Curr Microbiol DOI 10.1007/s00284-014-0576-x
Bacillus alkalicola sp. nov., An Alkaliphilic, Gram-Positive Bacterium Isolated from Zhabuye Lake in Tibet, China Lei Zhai • Yiwei Ma • Yanfen Xue Yanhe Ma
•
Received: 14 November 2013 / Accepted: 15 February 2014 Ó Springer Science+Business Media New York 2014
Abstract A Gram-positive, alkaliphilic bacterium, designated strain Zby6T, was isolated from Zhabuye Lake in Tibet, China. The strain was able to grow at pH 8.0–11.0 (optimum at pH 10.0), in 0–8 % (w/v) NaCl (optimum at 3 %, w/v) and at 10–45 °C (optimum at 37 °C). Cells of the isolate were facultatively anaerobic and spore-forming rods with polar flagellum. The predominant isoprenoid quinone was MK-7, and its cell wall peptidoglycan contained meso-diaminopimelic acid. The major cellular fatty acids were iso-C15:0, C16:0 and anteiso-C15:0. The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine. The genomic DNA G?C content of the isolate was 38.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Zby6T was a member of the genus Bacillus and most closely related to Bacillus
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Zby6 T is GU583650.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0576-x) contains supplementary material, which is available to authorized users. L. Zhai Y. Xue Y. Ma (&) State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China e-mail: [email protected] L. Zhai University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China e-mail: [email protected] Y. Ma School of Life Sciences, University of Sichuan, Chengdu 610065, People’s Republic of China
cellulosilyticus DSM 2522T (97.7 % similarity). The DNA– DNA relatedness value between strain Zby6T and B. cellulosilyticus DSM 2522T was 59.2 ± 1.8 %. Comparative analysis of genotypic and phenotypic features indicated that strain Zby6T represents a novel species of the genus Bacillus, for which the name Bacillus alkalicola sp. nov. is proposed; the type strain is Zby6T (=CGMCC 1.10368T = JCM 17098T = NBRC 107743T).
Introduction Alkaliphiles are defined as organisms that grow optimally at alkaline pH, with pH optima for growth being in excess of pH 8 (usually between 9 and 10), with some being capable of cultivation at pH values [11 [18]. Natural alkaline environments, in particular soda lakes, which are distributed worldwide, contain large numbers of alkaliphiles from diverse lineages [20, 25]. Soda lakes are naturally occurring, stable high alkaline environments often with pH values between 9 and 12 that are characterized by large concentrations of sodium carbonate minerals and NaCl. Phylogenetic surveys of prokaryotes indicate that soda lakes harbor considerable taxonomically diverse alkaliphilic populations. Alkaliphilic Bacillus species, as the major group of alkaliphiles, make up an extremely heterogeneous group of microorganisms and have attracted much attention with regard to their physiological adaptation to high pH [29] and biotechnological potential in the production of hydrolytic enzymes [18]. Based on comparative 16S rRNA sequence analysis, Bacillus species were divided into six rRNA groups, with the majority of alkaliphilic Bacillus species assigned to rRNA groups 1 and 6 [3, 26]. Their studies have provided a firm basis for the
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L. Zhai et al.: Bacillus alkalicola sp. nov.
subsequent taxonomic arrangement of the bacilli. In recent years, many novel alkaliphilic Bacillus species, e.g., Bacillus bogoriensis from Bogoria soda lake in Kenya [39], Bacillus chagannorensis from Lake Chagannor in China [8], Bacillus aurantiacus from soda ponds in Kiskunsag National Park, Hungary [6], Bacillus beveridgei from Mono Lake in USA [4], Bacillus marmarensis from mushroom compost in Turkey [11], Bacillus alkalisediminis from sediment of extremely shallow soda ponds [7], and Bacillus nanhaiisediminis from the South China Sea [41], have been described. Lake Zhabuye (31°270 N 83°570 E) is located on the Tibetan Plateau 4421 m above sea level, China. It is a typical saline soda lake with the pH value of 9.4 and salinity [25 %. During the course of studying the phylogenetic diversity of Zhabuye Lake (Chabyer Caka), alkaliphilic strain Zby6T was isolated from a sludge sample of lake edge by pure culture techniques. In this paper, the phenotypic, chemotaxonomic, and genotypic characteristics of alkaliphilic strain Zby6T are measured. Based on the 16S rDNA sequences, as well as phenotypic and chemotaxonomic properties, strain Zby6T appeared to represent a novel species of the genus Bacillus, for which the name Bacillus alkalicola sp. nov. is proposed. The most closely related Bacillus cellulosilyticus DSM 2522T was used as reference strains for comparative studies.
