Curr Microbiol (2014) 68:629–634 DOI 10.1007/s00284-014-0518-7

Oceanobacillus halophilum sp. nov. Isolated from a Mangrove Forest Soil Jia Tang • Guiqin Yang • Yueqiang Wang Chu Wu • Shungui Zhou

•

Received: 24 June 2013 / Accepted: 24 November 2013 / Published online: 16 January 2014 Ó Springer Science+Business Media New York 2014

Abstract A halophilic, aerobic bacterium, designated GD01T, was isolated from a mangrove forest soil near the South China Sea. Cells of strain GD01T were Gram staining positive, oxidase positive, and catalase positive. The strain was rod shaped and motile by means of peritrichous flagella and produced ellipsoidal endospores. The strain was able to grow with NaCl at concentrations of 0.5–12 % (optimum 3–5 %, w/v), at temperatures of 20–50 °C (optimum 30 °C), and at pH 6.0–8.5 (optimum pH 7.0). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain GD01T formed a cluster with O. profundus DSM 18246T (96.4 % 16S rRNA gene sequence similarity), O. caeni KCTC 13061T (95.4 %), and O. oncorhynchi JCM 12661T (94.5 %). The G?C content of strain GD01T was 38.7 mol%. The major respiratory quinone was MK-7. The major cellular fatty acids ([5 %) were anteiso-C15:0, iso-C16:0 (13.7 %), anteiso-C17:0 (12.6 %), iso-C15:0 (9.9 %), iso-C14:0 (9.5 %), and C16:0 (5.0 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, glycolipid, four unknown lipids, and four unknown phospholipids. Based on phenotypic characteristics, chemotaxonomic features, and phylogenetic analysis based on 16S rRNA gene sequences, the

strain was identified to represent a distinct novel species in the genus Oceanobacillus, and the name proposed is Oceanobacillus halophilum sp. nov. with type train GD01T (=CCTCC AB 2012863T = KCTC 33101T).

Introduction The genus Oceanobacillus includes 13 species and subspecies, which were isolated from various environments, such as algal mat in a sulfur spring [1, 2], biofilm on the mural painting [3, 4], chironomid egg mass [5], sediment core [6], and activated sludge [7]. The first proposed Oceanobacillus species is Oceanobacillus iheyensis, which was isolated from a deep-sea mud sample collected at a depth of 1,050 m on the Iheya Ridge of the Nansei Islands [8]. Bacteria belonging to the genus Oceanobacillus are rod shaped, endosporeforming, and moderately halophilic [7]. In this study, a Gram staining positive bacterium, GD01T, was isolated from a mangrove forest soil near the South China Sea. On the basis of polyphasic taxonomic studies, the strain is proposed to represent a novel species of the genus Oceanobacillus.

Jia Tang and Guiqin Yang have contributed equally to this study. The GenBank Accession Number for the 16S rRNA gene sequence of O. halophilum GD01T is JX274441.

Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0518-7) contains supplementary material, which is available to authorized users. J. Tang  G. Yang  Y. Wang  C. Wu  S. Zhou (&) Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650, People’s Republic of China e-mail: [email protected]

Materials and Methods Bacterial Isolation and Cultivation Strain GD01T was isolated from a soil sample which was taken from 20 cm below the top surface of a mangrove forest near the South China Sea in Shenzhen, Guangdong Province. The annual temperature is 22.5 °C, and the soil pH is 6.7. About 5.0 g of soil sample was suspended in

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50 ml sterilized phosphate buffer solution (PBS; 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4). The suspension was incubated at 30 °C with shaking for one night, and 200 ll of culture was taken out, serially diluted, spread onto nutrient agar (NA) plates, and incubated at 30 °C. After culture for 24 h, well-isolated colonies were picked using sterilized tooth picks and transferred to fresh NA plates. The colony isolation procedure was repeated until single colonies were obtained.

