Arch Microbiol DOI 10.1007/s00203-014-1032-9

ORIGINAL PAPER

Jilinibacillus soli gen. nov., sp. nov., a novel member of the family Bacillaceae Jingying Liu · Xiuran Wang · Meina Li · Qian Du · Qiyun Li · Pengda Ma 

Received: 5 June 2014 / Revised: 5 August 2014 / Accepted: 21 August 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract A Gram-positive, aerobic, rod-shaped, motile, endospore-forming bacterium, designated strain A12T, was isolated from a saline and alkali soil samples in Baicheng City, western of Jilin Province, China. Growth occurred in 15–45 °C (optimum, 30 °C) and at pH 7.0–11.5 (optimum, pH 9.0) and in the presence of 0–10 % (w/v) NaCl [optimum, 1–3 % (w/v) NaCl]. Meso-DAP was present in the peptidoglycan. The predominant menaquinone was MK-7. The major polar lipid profile was phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl inositol-methyl and phosphotidylinositol dimannosid.

Communicated by Erko Stackebrandt. Electronic supplementary material  The online version of this article (doi:10.1007/s00203-014-1032-9) contains supplementary material, which is available to authorized users. J. Liu · P. Ma (*)  College of Life Sciences, Northwest A&F University, Yangling 712100, People’s Republic of China e-mail: [email protected] X. Wang  College of Life Sciences, Jilin Agricultural University, Changchun 130118, People’s Republic of China M. Li  Chemical and Pharmaceutical Engineering Department, Southeast University Chengxian College, Nanjing 210088, People’s Republic of China Q. Du · Q. Li (*)  Institute of Plant Protection, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, People’s Republic of China e-mail: [email protected]

The major fatty acid (>10 % of total fatty acids) was anteiso-C15:0. DNA G + C content was 36.2 mol %. The level of 16S rRNA gene sequence similarity between strain A12T and other recognized species of the family was below 95.6 %. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the strain A12T fell with the family Bacillaceae and formed a distinct taxon. Based on physiological, chemotaxonomic and phylogenetic analyses, strain A12T was considered to represent a novel species of a new genus, for which the name Jilinibacillus soli gen. nov., sp. nov. was proposed. The type strain of Jilinibacillus soli was A12T (=GIMN1.014T = CCTCC M2011164T = KCTC 33417T). Keywords  Jilinibacillus soli · Bacillaceae · Halophilic alkaliphilic bacterium · 16S rRNA · Phylogenetic analysis

Introduction Moderately aerobic or facultatively anaerobic, Gram-positive or Gram-variable, endospore-forming, rod-shaped bacteria have been isolated from various environments. They were originally assigned to the genus Bacillus (Claus and Berkeley 1986), but, on the basis of phylogenetic and chemotaxonomic analyses, the family Bacillaceae has been reclassified. At present, the family Bacillaceae has 51 genera on the level with Bacillus (http://www.bacterio. net/). Saline and alkali soils host dense populations of microbes and, as such, provide vast potentially useful enzymes for an industry and stress-resistance genes for genetic modification of plants. Here, we describe the characterization of another strain, A12T, from a saline and alkali soil sample.

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Materials and methods Strain isolation Stain A12T was isolated from a saline and alkali soil sample collected in Baicheng City (45° 37′N 122° 50′E), western of Jilin Province, China, by the standard dilution plating technique on beef extract peptone agar [10 g beef extract, 10 g peptone, 5 g NaCl, 20 g agar and 1 L distilled water (pH 7.0)]. Plates were incubated for 7 days at 30 °C. Pure cultures were maintained as 30 % (v/v) glycerol suspensions at −70 °C.

Arch Microbiol

spectrophotometer fitted with a temperature program accessory (Marmur and Doty 1962). The Tm was determined by the graphical method described by Ferragut and Leclerc (1976), and the G + C content was estimated from this temperature using the equation of Owen & Hill (1979). Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA and purification of PCR products were performed as described by Cui et al. (2001). The resulting 16S rRNA gene sequence of strain A12T was compared with

Morphological and physiological characteristics Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy (HITACHI H7650). Gram reaction, motility, endospore observation and anaerobic growth used for determination were those described by Chen et al. (2008). Optimum growth was tested at different temperatures (4, 10, 20, 25, 30, 45, 50 and 55 °C) and different salt concentrations [0, 0.5, 1, 2, 3, 4, 5, 10, 15, 20 and 25 % (w/v) NaCl] on beef extract peptone medium and at different pH values (4, 5, 6, 7, 8, 9, 10, 11, 12, 13) in beef extract peptone medium. Catalase activity was determined by bubble production from 3 % (v/v) H2O2, and oxidase activity was determined using 1 % (w/v) tetramethylp-phenylenediamine. Enzyme activity was determined using the API ZYM system (bioMérieux). Carbon source utilization was tested using Biolog GEN III MicroPlate™. Acid production from carbohydrates, nitrated reduction and other physiological characteristics were carried out using API 50 CH system, API 20E system (bioMérieux).