Materials and Methods Bacterial Isolation and Cultivation The sample for bacterial isolation was obtained from sludge of Lake Zhabuye (31°270 N 83°570 E) in Tibet, China. About 5 g sludge was suspended in 45 ml sterilized Horikoshi I medium. Horikoshi I medium contained the following: 10 g glucose, 5 g polypeptone (NIHON SEIYAKU), 5 g yeast extract (OXOID), 50 g NaCl, 1 g K2HPO4, 0.2 g MgSO4 7H2O, and 900 ml distilled water, to which 100 ml 10 % Na2CO3 solution was added aseptically after autoclaving [19]. The pH of Horikoshi medium I was 10.0. Isolation was performed using the dilution plate method. The enriched population was serially diluted and spread on the surface of Horikoshi I agar plates. Colonies were picked and sub-cultivated on Horikoshi I agar plates again; Strain Zby6T was maintained in liquid Horikoshi I medium supplemented with 30 % (v/v) glycerol at -80 °C. Unless otherwise stated, strain Zby6T was grown in modified Horikoshi medium I with 3 % (w/v) NaCl at 37 °C for 20 h. Solid media were prepared by the addition of 2 % agar. Bacillus cellulosilytics DSM 2522T was used as a reference strain and grown in modified Horikoshi medium I with 6 % (w/v) NaCl at 37 °C.
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Morphological and Phenotypic Characteristics Cellular morphology was determined with a transmission electron microscopy (TEM) after growth on modified Horikoshi I agar at 37 °C for 20 h. The preparation of bacterial cells for TEM was pretreated described by Wu et al. [40]. The motility of strain was tested using semisolid stab method. Endospores were observed by phasecontrast microscopes after staining cultures heated at 100 °C for 10 min. Gram staining was performed as described previously [15], in parallel with KOH lysis method [16]. Salt requirement for growth was tested by Horikoshi medium with varied NaCl concentrations ranging from 0 to 20 % (w/v) with increments of 5 %. Then, NaCl concentrations ranging from 0 to 10 % (w/v) with increments of 1 % were used to determine the optimum NaCl requirement. The pH range for growth was tested at intervals of 0.5 pH unit in modified Horikoshi medium I according to the previous methods [27]. Growth at different temperatures (4, 10, 15, 20, 25, 30, 37, 40, 45, and 50 °C) was determined in modified Horikoshi liquid medium I. Anaerobic cultivation was performed in Horikoshi I medium using the Oxoid AnaeroGen system General physiological, and biochemical tests were performed according to previously described methods [37]. Catalase activity was tested by a bubble production assay with 3 % (v/v) H2O2 [28], and oxidase activity was determined using an oxidase reagent (BioMerieux). The Voges–Proskauer reaction, reduction of nitrate, and urease activity were determined according to the methods described by Pettersson et al. [30]. Hydrolysis of gelatin, casein, and starch, indole production, citrate utilization. M.R. test, and H2S production were tested as described by Dong and Cai [12]. Susceptibility to antibiotics was tested using antibiotic disks method described by Romano (1993) [31]. Utilization of carbon sources was determined by growth in modified Horikoshi I medium containing 1 g yeast extract l-1 and 1 g polypeptone l-1 with a variety of substrates (added at 0.5 %, w/v); controls were grown without addition of any substrate. All tests were carried out in triplicate. Chemotaxonomic Properties The cells of Strain Zby6T were grown in Horikoshi I medium with 3 % (w/v) NaCl at 37 °C for 20 h and harvested by centrifugation, washed with distilled water, and freeze-dried. Determination of the cell wall peptidoglycan composition was performed as described in previous methods [36]. Respiratory quinones were extracted with chloroform/methanol (2:1) from dried cells and purified on TLC [9]. The purified contents were analyzed by reversephase HPLC [17]. For analysis of fatty acids composition, cell lipids were extracted and converted to fatty acid