J. Tang et al.: Oceanobacillus halophilum sp. nov.

[17] after multiple alignment of the sequence data with CLUSTAL_X [18]. Distances were calculated using distance options according to Kimura’s two-parameter model [19], and clustering was performed with the neighborjoining method [20] and minimum-evolution method [21]. Statistical support for the branches of phylogenetic trees was determined using bootstrap analysis (based on 1,200 resamplings) [22]. Chemotaxonomic Characterization

Morphology and Phenotypic Studies Cell morphology was determined with a JEM 1400 transmission electron microscopy (JEQL, Japan) after cultivation on NA plates at 30 °C for 12 h. In preparation for electron microscopy, the bacterial cells were suspended in PBS, dried on a nickel-coated mesh, and negatively stained with phosphotungstic acid. The motility of cells was tested by observing the growth spread in a test tube containing semi-solid NA medium. Endospores were observed with a transmission electron microscope after cells were grown for 3 days according to [9]. The Gram staining reaction was carried out according to Smibert and Krieg [10]. The pH range (pH 5.0–10.0 at intervals of 0.5 pH unit) for growth was determined in NB buffered with citrate/phosphate buffer or Tris/hydrochloride buffer [11]. Tolerance of NaCl was determined in NB containing 0.5–15 % NaCl (w/ v) with increments of 0.5 %. Growth was examined under various temperatures (10, 15, 20, 25, 30, 35, 40, 45, and 50 °C) in NB. Anaerobic growth was assessed on NA under an atmosphere of 100 % N2. Growth on MacConkey agar was also tested. Catalase and oxidase ativities were tested as described by McCarthy and Cross [12]. Other physiological and biochemical properties were determined according to Dong and Cai [13]. Carbon metabolic, enzyme activities, and acid production characteristics were examined with the ID 32GN, API 20E, and API 50CH systems (bioMe´rieux) according to the manufacturer’s instructions. Phylogenetic Analysis Genomic DNA was extracted according to standard procedures [14]. The 16S rRNA gene was PCR amplified from genomic DNA using two bacterial universal primers (27f and 1492r) [15]. The PCR product was gel purified using Gel Extraction Kit D2500-01 (Omega Bio-tek) and sequenced by Sangon (Shanghai, China) using the 27f and 1492r primer pair as the sequencing primers. Pairwise sequence similarity was calculated using a global alignment algorithm implemented at the EzTaxon server (http:// eztaxon-e.ezbiocloud.net/) [16]. Phylogenetic analysis was carried out using the software package MEGA version 4.0

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The G?C content of the genomic DNA was determined by HPLC according to the method of Mesbah et al. [23]. For cellular fatty acid analysis, cells of strain GD01T and the reference strains were grown on NA at 30 °C for 24 h to exponential phase. Fatty acids in whole cells were saponified, methylated, and extracted using the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B). The fatty acids were analyzed with GC (Agilent Technologies 6850) and identified using the TSBA6.0 database of the Microbial Identification System [24]. Respiratory quinones were extracted according to Collins et al. [25] and analyzed with HPLC as described by Tamaoka et al. [26]. Polar lipids were extracted, examined by two-dimensional TLC, and identified using previously described procedures [27].

Results and Discussion The isolate was halophilic and aerobic, and the cells were Gram positive, catalase positive, and oxidase positive. Gelatin and casein were not hydrolyzed. Cells of strain GD01T were motile straight rods (0.2–0.5 lm wide and 1.2–2.0 lm long) with peritrichous flagella (Fig. S1). The cells have ellipsoidal endospores which were produced centrally, causing swelling of the sporangia (Fig. S1). Colonies were white pigmented and circular with entire edges. The strain grew in the presence of 0.5–12 % (w/v) NaCl with optimum growth at 3–5 % (w/v) NaCl, at 20–50 °C (optimum 30 °C), and at pH 6.0–8.5 (optimum pH 7.0). Detailed morphological, physiological, and biochemical characteristics were summarized in the description of species and Table 1 below. A nearly complete 16S rRNA gene sequence was determined for GD01T (1,462 bp). Phylogenetic analysis based on 16S rRNA gene sequence revealed that the strain GD01T was most closely related to O. profundus DSM 18246T (96.4 % 16S rRNA gene sequence similarity), followed by O. caeni KCTC 13061T (95.4 %) and O. oncorhynchi JCM 12661T (94.5 %), and formed a cluster with them in the neighbor-joining tree (Fig. 1) and minimumevolution tree (Fig. S2). Given that the levels of 16S rRNA

