and transmission electron microscopy (JEM-2100, JEOL) using cultivated on TSA at 37 °C for 2 days. The presence of endospores was investigated by using the phase contrast microscope (BX51M; Olympus) and transmission electron microscopy (JEM-2100, JEOL) when cells were cultivated on TSA plates at 37 °C for 10 days. Gram staining was performed by the Burke method (Nam et al. 2008) and the result was confirmed by the KOH test (Baron and Finegold 1990). Motility was analysed by the wet-mount method (Nam et al. 2008). Anaerobic growth was determined using the GasPak anaerobic system (BBL) according to the manufacturer’s instructions. Growth at different temperatures (4, 10, 15, 20, 25, 30, 35, 37, 40, 45, 50 and 55 °C) and NaCl concentrations (0–10%, w/v, at 1% intervals) were examined by using TSA medium at 37 °C for 14 days. The ability of the strain to grow at different pH values (4.0–10.0, at 0.5 intervals by using the buffer system described by Tang et al. 2010). Catalase activity was determined by production of bubbles after adding 3% H2O2 to the tested bacteria (Tarrand and Gröschel 1982). Tests for hydrolysis of gelatin, starch, Tween 20, 40 and 80, urease activity, nitrate reduction, and hydrogen disulphide production were determined by using traditional methods (Dong and Cai 2001). Carbon sources utilization and chemical sensitivity were determined by using BIOLOG GEN III MicroPlate. Using API ZYM systems tested enzyme activities. Acid production was detected by 50CH systems. Additional biochemical tests were performed using two Gallery systems (bioMérieux): API 20 NE and API 20 E according to the manufacturer’s instructions. Antibiotic susceptibility tests were performed using discs impregnated with various antimicrobial compounds.

Chemotaxonomic characteristics

Biomass for chemical and molecular studies was obtained by cultivation in TSA without agar (pH 7.0) at 37 °C and collected when the bacteria reached their mid-exponential phase. Analyses of diaminopimelic acid in the cell wall and sugars of whole-cell hydrolysates were performed according to the procedures described by Lechevalier and Lechevalier (1970) and Tang et al. (2009). Cellular fatty acids were extracted, methylated and analysed by using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions. Fatty acid methyl esters were analysed by using the Microbial Identification software package (Sherlock Version 6.1; MIDI databaseTSBA6) (Sasser 1990). The respiratory quinones of YIM B00363T were extracted from lyophilized cells (Collins et al. 1977), purified by TLC and then analysed by HPLC according to the methods of Xie and Yokota (2003). Polar lipids were extracted, examined by two-dimensional TLC and identified using the procedures described by Collins and Jones (1980) and Minnikin et al. (1979).

Results and discussion

Molecular phylogenetic analysis

An almost complete 16S rRNA gene sequence (1536 bp) of strain YIM B00363T was generated. YIM B00363T was most moderately related to P. hodogayensis SGT (Takeda et al. 2005) and P. ginsengarvi Gsoil 139T (Yoon et al. 2007) with 93.8% sequence similarity, which is far below 97%. The available data indicate that organisms having less than 97.0% 16S rRNA gene sequence similarity will not exhibit more than 60% reassociation, irrespective of the hybridization method applied (Stackebrandt and Goebel 1994; Keswani and Whitman 2001). Therefore, neither wet lab nor in-silico DNA-DNA hybridization were carried out. Meanwhile, these strains clustered in the genus Paenibacillus stably after phylogenetic analyses of the 16S rRNA gene sequences (Fig. 1, S1, S2). The NJ tree clustered YIM B00363T with P. residui MC-246T (93.2%), P. senegalensis JC66T (92.8%) and P. thermoaerophilus TC22-2bT (92.8%) (Fig. 1). The ML and MP trees showed similar results (Figs S1 and S2). According to 16S rRNA gene sequence similarities and topology of the phylogenetic trees, the literature data of above five strains, P. hodogayensis, P. ginsengarvi, P. residui, P. senegalensis and P. thermoaerophilus, were selected for comparison in this study. The DNA G + C content of strain YIM B00363T was 50.4 mol%. These data supported the finding that strain YIM B00363T represents a different genomic species of the genus Paenibacillus.

Fig. 1
figure 1

Neighbour-joining tree based on 16S rRNA gene sequences showing the relationships between strain YIM B00363T and some members of the genus Paenibacillus. Bootstrap values (> 50%) based on 1000 replicates are shown at the branch nodes. Asterisks indicate that the corresponding branches were also recovered in trees generated with the maximum parsimony and maximum likelihood methods. Bacillus subtilis DSM 10T was used as an outgroup. Bar, 1% sequence divergence

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Phenotypic, physiological and biochemical characteristics

YIM B00363T was Gram positive, aerobic, rod shaped without peritrichous flagella (Fig S3a), swollen by ellipsoidal spores and approximately 2–3 µm long and 0.5–1.5 µm wide (Fig S3b). It could grow at 20–45 °C (optimum at 37 °C), at pH 6.0–9.5 (optimum at 7.5–8.0) and in the presence of 0–5% (w/v) NaCl (optimum without NaCl). Other physiological characteristics are given in Table 1, Table S1 and in the species description.