L. Zhai et al.: Bacillus alkalicola sp. nov.
methyl esters (FAMEs). FAMEs were analyzed with GC (Agilent Technologies 6850) and identified using the TSBA 6.0 database of the Microbial Identification System [35] at the Identification Service of China General Microbiological Culture Collection Center (Beijing, China). Harvested cells of 20 h cultivation were used to measure polar lipid compositions. Polar lipids were extracted, separated by two-dimensional thin-layer chromatography, and identified according to the previous method [32]. Molecular Analyses Chromosomal DNA was extracted using a standard method [23]. The genomic DNA G?C content was determined by the thermal denaturation method [24] and DNA–DNA hybridization (n = 4) was measured using the previous method [10]. The 16S rRNA gene of strain Zby6T was amplified by PCR using the universal primers 27F (50 AGAGTTTGATCATGGCTCAG-30 ) and 1541R (50 AAGGAGGTGATCCAGCC-30 ) [5]. Identification of phylogenetic neighbors and calculation of levels of pairwise 16S rRNA gene sequence similarity were achieved by EzTaxon-e [21]. Multiple alignments with sequences from closely related species were performed using the program CLUSTALW in MEGA5 [38]. Evolutionary distances were calculated using Kimura’s two-parameter model [22]. Phylogenetic trees were constructed with the neighborjoining [34], minimum-evolution [33], and maximumlikelihood [13] algorithms in MEGA5 [38]. Topologies of the resultant trees were evaluated by bootstrap analyses based on 1000 resamplings [14]. Results and Discussions Strain Zby6T showed a range of phenotypic characteristics typical of those of the genus Bacillus. Cells of the strain were Gram-positive, rod-shaped (3.0–4.0 lm in length and 0.6–0.8 lm in width), facultative anaerobic, and motile by polar flagellum. Oval endospores formed at terminal positions in swollen sporangia. (Fig. 1). Colonies that formed after incubation on Horikoshi I agar plate with 3 % (w/v) NaCl at 37 °C for 20 h were circular, smooth, and cream-white with entire margins (3–4 mm in diameter). The temperature range for growth was 10–45 °C with an optimum of around 37 °C. No growth occurred at 4, 45, and 50 °C. Strains grew in medium containing 0–8 % (w/ v) NaCl with optimum NaCl concentration of 3 % indicating that Na? was required but not an essential for the growth. The pH range for growth of strain Zby6T was from 8.0 to 11.0, with an optimum of 10.0. Therefore, strains Zby6T are obligate alkaliphilic and slightly halophilic. Strain Zby6T was sensitive to specinomycin (100 lg), rifampicin (5 lg), penicillin (10 lg), medemycin (15 lg),
ampicillin (10 lg), josamycin (15 lg), vancomycin (30 lg), oxacillin (1 lg), chloramphenicol (30 lg), erythrocin (15 lg), kanamycin (30 lg), ciprofiloxacin (5 lg), spiramycin (15 lg), azithromycin (15 lg), kitasamycin (15 lg), and nitrofurantoin (300 lg). The specific characteristics of strain Zby6T are summarized in Table 1. Chemotaxonomic analyses indicated that strain Zby6T contained meso-diaminopimelic acid as a diagnostic diamino acid in the cell wall peptidoglycan. Phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine were the major polar lipidand, and MK7 was the major isoprenoid quinone. The cellular fatty acid profiles of strain Zby6T were iso-C15: 0 (27 %), anteiso-C15: 0 (26.7 %), C16: 0 (10.4 %) and iso-C16: 0 (7.6 %), anteiso-C17: 0 (7.0 %), iso-C17: 0 (6.1 %), iso-C14: 0 (4.3 %), C16: 1 x11c (3.2 %), and C16: 1 x7c (1.5 %). These chemotaxonomic features of strain Zby6T are typical of those found in members of the genus Bacillus. The nearly complete nucleotide sequence (1458 bp) was determined by direct sequencing, and the EZTaxon analysis showed that strain Zby6T was most closely related to B. cellulosilyticus DSM 2522T (16S rRNA gene sequence similarity of 97.7 %) and had 96.8 % sequence similarity to B. vedderi, 96.1 % sequence similarity to Bacillus clarkii DSM 8720T, and less than 95.9 % sequence