Fig. 1  Transmission electron micrograph of strain A12T. Bar, 1 µm

Chemotaxonomy characteristics Diaminopimelic acid (DAP) isomers were analyzed using the method of Komagata and Suzuki (1987). Menaquinones were detected by HPLC according to Nishijima et al. (1997). Extraction and analysis of polar lipids by two-dimensional TLC were performed according to the methods of Minnikin et al. (1984). For fatty acids analysis, strain A12T was cultured on beef extract peptone medium for 48 h at 30 °C. Cellular fatty acid analysis was performed as described by Sasser (1990) using the Microbial Identification System (MIDI). GC content, PCR amplification, sequencing and phylogenetic analysis DNA was purified using the method of e midpoint value (Tm) of the thermal denaturation profile obtained at 260 nm with a Perkin-Elmer Lambda35 UV/Vis

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Fig. 2  Two-dimensional thin-layer chromatograms of polar lipids of strain A12T. PE phosphatidylethanolamine, PG phosphatidylglycerol, PI phosphatidylinositol, PIM phosphatidyl inositol-methyl, PIMD phosphotidylinositol dimannoside

Arch Microbiol Fig. 3  A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of strain A12T and related taxa. Bootstrap values are shown in percentages of 1,000 replicates, when more than 50 %. Bar, 0.01 substitutions per nucleotide position

sequences obtained from public databases to find its most closely related phylogenetic neighbors. Phylogenetic trees were generated using the neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Kluge and Farris 1969) methods in the MEGA program version 6.05. A bootstrap test with 1,000 replicates was used to determine the confidence of the branching patterns of the trees created (Felsenstein 1985). Nucleotide sequence accession number: The GenBank/ EMBL/DDBJ accession number of the 16S rRNA gene sequence of strain A12T is HQ693527.

Results and Discussion Cells of strain A12T were rod-shaped (0.6–0.7 × 1.5– 5.0 µm), Gram-positive, aerobic and motile (Fig. 1).

Colonies were cream in color, circular, flat and translucent after 3 days on beef extract peptone medium plates at 30 °C. Growth was observed at 15–45 °C (optimum, 30 °C) and at pH 7–11.5 (optimum, pH 9) and in the presence of 0–10 % (w/v) NaCl [optimum, 1–3 % (w/v) NaCl. Catalase was positive and oxidase was negative. Nitrate was reduced. Other physiological properties were given in the species description. Strain A12T possessed meso-DAP in the cell wall peptidoglycan. The predominant quinone detected was MK-7. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl inositol-methyl and phosphotidylinositol dimannosid (Fig.  2). The fatty acid profile contained anteiso-C15:0 (68.1 %), anteiso-C17:0 (9.7 %), iso-C15:0 (7.1 %), C16:0 (5.6 %), iso-C16:0 (2.8 %), iso-C17:0 (2.1 %) and iso-C14:0 (1.4 %).

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Table 1  Characteristics of used to distinguish strain A12T from its close phylogenetic relatives Characteristics

1

2

3

4

5

Colonial pigmentation

Cream

Creamy white

Beige

Orange to brownish

White

Spore position Anaerobic growth Salinity range for growth (optimum)  % pH range for growth (optimum) Temperature range for (optimum) (°C) H2S production

C − 0–10 (1–3)

T − 0.5–12.5(5–7.5)

C − 0–6(ND)

T + 0–6(ND)

C + 3–12(3)

7–11.5 (9)

6.5–8.5(7.5–8)

6.5–9.5(7–9)

7–10(7.5–8.5)

7–12(9)

15–45 (30)

20–55(35–40)

20–45(30)

15–40(25–30)

5–48(35)

+ + − +

− − − −

− + + ND

− + + +

+ + + ND

+ + − − − +

+ + ND + ND +

+ ND ND w ND ND

w − − − ND ND

+ + + + − +

+ + +

+ − − A4β(L-Orn–d-Asp) 36.9

ND ND ND A4β(L-Orn–d-Asp)

