Introduction

The genus Paenibacillus was proposed by Ash et al. (1993) on the basis of analysis of the 16S rRNA gene sequences of group 3 bacilli, with Paenibacillus polymyxa as the type species. At the time of writing, the genus Paenibacillus comprises more than 150 recognised species (http://www.bacterio.net/p/paenibacillus.html),some (flagged→) at LPSN are reclassified. Members of the genus Paenibacillus are either Gram-stain positive or negative, facultatively anaerobic or strictly aerobic, non-pigmented, rod-shaped, motile and produce ellipsoidal spores (Ash et al. 1993; Osman et al. 2006; Priest 2009). The DNA G+C content ranges from 39 to 59 mol% (Yao et al. 2014). The predominant menaquinone is MK-7 and the main cellular fatty acid is anteiso-C15:0 (Ludwig et al. 2009).

Some Paenibacillus species have shown the considerable potential applications in agricultural production because they can fix nitrogen, produce various enzymes (Choi et al. 2008; Li et al. 2014) and some antimicrobial substances (Jin et al. 2011; Xie et al. 2012). During the course of screening of anaerobic petroleum-degrading bacteria from saline silt samples collected from the Chinese Sea of Death located in Suining city, Sichuan province, China, a Paenibacillus-like strain, designated strain LAM0A28T was isolated. By using a polyphasic taxonomic approach, we conclude here that strain LAM0A28T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus salinicaeni sp. nov. is proposed.

Materials and methods

Isolation and culture of bacterial strains

Strain LAM0A28T was isolated from a sea silt sample collected from the Chinese Sea of Death located in Suining city, Sichuan province, China. An inorganic salt medium (ISM) was used for this study and consisted of the following materials (per liter): 1 g of NH4NO3, 0.2 g of MgSO4·7H2O, 0.03 g of CaCl2·2H2O, 1 g of K2HPO4, 1 g of KH2PO4, 0.25 g l-Cysteine HCl, 1 mg resazurin, pH adjusted to 7.5 with 1 N NaOH. The medium was sterilised by autoclaving at 121 °C for 20 min. The enrichment and isolation medium was ISM supplemented with 1 % (v/v) petroleum or hexadecane. The anaerobic technique of Hungate (Hungate, 1969; Bryant 1972; Miller and Wolin 1974) was used throughout the enrichment and isolation. One of the isolates obtained, designated strain LAM0A28T, was purified at least twice before being preserved in 25 % (v/v) glycerol at -80 °C for further study.

Biomass for chemotaxonomic and molecular studies was obtained from TSB medium (BD/Difco 211825, Sparks, MD, USA) at 30 °C for 2 days. The proposed minimal standards for the description of aerobic, endospore-forming bacteria (Logan et al. 2009) were followed. The reference type strains Paenibacillus jilunlii DSM 23019T and Paenibacillus graminis DSM 15220T were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ; Germany). All strains were cultured under the same conditions for comparative analyses.