similarity to other recognized species of Bacillus.. The phylogenetic tree based on the maximum-likelihood algorithm (Fig. 1) showed that strain Zby6T formed a phylogenetic group with B. cellulosilyticus DSM 2522T within the genus Bacillus and was distantly related to other members of the genus. Similar tree topologies were obtained by the minimum-evolution method (Supplementary Fig. S3). The genomic DNA G?C content of strain Zby6T was 38.9 mol %. The DNA–DNA relatedness value between strain Zby6T and the most closely relative B. cellulosilyticus DSM 2522T was 59.2 ± 1.8 % (n = 4). Strain Zby6T could be distinguished by several reactions from each of its phylogenetic relatives, including oxidase and catalase activities, hydrolysis of starch, casein and cellulose, nitrate reduction, indole production, and utilization of several carbon sources (Table 1). Furthermore, major cellular fatty acids of strain Zby6T and B. cellulosilyticus DSM 2522T are both iso-C15:0, anteiso-C15:0, and C16:0; however, the amount of the fatty acids differs widely (Supplementary Table 1). Therefore, on the basis of the taxonomic data presented here, it is suggested that the isolate represents a novel species for which the name Bacillus alkalicola sp. nov. is proposed. Description of Bacillus alkalicola sp. nov. Bacillus alkalicola (al.ka.li’co.la. N.L. n. alkali (from Arabic article al, the; Arabic n. qaliy, ashes of saltwort), alkali; L. suff. -cola (from L. n. incola), inhabitant,
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L. Zhai et al.: Bacillus alkalicola sp. nov. Table 1 Differential characteristics of strain Zby6T and phylogenetically related Bacillus species Characteristic
1
2
3
4
5
Range
15–40
20–40
15–45
5–47
ND-50
Optimum
37
37
30
19–31
40
Growth temperature (°C)
Growth pH Range
8.0–11.0
8.0–10.0
7.5–11.0
8.0–12.0
8.9–10.5
Optimum
10.0
10.0
10.0
10.0
10.0
0–8
0–12
0–15
3–14
0–7
3
6
8
5
ND
NaCl concentration for growth (%, w/v) Range Optimum Oxidase
?
–
?
–
?
Catalase Nitrate reduction
– –
? ?
? –
? ?
– ND
Indole production
–
–
?
–
ND –
Hydrolysis of Starch
–
?
?
–
Casein
–
–
?
?
–
Cellulose
–
?
–
–
–
D-Xylose
?
–
?
?
–
D-Lactose
?
?
?
–
–
D-Galactose
?
?
?
–
–
Ribose
?
–
?
–
–
D-Mannose
–
?
?
?
?
Inulin
–
–
?
–
–
Glycerol
–
–
?
?
–
Utilization of
Mannitol
–
–
?
?
?
Sorbitol Inositol
? –
– ?
? –
– –
– –
–
?
?
–
–
38.9
40.3
42.8
42.9
38.3
Salicin DNA G?C content (mol%)
Strain 1 Zby6T, 2 Bacillus cellulosilyticus DSM 2522T, 3 Bacillus clarkii DSM 8720T (strain 1–3 from this study), 4 Bacillus polygoni JCM 14604T [2], 5 Bacillus vedderi DSM 9768T [1]. All strains are motile, Gram-positive rods and facultatively anaerobic. All strains are positive for citrate utilization, Voges–Proskauer reaction, and glucose fermentation, utilization of D-glucose, D-fructose, D-sucrose, and D-raffinose as sole carbon sources. Negative for utilization of ethanol, erythritol, rhamnose and dulcose, H2S production, urease activity, methyl red test, hydrolysis of Tween 80, gelatin, and xylan. All strains have phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine as the predominant polar lipids, meso-diaminopimelic acid as diagnostic diamino acid, and MK-7 as the main isoprenoid quinone. Data are from this study. ? Positive, - negative
dweller; N.L. n. alkalicola, an inhabitant of alkaline environments). Cells are Gram-positive rods (3.0–4.0 lm in length and 0.6–0.8 lm in width), facultatively anaerobic and motile by polar flagellum. Oval endospores form at terminal positions in swollen sporangia. Colonies are circular and white-creamy, 3–4 mm in diameter with regular margins after cultivation at 37 °C on Horikoshi agar medium I with 3 % NaCl for 24 h. Growth occur between 15 and 40 °C, with the optimum growth at 37 °C. The pH range for growth is from 8.0 to 11.0, with the optimum growth at pH 10.0. Cells are able to grow at NaCl concentration of