− + − ND

ND

ND

+ − + Alγ(meso-DAP) 40.6

ai-C15:0

i-C15:0, ai-C15:0, i-C16:0, ai-C17:0,

i-C15:0, ai-C15:0, ai-C17:0,

ai-C15:0, i-C15:0

i-C15:0, ai-C15:0, C16:0, ai-C17:0,

PE, PG, PI, PIM, PIMD

PG, DPG, PLs

PG, DPG, PLs

PE, PG, DPG, PLs

PE, PG, DPG, PLs

Catalase Oxidase Nitrate reduction Acid production from: d-Glucose d-Fructose d-Mannose d-Mannitol d-Melibiose Glycerol Hydrolysis of: Aesculin Gelatin Casein Peptidoglycan type DNA G + C content (mol %) Major fatty acids (> 10 % of total) Polar lipids

Alγ(meso-DAP) 36.2

T

Taxa: 1, strain A12   (data from this study); 2, Ornithinibacillus halophilus (Sánchez-Porro and Ventosa 2013); 3, Ornithinibacillus contaminans (Kämpfer et al. 2010); 4, Virgibacillus soli (Kämpfer et al., 2011); 5, Oceanobacillus polygoni (Hirota et al., 2013) +, positive; −, negative; w, weakly positive; ND, no data available; ai, anteiso-branched; i, iso-branched; C, central; T, terminal; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PI, phosphatidylinositol; PIM, phosphatidyl inositol-methyl; PIMD, phosphotidylinositol dimannoside; DPG, diphosphatidylglycerol; PLs, unidentified phospholipids; GL, unidentified glycolipid; DAP, diaminopimelic acid

The DNA G + C content was 36.2 mol %. 16S rRNA gene sequence-based phylogenetic analysis of strain A12T demonstrated a close relationship to members of the family Bacillaceae (Fig. 3, Supplementary Fig. S1 and S2). The highest level of 16S rRNA gene sequence similarity was found with Ornithinibacillus contaminans G8BT (95.6 %), Virgibacillus soli CC-YMP-6T (94.9 %) and Oceanobacillus polygoni SA9T (94.9 %). The values for sequence similarity between stain A12T and the type strains of other Virgibacillus, Oceanobacillus and Ornithinibacillus were 90.8–94.8, 91.7–94.7 and 94–94.8 %, respectively. A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain A12T is

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a member of the family Bacillaceae and with the isolated grouping on a sub-branch with Ornithinibacillus halophilus G8BT (Fig. 3). Phylogenetic trees based on maximumlikelihood and maximum-parsimony algorithms were also constructed, and although they demonstrated a different tree topology, the relationships among the members of the family Bacillaceae remained similar (Supplementary Fig. S1 and S2). In general, organisms sharing less than 97 % 16S rRNA gene sequence similarity do not have DNA– DNA reassociation values >70 % (a cutoff point representing (a criterion for delineating a genomic species; Stackebrandt and Goebel 1994), so DNA–DNA hybridization was not performed.

Arch Microbiol

Jilinibacillus soli (so’li. L. neut. gen. n. soli of soil)