Morphological, physiological and biochemical characteristics

Cell morphology of an exponentially growing culture of strain LAM0A28T were examined using light microscope (Nikon 80i, Tokyo, Japan) and transmission electron microscope (Hitachi 7500, Tokyo, Japan) (Chen et al. 2015; Han et al. 2015). Gram-staining reaction was carried out according to the method described by Smibert and Krieg (1994). Cell motility was examined in semi-solid TSB medium (0.4 % agar added). The pH, temperature and NaCl ranges for growth were determined in LD medium (per litre water: 10 g tryptone, 5 g yeast extract, 2.5 g NaCl, pH 7.0). Growth characteristics of strain LAM0A28T were determined at various temperatures (4, 10, 15, 20, 25, 30, 35, 40, 45 and 50 °C) and pH values (4.0-11.0, at 0.5 unit intervals) in LD medium for 5 days. Tolerance of NaCl was tested at different salt concentrations [0-3 % (w/v) at 0.5 unit intervals and 3–8 % (w/v) at 1 unit intervals]. The medium was adjusted to the desired pH by using sterile solutions of citric acid/Na2HPO4 (pH 4.0–5.0), Na2HPO4/NaH2PO4 buffer (pH 6.0–8.0), NaHCO3/Na2CO3 buffer (pH 9.0–10.0) or Na2HPO4/NaOH buffer (pH 11.0) (Ruan et al. 2014). Catalase activity was detected by placing drops of 3 % (v/v) H2O2 onto plate grown cultures and observing the production of oxygen bubbles. Oxidase activity was determined by using 1 % (w/v) tetramethyl-p-phenylenediamine. Examinations of antibiotic susceptibility were performed on TSA medium by using susceptibility discs with various antibiotics and the inhibition zones were judged according to the manufacturer’s instruction. The following biochemical characteristics were studied using the methods described by Smibert and Krieg (1994): starch hydrolysis, gelatin liquefaction, Tween 20, Tween 60 and Tween 80 digestion, methyl red tests. More biochemical properties were determined by using the Biolog GP2 MicroPlates (Biolog, Hayward, CA, USA) and API 20NE, API ZYM, API 50CH test systems (bioMérieux, L´Etoile, France) according to the manufacturers’ instructions. The nitrogenase activity of strain LAM0A28T was tested with the acetylene reduction assay method (Berge et al. 2002).

Phylogenetic and genomic related analyses

Extraction and purification of genomic DNA from strain LAM0A28T was performed as described by Mandel and Marmur (1968). The 16S rRNA gene was amplified by PCR using prokaryotic 16S rRNA universal primers 27F and 1492R (Weisburg et al. 1991). The nifH gene was amplified using PolF and PolR (Poly et al. 2001). Sequence similarity and multiple sequence alignment were analysed using the EzTaxon-e service (Kim et al. 2012), NCBI BLAST program (http://www.ncbi.nlm.nih.gov) and CLUSTAL W (Thompson et al. 1994). Phylogenetic trees were constructed using MEGA 6 software (Tamura et al. 2013). Tree topology was evaluated by neighbor-joining method (Saitou and Nei 1987), maximum likelihood (Felsenstein 1981) and maximum-parsimony method (Fitch 1971) with 1000 replications bootstrap analysis.

The genomic DNA G+C content was determined by the thermal denaturation method (Marmur and Doty 1962) using a Beckman DU 800 spectrophotometer (Beckman Coulter, Brea, CA, USA). Escherichia coli K-12 was used as the reference strain. The DNA–DNA reassociation values were detected by measuring the renaturation rates of the denatured DNAs as described by De Ley et al. (1970) and Huss et al. (1983). The experiments were carried out in quintuplicate.

Chemotaxonomic characterisation

Chemotaxonomic analyses were executed on the strain LAM0A28T and the reference strains under the same conditions. All strains were incubated in TSB medium. Cells were harvested in the late exponential phase of growth at 30 °C. The cellular fatty acid analyses were performed on strain LAM0A28T, P. jilunlii DSM 23019T and P. graminis DSM 15220T as described by Sakamoto et al. (2002). Identification and quantification of the cellular acids were performed using the Sherlock Microbial Identification System with the standard MIS Library Generation Software (Version 6.0 and Date 4, Microbial ID Inc., Newark, DE, USA). The respiratory quinones were analysed from strain LAM0A28T by using reversed-phase HPLC as described previously (Komagata and Suzuki 1987). The polar lipids of strain LAM0A28T were extracted and separated on silica gel plate (10 × 10 cm, Merck 5554) (Kates 1986) and further analyzed with the method described by Minnikin et al. (1984) and Xu et al. (2011). Molybdatophosphoric acid was used to reveal total polar lipids. Aminolipids were determined using ninhydrin reagent and phospholipids were identified by Zinzadze reagent. The results were analysed as described by Fang et al. (2012). The cell wall peptidoglycan structure of strain LAM0A28T was analysed by TLC (Komagata and Suzuki 1987) with the methods described by Schleifer (1985). The sugar profile of strain LAM0A28T was analysed according to the method described by Lechevalier and Lechevalier (1980).