J. Tang et al.: Oceanobacillus halophilum sp. nov. Table 1 Distinctive characteristics of strain GD01T and its closest phylogenetic neighbors

631

1

2

3

4

Flagella

Peritrichous

Subpolar

Peritrichous

Peritrichous

Endospore position

C

C

T

ST

pH range

6.0–8.5

6.0–9.0

6.5–9.5

9.0–10.0

pH optimum

7.0

7.0

7.5-8.5

9.5

NaCl range (%, w/v)

0.5–12

0–10

0–14

0–22

NaCl optimum (%, w/v)

3–5

2–5

1–3

7

Growth at 15 °C

–

–

?

?

50 °C

?

–

–

–

Temperature optimum (°C)

30

30–40

35

30–36

–

–

–

?

Anaerobic growth Hydrolysis of Data were taken from this study unless indicated

Gelatin

–

–

?

–

Casein

–

ND

?

–

–

?

?

–

D-Xylose

–

?

?

–

D-Galactose

–

–

?

?

D-Glucose

–

?

?

?

D-Fructose

–

?

?

?

D-Mannose

–

–

?

?

D-Dorbitol

–

?

–

–

D-Maltose

–

?

?

?

Data taken from Nam et al. [7]

D-Melibiose

–

w

–

?

–

–

–

?

b

D-Trehalose

–

–

–

?

D-Raffinose

–

–

–

?

38.7

33.6a

40.2b

38.5c

Strains 1 Oceanobacillus halophilum sp. nov. GD01T, 2 Oceanobacillus caeni KCTC 13061T, 3 Oceanobacillus profundus DSM 18246T, 4 Oceanobacillus oncorhynchi JCM 12661T. ? positive, - negative, w weakly positive, ND not determined. For endospore position C central, ST subterminal, T terminal, PC paracentral a

Data taken from Kim et al. [6]

c

Data taken from Yumoto et al. [2]

Acid production from D-Ribose

D-Saccharose

G?C (mol%)

gen sequence similarity were all lower than 97 % between strain GD01T and the phylogenetic neighbors, DNA–DNA hybridization studies were not carried out. It was clear from the phylogenetic analysis based on 16S rRNA gene sequence that the new isolate can be grouped as a member of Oceanobacillus and represents a distinct phyletic line that can be considered as a separate genomic species [28]. The G?C content of the genomic DNA of strain GD01T was determined to be 38.7 mol%. The quinone system of the strain contained MK-7 as the predominant component. Cellular fatty acid analysis revealed that the major fatty acids were anteiso-C15:0 (34.5 %), iso-C16:0 (13.7 %), anteiso-C17:0 (12.6 %), iso-C15:0 (9.9 %), iso-C14:0 (9.5 %), and C16:0 (5.0 %). The fatty acid composition of the isolate was similar to that of other species of the genus Oceanobacillus in that anteiso-C15:0 is the predominant fatty acid [29], but there were differences in the presence of C17:0, anteiso-C13:0, and anteiso-C17:0 (Table 2). The polar lipids consisted of major amounts of diphosphatidylglycerol and phosphatidylglycerol, minor amounts of glycolipid, four unknown lipids, and four unknown phospholipids (Fig. S3).