NaCl (optimum, 1–3 % (w/v) NaCl). Positive for esterase (C4), esterase lipase (C8), leucine arylamidase, trypsin and α-chymotrypsin; negative for alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-bglucosaminidase, α-mannosidase, α-fucosidase. Utilizes dextrin, D-trehalose, sucrose, D-turanose, α-D-glucose, D-fructose, D-galactose, glycerol, D-serine, gelatin, L-histidine, pectin, D-lactic acid methyl ester, L-lactic acid, α-keto-glutaric acid, D-malic acid, tween 40, γ-amino-butryric acid, α-hydroxybutyric acid, β-hydroxy-D,L-butyric acid, α-keto-butyric acid, acetoacetic acid, acetic acid, formic acid, but does not utilize D-trehalose, D-cellobiose, gentiobiose, stachyose, D-raffinose, α-D-lactose, D-melibiose, β-methyl-Dglucoside, D-salicin, N-acetyl-D-glucosamine, N-acetylβ-D-mannosamine, N-acetyl-D-galactosamine, N-acetyl neuraminic acid, D-mannose, 3-methyl glucose, D-fucose, L-fucose, L-rhamnose, inosine, D-sorbitol, D-mannitol, D-arabitol, myo-inositol, D-glucose-6-PO4, D-fructose6-PO4, D-aspartic acid, glycyl-L-proline, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, L-serine, D-galacturonic acid, L-galactonic acid lactone, D-gluconic acid, D-glucuronic acid, glucuronamide, mucic acid, quinic Acid, D-saccharic acid, p-hydroxy-phenylacetic acid, methyl pyruvate, citric acid, L-malic acid, bromo-succinic acid, propionic acid. Acid is produced from glycerol, D-galactose, D-glucose, D-fructose, aesculin, D-maltose, D-lactose, sucrose, D-melezitose, D-turanose, but not from erythritol, D-arabinose, L-arabinose, D-ribose, D, L-xylose, D-ardonitol, mehyl-β-D-xylopyranoside, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, mehyl-α-D-mannopyranoside, mehyl-α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-celobiose, D-melibiose, D-trehalose, inulin, D-raffinose, starch, glycogen, xylitol, gentiobiose, D-lyxose, D-tagatose, D, L-fucose, D-arabitol, L-rarabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate. The type stain is A12T (= GIMN1.014T  = CCTCC M2011164T  = KCTC 33417T), isolated from saline and alkali soil sample collected in Jilin Province, China.

Display the following characteristics in addition to those described for the genus. Cells are rod-shaped (0.6– 0.7×1.5–5.0 µm), are cream in color, circular, flat and translucent after 3 days on beef extract peptone medium plates at 30 °C. Catalase is positive and oxidase is negative. Nitrate is reduced. Indole and H2S are produced. Aesculin, casein and gelatin are hydrolyzed. Growth occurs in 15–45 °C (optimum, 30 °C) and at pH 7.0–11.5 (optimum, pH 9.0) and in the presence of 0–10 % (w/v)

Acknowledgments  We thank Prof. H.H. Zhu (Guangdong Provincial Microbial Culture Collection and Application Key Laboratory, Guangdong Institute of Microbiology) for transmission electron microscopy and phylogenetic analysis. This work was supported by Chinese Universities Scientific Fund (QN2013035 and 2014YB044), Natural Science Basic Research Plan in Shaanxi Province of China (2013JQ3006), Specialized Research Fund for the Doctoral Program of Higher Education (20130204120018 and 20130204120038), Agromicrobiology Research and Utilization Innovative Research Team in Jilin Province (20121812), National Natural Science Foundation of China (31401839).

In terms of phenotypic characteristics, there were differences between strain A12T and the genus two most-related species of the genus Ornithinibacillus (Kämpfer et al. 2010; Sánchez-Porro and Ventosa 2013), V. soli (Kämpfer et al. 2011) and O. polygoni (Hirota et al. 2013) as summarized in Table 1. Strain A12T could also be distinguished from O. halophilus G8BT and O. contaminans CCUG 53201T by H2S production, peptidoglycan type, major fatty acids and polar lipids. Difference between strain A12T and Virgibacillus albus YIM 93624T occurred in colonial pigmentation, spore position, anaerobic growth, H2S production, oxidase reaction, acid production from d-fructose, hydrolysis of aesculin and casein, major fatty acids and polar lipids. Strain A12T was distinguishable from O. polygoni SA9T as its DNA G + C content was 36.2 lower than O. polygoni SA9T. Furthermore, there were differences in colonial pigmentation, anaerobic growth, oxidase reaction, acid production from d-mannose and d-mannitol, hydrolysis of gelatin, major fatty acids and polar lipids. On the basis of phenotypic, chemotaxonomic and genotypic data presented, strain A12T represents a novel species of a new genus, for which the name Jilinibacillus soli gen. nov., sp. nov. is proposed. Description of Jilinibacillus gen. nov. Jilinibacillus (Ji.li.ni.ba.cil’lus. L. masc. n. bacillus small rod; N.L. masc. n. jilinibacillus a rod from Jilin Province, China). Cells are Gram-positive, aerobic and motile. mesoDAP is present in the peptidoglycan. The predominant menaquinone is MK-7. The major polar lipid profile is phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl inositol-methyl and phosphotidylinositol dimannosid. The major fatty acid (>10 % of total fatty acids) is anteiso-C15:0. The DNA G + C content is 36.2 mol %. The phylogenetic position based on 16S rRNA gene sequence analysis is within the order Bacillaceae. The type species is Jilinbacillus soli. Description of Jilinibacillus soli sp. nov

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