Results and discussion

Morphological, physiological and biochemical characteristics

Cells of LAM0A28T were observed to be motile, straight-rod shaped with a cell size of 0.6–1.0 µm in width and 2.2–12.6 µm in length (Fig. 1). The isolate was found to be Gram-stain positive, facultatively aerobic and spore-forming (Fig. S1). Colonies of strain LAM0A28T cultivated on TSA for 72 h at 30 °C were observed to be milky, circular, convex, semi-translucent and smooth. Growth was observed at 15–45 °C (optimum 30–35 °C), pH 5.0–10.0 (optimum pH 7.5) and 0–5 % (w/v) NaCl (optimum 0.5 %). Growth was not inhibited by 0.001 % (w/v) lysozyme. Strain LAM0A28T was found to be catalase positive and oxidase negative. Cells were found to be positive for the methyl red test. Strain LAM0A28T was found to be resistant to (µg per disc unless otherwise indicated) carbenicillin (100), chloramphenicol (30), kanamycin (30), streptomycin (10) and tetracycline (30), but sensitive to ampicillin (10), gentamicin (10), neomycin (30) and polymixin B (300 U). In API ZYM tests, strain LAM0A28T was found to be positive for alkaline phosphatase, leucine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase; and weakly positive for esterase (C4), esterase lipase (C8), valine arylamidase and β-glucosidase. In the API 20NE system, the hydrolysis of esculin and 4′-nitrophenyl-β-d-galactopyranoside, and the assimilation of d-glucose, l-arabinose, d-mannose, mannitol, N-acetyl-glucosamine, d-maltose and potassium gluconate were found to be positive. Nitrate was reduced to nitrite. With API 50CH, acid was found to be produced from glycerol, l-arabinose, d-ribose, d-xylose, d-galactose, d-glucose, d-fructose, d-mannose, inositol, d-mannitol, amygdalin, arbutin, aesculin, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-sucrose, d-trehalose, d-raffinose, starch, glycogen, d-gentiobiose, d-turanose, d-fucose and potassium gluconate. The differences in the physiological and biochemical characteristics between strain LAM0A28T and its relatives are shown in Table 1. The detailed results obtained from the API system are shown in Table S1. The result of nitrogenase activity assay (Table 2) indicated that strain LAM0A28T exhibits a relatively high nitrogenase activity compared to the reference strains.

Fig. 1
figure 1

Transmission electron micrograph of cells of strain LAM0A28T. Bar, 1 μm

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Table 1 Differential characteristics of strain LAM0A28T and its close phylogenetic relatives
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Table 2 Nitrogenase activity of strain LAM0A28T compared with the reference type strains
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Molecular analyses

Phylogenetic analysis based on the nearly complete 16S rRNA gene sequence of strain LAM0A28T (1476 nt, GenBank accession number KM260652) indicated that the strain is a member of the genus Paenibacillus and closely related to strains P. jilunlii DSM 23019T and P. graminis DSM 15220T with 97.5 and 97.2 % sequence similarity, respectively (Fig. 2). Phylogenetic trees constructed based on the maximum-likelihood and maximum-parsimony algorithms also supported this conclusion (Fig. S2 and S3). Levels of 16S rRNA gene sequence similarity between strain LAM0A28T and other recognised members of the genus Paenibacillus were below 97.0 %. The DNA–DNA hybridization values between strain LAM0A28T and P. jilunlii DSM 23019T and P. graminis DSM 15220T were 30.2 ± 1.6 and 44.7 ± 2.1 %, respectively. The DNA G+C content of strain LAM0A28T was determined to be 51.2 mol%, which is within the range of values reported for members of the genus Paenibacillus. A 365 bp segment of the nifH gene (GenBank accession number KU171013) was amplified from strain LAM0A28T. The nifH gene sequence similarities between strain LAM0A28T and P. graminis DSM 15220T, P. riograndensis SBR5T, P. sonchi X19-5T, P. jilunlii DSM 23019T are 97, 92, 91 and 91 %, respectively. Phylogenetic analysis based on nifH gene sequences revealed that strain LAM0A28T clusters with species of the genus Paenibacillus (Fig. S4, S5 and S6).