With a G?C content of 38.7 mol%, stain GD01T contains MK-7 as the major respiratory quinone, and anteisoC15:0 and iso-C16:0 as the predominant fatty acids. This and its position in the phylogenetic trees support the affiliation of strain GD01T with the genus Oceanobacillus. However, it can be differentiated clearly from its closely related species in genus Oceanobacillus by characteristics such as flagella position, temperature range for growth, and some chemotaxonomic feature. In detail, the flagella position of strain GD01T was similar to O. profundus and O. oncorhynchi but not to O. caeni. Strain GD01T can grow at 50 °C but not at 15 °C, but its closely related strains present in the opposite phenomenon. The strain GD01T cannot hydrolyze gelatin and casein. In addition, acid is not produced from D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-dorbitol, D-maltose, D-melibiose, T D-saccharose, D-trehalose, or D-raffinose for strain GD01 . But at least one of the reference strains is positive for these items. Moreover, strain GD01T showed \97 % 16S rRNA gene sequence similarity to its closest relatives, and other phenotypic and chemotaxonomic differences such as the

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J. Tang et al.: Oceanobacillus halophilum sp. nov. Oceanobacillus oncorhynchi subsp. oncorhynchi LMG 23637T (AB188089)

82

Oceanobacillus oncorhynchi subsp. oncorhynchi LMG 23637T (AB188089)

99

Oceanobacillus sojae Y27T (AB473561) 92

64

Oceanobacillus neutriphilus A1gT (EU709018) Oceanobacillus kimchii X50T (GU784860)

100

Oceanobacillus iheyensis HTE831T (BA000028) Oceanobacillus indicireducens A21T (AB623011)

80

Oceanobacillus chironomi LMG23627T (DQ298074) Oceanobacillus halophilum GD01T (JX274441)

73

Oceanobacillus caeni S-11T (AB275883) Oceanobacillus profundus CK-MP28T (DQ386635) Ornithinibacillus contanimans CCUG 53201T(FN597064)

52

Oceanobacillus picturae LMG19492T (AJ315060)

88

Oceanobacillus kapialis SSK2-2T (AB366005)

98 96

Oceanobacillus manasiensis YD3-56T (FJ386518) Bacillus koreensis BR030T(AY667496)

Ornithinibacillus halophilus G8BT(HQ433440) Ornithinibacillus bavariensis WSBC 24001T(Y13066) 78

Paucisalibacillus globules B22T(AM114102) Virgibacillus chiguensis NTU-101T(EF101168) Paraliobacillus quinghaiensis YIM-C158T(EU135728) 100

Streptohalobacillus salinus H96B60T(FJ746578)

0.005

Fig. 1 Phylogenetic tree constructed using the neighbor-joining method based on 16S rRNA gene sequences. Bootstrap values, generated from 1,200 re-samplings, at or above 50 % are indicated at the branching points. Bar 0.005 nucleotide substitutions per nucleotide position

proportions of some fatty acids also confirmed that this strain represents a species that differs from the recognized Oceanobacillus species. Therefore, on the basis of the data presented, strain GD01T is considered to represent a novel species of Oceanobacillus for which the name Oceanobacillus halophilum sp. nov. is proposed.

Description of Oceanobacillus halophilum sp. nov. Oceanobacillus halophilum (ha.lo0 phi.lum. Gr. n. halos salt; Gr. adj. philos loving; N.L. neut. adj. halophilum salt-loving). Cells are halophilic, aerobic, and Gram staining positive rods (0.2–0.5 lm wide and 1.2–2.0 lm long). Cells are motile with peritrichous flagella. Endospores are ellipsoidal and occur centrally in swollen sporangia. Colonies are circular, white, and smooth with regular margins after 24 h of aerobic growth on NA at 30 °C. The strain is catalase positive and oxidase positive. Growth of strain GD01T occurs in the presence of 0.5–12 % (w/v) NaCl with the optimal growth at 3–5 % (w/v) NaCl, at 20–50 °C