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0–8 % (w/v), with the optimum growth at 3 %. Positive for oxidase, citrate utilization, and VP test, but negative for hydrolysis of starch, gelatin, casein, xylan, cellulose and Tween 80, catalase, urease activity, indole test, nitrate reduction, H2S production, and M.R. test. Strain Zby6T is able to grow using D-glucose, D-lactose, D-fructose, D-mannose, D-xylose, D-ribose, glucosamine, gluconate, D-raffinose, D-cellobiose, D-galactose, D-sucrose, maltose, melitose, melicitose, inulin, mannan, D-trehalose, and esculin as sole carbon sources in modified Horikoshi medium I. Acids are produced fermentatively from D-glucose. The major cellular fatty acids are iso-C15:0, anteiso-
L. Zhai et al.: Bacillus alkalicola sp. nov. Fig. 1 Phylogenetic tree showing the relationship between strain Zby6T and related strains based on 16S rRNA gene sequences. Numbers at nodes represent the levels of bootstrap support (%) based on a maximum-likelihood analysis of 1000 resampled datasets, only values [50 % are given. Nucleotide sequence accession numbers are given in parentheses. Bar 1 % sequence divergence
C15:0, and C16:0. Phosphtidyglycerol, diphosphatidylglycerol, and phosphoethanolamine are the predominant polar lipids. MK-7 is the predominant isoprenoid quinone, and meso-diaminopimelic acid is a diagnostic diamino acid in the cell wall peptidoglycan. The accession number for the 16S rRNA gene sequence of strain Zby6T is GU583650. Strain Zby6T was obtained from sludge of Lake Zhabuye of Tibet, China. The type strain is Zby6T (=CGMCC 1.10368T = JCM 17098T = NBRC 107743T), isolated from a water sample of Zhabuye Lake of Tibet, China. The genomic DNA G?C content of the type strain is 38.9 mol% (Tm). Acknowledgments This work was supported by the Grants from the Chinese Academy of Sciences (Knowledge Innovation Program, KSCX2-EW-G-3) and the Ministry of Science and Technology of China (2013CB733900 and 2012AA022100).
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Bacillus alkalicola sp. nov., An Alkaliphilic, Gram-Positive Bacterium Isolated from Zhabuye Lake in Tibet, China Lei Zhai • Yiwei Ma • Yanfen Xue Yanhe Ma
•
Received: 14 November 2013 / Accepted: 15 February 2014 Ó Springer Science+Business Media New York 2014
Abstract A Gram-positive, alkaliphilic bacterium, designated strain Zby6T, was isolated from Zhabuye Lake in Tibet, China. The strain was able to grow at pH 8.0–11.0 (optimum at pH 10.0), in 0–8 % (w/v) NaCl (optimum at 3 %, w/v) and at 10–45 °C (optimum at 37 °C). Cells of the isolate were facultatively anaerobic and spore-forming rods with polar flagellum. The predominant isoprenoid quinone was MK-7, and its cell wall peptidoglycan contained meso-diaminopimelic acid. The major cellular fatty acids were iso-C15:0, C16:0 and anteiso-C15:0. The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine. The genomic DNA G?C content of the isolate was 38.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Zby6T was a member of the genus Bacillus and most closely related to Bacillus
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Zby6 T is GU583650.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0576-x) contains supplementary material, which is available to authorized users. L. Zhai Y. Xue Y. Ma (&) State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China e-mail: [email protected] L. Zhai University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China e-mail: [email protected] Y. Ma School of Life Sciences, University of Sichuan, Chengdu 610065, People’s Republic of China
cellulosilyticus DSM 2522T (97.7 % similarity). The DNA– DNA relatedness value between strain Zby6T and B. cellulosilyticus DSM 2522T was 59.2 ± 1.8 %. Comparative analysis of genotypic and phenotypic features indicated that strain Zby6T represents a novel species of the genus Bacillus, for which the name Bacillus alkalicola sp. nov. is proposed; the type strain is Zby6T (=CGMCC 1.10368T = JCM 17098T = NBRC 107743T).