Fig. 2
figure 2

Neighbor-joining phylogenetic tree based on a comparison of the 16S rRNA gene sequences of strain LAM0A28T and its closest relatives. Genbank accession numbers are given in parentheses. Bar, 5 nucleotide changes per 1000 nucleotides. Only bootstrap values >50 % are shown at the branch points

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Chemotaxonomic characteristics

The major fatty acids of strain LAM0A28T were identified as anteiso-C15:0 (35.8 %), C16:0 (17.9 %), iso-C16:0 (14.0 %) and C14:0 (10.9 %). The detailed fatty acid compositions of strain LAM0A28T, P. jilunlii DSM 23019T and P. graminis DSM 15220T are shown in Table 3. The predominant menaquinone was identified as MK-7. The major polar lipids of strain LAM0A28T were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids and six unidentified lipids (Fig. S7). The diamino acid of the cell wall peptidoglycan was determined to be meso-diaminopimelic acid. The sugar profile was found to contain xylose, ribose and traces of galactose (Fig. S8).

Table 3 Cellular fatty acid contents of strain LAM0A28T and the related type strains of the genus Paenibacillus
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Taxonomic conclusion

Strain LAM0A28T was observed to be Gram-positive, facultatively aerobic, motile, endospore-forming and rod-shaped. The predominant fatty acids and menaquinone were anteiso-C15:0 and MK-7, respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids and six unidentified lipids. In combination with the analysis of the 16S rRNA gene sequence, nifH gene sequence and DNA G+C content, it is concluded that strain LAM0A28T is a member of the genus Paenibacillus. However, evident distinctions were exhibited between strain LAM0A28T and the closely related strains P. jilunlii DSM 23019T and P. graminis DSM 15220T in morphological features, growth conditions, catalase activity, hydrolysis of esculin, antibiotic resistance, enzyme activities, and acid production from carbohydrates (Table 1). The predominant fatty acids in strain LAM0A28T were the same as or similar to the reference strains in this study (Table 3), although C16:1ω11c was found only in strain LAM0A28T. Differences in the nitrogenase activity were also found between strain LAM0A28T and the reference strains. The low DNA–DNA hybridization values (30.2 ± 1.6 and 44.7 ± 2.1 %) between the novel strain and its close relatives precludes genomic relatedness and supports the designation of strain LAM0A28T as the type strain of a novel species within the genus Paenibacillus (Stackebrandt and Goebel 1994). Based on the phylogenetic, phenotypic, and chemotaxonomic characteristics, strain LAM0A28T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus salinicaeni sp. nov. is proposed.

Description of Paenibacillus salinicaeni sp. nov

Paenibacillus salinicaeni (sa.li.ni.cae’ni. L. adj. salinus saline; L. neut. n. caenum mud, silt; N.L. gen. n. salinicaeni of saline silt).

Cells are Gram-stain positive, facultatively anaerobic, motile and straight-rod shaped. Ellipsoidal terminal spores are formed in swollen sporangia. Growth occurs with 0–5 % (w/v) NaCl (optimum 0.5 %), at pH 5.0–10.0 (optimum pH 7.5) and at 15–45 °C (optimum 30–35 °C). Catalase positive and oxidase negative. Methyl red reaction is positive. Growth is not inhibited by 0.001 % (w/v) lysozyme. Nitrate is reduced to nitrite. Starch, esculin and 4′-nitrophenyl-β-d-galactopyranoside are hydrolysed but gelatin, urea, Tween 20, Tween 60 and Tween 80 are not. The major fatty acids (>10 % of total) are anteiso-C15:0, C16:0, iso-C16:0 and C14:0. The cell wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinone is MK-7. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids and six unidentified lipids. The genomic DNA G+C content is 51.2 mol% as determined by the T m method.

The type strain is LAM0A28T (=ACCC 00741T = JCM 30850T), which is isolated from a saline silt sample collected from Chinese Sea of Death located in Suining city, Sichuan province, China. The 16S rRNA gene sequence of strain LAM0A28T has been deposited in GenBank under accession number KM260652.