Table 1 Phenotypic characteristics of strain YIM B00363T and the type strains of closely related species of the genus Paenibacillus
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Chemotaxonomic characteristics

Amino acids contained meso-diaminopimelic acid as the cell wall diamino acid, with glucose (11.2%), galactose (10.8%), xylose (12.7%), arabinose (20%) and fucose (18.1%) as the major whole-cell sugars (> 10%). The predominant menaquinone was MK-7. The polar lipids were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), unidentified phospholipids (PL), unidentified glycolipids (GL) and unknown lipids (UL). (Fig S4). The cellular fatty acid profile of strain YIM B00363T contained anteiso-C15:0 (53.52%), iso-C15:0 (15.04%) and C16:0 (12.76%) as major fatty acids (> 10%), and C14:0 (5.62%), iso-C16:0 (3.66%), anteiso-C17:0 (2.07%), iso-C14:0 (2.00%), iso-C17:0 (1.95%) and C12:0 (1.52%) as minor fatty acids. Compared with the data of other two reference strains, iso-C16:0 was minor fatty acid of YIM B00363T, but major fatty acid (> 10%) of others (Table 2).

Table 2 Cellular fatty acid profile of strain YIM B00363T and type strains of phylogenetically related species of the genus Paenibacillus
Full size table

All chemotaxonomic data conform to the characteristics of the genus Paenibacillus, which was described by Logan et al. (2009) and Tindall et al. (2010). However, there are some differences between strain YIM B00363T and four reference strains, for example, the content of iso-C16:0. Based on the phenotypic, genotypic, phylogenetic and chemotaxonomic data presented here, strain YIM B00363T should belong to the genus Paenibacillus. Thus, it represents a novel species, for which the name Paenibacillus turpanensis sp. nov. is proposed.

Description of Paenibacillus turpanensis sp. nov.

Paenibacillus turpanensis (tur.pan.en’sis. N.L. masc. adj. turpanensis relating to Turpan, the name of a city in Xinjiang, China, the geographical origin of isolation of the type strain).

Paenibacillus turpanensis is Gram stain-positive, non-motile, aerobic, rod-shaped, spore-forming bacterium. Cells are rods with a width of 0.5–1.5 μm and length of 2–3 μm. It can grow at 20–45 °C (optimum 37 °C), at pH 6–9.5 (optimum pH 7.5–8) and in the presence of 0–5% (w/v) NaCl (optimum without NaCl). It is positive for catalase activity, nitrate reduction, aesculin ferric citrate, hydrolysis of starch, and Tween 20, 40 and 80 and negative for production of H2S, production of indole, Voges–Proskauer test, hydrolysis of gelatin, l-arginine and urea. d-glucose, l-arabinose and maltose are assimilated. For enzyme activities (API ZYM system), it is positive for alkaline phosphatase, esterase C4, esterase lipase C8, lipase C14, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase. For the utilization of carbon sources (API Biolog GEN III systems), the following substrates are utilized for growth: dextrin, d-maltose, d-trehalose, d-cellobiose, gentiobiose, sucrose, d-turanose, Stachyose, d-raffinose, α-d-lactose, d-melibiose, d-salicin, N-acetyl-d-glucosamine, α-d-glucose, d-fructose, d-galactose, l-rhamnose, pectin, d-galacturonic acid, l-galactonic acid lactone, d-gluconic acid, d-glucuronic acid, glucuronamide and acetic acid as substrates. With API 50CH, acid is produced from glycerol, l-arabinose, d-ribose, d-xylose, methyl-βd-xylopyranoside, d-galactose, d-glucose, fructose, d-mannose, l-rhamnose, methyl-αd-glucopyranoside, aesculin, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-saccharose, d-trehalose, d-melezitose, d-raffinose, amidon (starch), glycogen, d-gentiobiose, d-turanose, d-tagatose and potassium 5-ketogluconate and weakly produced from l-sorbose, amygdalin, d-lyxose and l-fucose. It is sensitive to kanamycin (30 μg), gentamicin (10 μg), neomycin (30 μg), amoxicillin/clavulanic acid (10 μg), erythromycin (5 μg), carbenicillin (100 μg), chloramphenicol (30 μg), tetracycline (30 μg), novobiocin (5 μg) and streptomycin (300 μg). The whole-cell hydrolysates contain meso-diaminopimelic acid as the cell wall diamino acid. Glucose, galactose, xylose, arabinose and fucose are the major whole-cell sugars. The polar lipids are phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, unidentified glycolipids and unknown lipids. The predominant menaquinone is MK-7. Anteiso-C15:0 (53.52%), iso-C15:0 (15.04%) and C16:0 (12.76%) are the major fatty acids. The DNA G + C content of the type strain is 50.4 mol%.

The type strain is YIM B00363T (= CGMCC 1.17507T = KCTC 43184T), isolated from the saline soil of Turpan city in Xinjiang province, north-west China. The GenBank accession numbers of the 16S rRNA gene and the genome sequence of YIM B00363T are MT032315 and WTLI00000000, respectively.