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(optimum 30 °C), and at pH 6.0–8.5 (optimum 7.0). Results were positive for catalase and oxidase. Results were negative for hydrolysis of gelatin and casein, nitrate reduction, b-galactosidase, lysine decarboxylase, ornithine decarboxylase, H2S, and indole production. Acid is produced from L-arabinose, dulcitol, methyl-aD-mannopyranoside, salicin, xylitol, D-lyxose, and potassium 2-ketogluconate but not from glycerol, erythritol, D-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl-bDxylopyranoside, D-galactose, D-glucose, D-fructose, Dmannose, L-sorbose, L-rhamnose, inositol, D-mannitol, Ddorbitol, methyl-aD-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, Dcellobiose, D-maltose, D-lactose, D-melibiose, D-saccharose, D-trehalose, inulin, Dmelezitose, D-raffinose, starch, glycogen, gentiobiose, Dturanose, D-tagatose, D-fucose, L-fucose, potassium gluconate, and potassium 5-ketogluconate. The predominant quinone system is menaquinone MK-7. The major cellular fatty acids ([5 %) are anteiso-C15:0 (34.5 %), C16:0 (12.3 %), iso-C16:0 (13.7 %), anteiso-C17:0 (12.6 %), isoC15:0 (9.9 %), iso-C14:0 (9.5 %), and C16:0 (5.0 %). The

J. Tang et al.: Oceanobacillus halophilum sp. nov.

633

Table 2 Cellular fatty acid profiles of strain GD01T and its closely related species of the genus Oceanobacillus Fatty acid (%)

1

2

3

4

Acknowledgments This work was supported by the Key Projects of the National Science & Technology Pillar Program of China (2012BAD14B16) and the Science and Technology Planning Project of Guangdong Province, China (2012B010500035 and 2012B010500036).

Saturated straight-chain C10:0

–

0.2

–

0.2

C12:0

1.3

0.4

0.3

–

C14:0

1.8

0.9

0.7

2.8

C16:0

5.0

7.8

3.8

11.6

C17:0

–

0.3

0.1

0.4

C18:0 C16:0 2-OH

2.6 3.1

0.5 –

– 0.2

6.1 –

C14:1x5c

–

–

–

0.2

C16:1x11c

–

–

–

0.4

Unsaturated straight-chain

C16:1x7c alcohol

–

–

0.4

–

C18:1x9c

1.6

1.7

1.0

5.1

Summed feature 3a

0.8

2.8

2.1

0.4

Summed feature 8a

1.2

0.6

0.6

–

Iso-C11:0

–

0.1

–

–

Iso-C12:0

–

0.1

–

–

Iso-C13:0

–

0.2

0.1

0.3

Iso-C14:0

9.5

0.9

10.3

5.6

Saturated branched-chain

Iso-C15:0

9.9

16.1

5.2

20.1

Iso-C16:0 Iso-C17:0

13.7 2.5

21.8 14.7

13.1 1.1

2.4 1.5

Iso-C18:0

–

0.4

–

–

Iso-C19:0

–

–

–

0.3

Anteiso-C11:0

–

0.2

0.1

–

Anteiso-C13:0

–

0.4

0.5

0.2

Anteiso-C15:0

34.5

16.2

48.5

32.2

Anteiso-C17:0

12.6

14.0

11.8

3.3

Anteiso-C17:1x9c

–

–

–

0.2

Summed feature 4a

–

–

–

0.2

Unsaturated branched-chain

Data were taken from this study. Values were percentages of the total fatty acids. Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system Strains 1 Oceanobacillus halophilum sp. nov. GD01T, 2 Oceanobacillus caeni KCTC 13061T, 3 Oceanobacillus profundus DSM 18246T, 4 Oceanobacillus oncorhynchi JCM 12661T, – not detected a

Summed feature 3 comprises C16:1x7c and/or C16:1x6c. Summed feature 4 comprises iso-C17:1 I and/or anteiso-C17:1 B. Summed feature eight comprises C18:1x7c and/or C18:1x6c

DNA G?C content of this strain is 38.7 mol%. The polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol, glycolipid, four unknown lipids, and four unknown phospholipids. The type strain GD01T (=CCTCC AB2012863T = KCTC 33101T) was isolated from a mangrove forest soil near the South China Sea.

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