Introduction Alkaliphiles are defined as organisms that grow optimally at alkaline pH, with pH optima for growth being in excess of pH 8 (usually between 9 and 10), with some being capable of cultivation at pH values [11 [18]. Natural alkaline environments, in particular soda lakes, which are distributed worldwide, contain large numbers of alkaliphiles from diverse lineages [20, 25]. Soda lakes are naturally occurring, stable high alkaline environments often with pH values between 9 and 12 that are characterized by large concentrations of sodium carbonate minerals and NaCl. Phylogenetic surveys of prokaryotes indicate that soda lakes harbor considerable taxonomically diverse alkaliphilic populations. Alkaliphilic Bacillus species, as the major group of alkaliphiles, make up an extremely heterogeneous group of microorganisms and have attracted much attention with regard to their physiological adaptation to high pH [29] and biotechnological potential in the production of hydrolytic enzymes [18]. Based on comparative 16S rRNA sequence analysis, Bacillus species were divided into six rRNA groups, with the majority of alkaliphilic Bacillus species assigned to rRNA groups 1 and 6 [3, 26]. Their studies have provided a firm basis for the
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subsequent taxonomic arrangement of the bacilli. In recent years, many novel alkaliphilic Bacillus species, e.g., Bacillus bogoriensis from Bogoria soda lake in Kenya [39], Bacillus chagannorensis from Lake Chagannor in China [8], Bacillus aurantiacus from soda ponds in Kiskunsag National Park, Hungary [6], Bacillus beveridgei from Mono Lake in USA [4], Bacillus marmarensis from mushroom compost in Turkey [11], Bacillus alkalisediminis from sediment of extremely shallow soda ponds [7], and Bacillus nanhaiisediminis from the South China Sea [41], have been described. Lake Zhabuye (31°270 N 83°570 E) is located on the Tibetan Plateau 4421 m above sea level, China. It is a typical saline soda lake with the pH value of 9.4 and salinity [25 %. During the course of studying the phylogenetic diversity of Zhabuye Lake (Chabyer Caka), alkaliphilic strain Zby6T was isolated from a sludge sample of lake edge by pure culture techniques. In this paper, the phenotypic, chemotaxonomic, and genotypic characteristics of alkaliphilic strain Zby6T are measured. Based on the 16S rDNA sequences, as well as phenotypic and chemotaxonomic properties, strain Zby6T appeared to represent a novel species of the genus Bacillus, for which the name Bacillus alkalicola sp. nov. is proposed. The most closely related Bacillus cellulosilyticus DSM 2522T was used as reference strains for comparative studies.
Materials and Methods Bacterial Isolation and Cultivation The sample for bacterial isolation was obtained from sludge of Lake Zhabuye (31°270 N 83°570 E) in Tibet, China. About 5 g sludge was suspended in 45 ml sterilized Horikoshi I medium. Horikoshi I medium contained the following: 10 g glucose, 5 g polypeptone (NIHON SEIYAKU), 5 g yeast extract (OXOID), 50 g NaCl, 1 g K2HPO4, 0.2 g MgSO4 7H2O, and 900 ml distilled water, to which 100 ml 10 % Na2CO3 solution was added aseptically after autoclaving [19]. The pH of Horikoshi medium I was 10.0. Isolation was performed using the dilution plate method. The enriched population was serially diluted and spread on the surface of Horikoshi I agar plates. Colonies were picked and sub-cultivated on Horikoshi I agar plates again; Strain Zby6T was maintained in liquid Horikoshi I medium supplemented with 30 % (v/v) glycerol at -80 °C. Unless otherwise stated, strain Zby6T was grown in modified Horikoshi medium I with 3 % (w/v) NaCl at 37 °C for 20 h. Solid media were prepared by the addition of 2 % agar. Bacillus cellulosilytics DSM 2522T was used as a reference strain and grown in modified Horikoshi medium I with 6 % (w/v) NaCl at 37 °C.
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Morphological and Phenotypic Characteristics Cellular morphology was determined with a transmission electron microscopy (TEM) after growth on modified Horikoshi I agar at 37 °C for 20 h. The preparation of bacterial cells for TEM was pretreated described by Wu et al. [40]. The motility of strain was tested using semisolid stab method. Endospores were observed by phasecontrast microscopes after staining cultures heated at 100 °C for 10 min. Gram staining was performed as described previously [15], in parallel with KOH lysis method [16]. Salt requirement for growth was tested by Horikoshi medium with varied NaCl concentrations ranging from 0 to 20 % (w/v) with increments of 5 %. Then, NaCl concentrations ranging from 0 to 10 % (w/v) with increments of 1 % were used to determine the optimum NaCl requirement. The pH range for growth was tested at intervals of 0.5 pH unit in modified Horikoshi medium I according to the previous methods [27]. Growth at different temperatures (4, 10, 15, 20, 25, 30, 37, 40, 45, and 50 °C) was determined in modified Horikoshi liquid medium I. Anaerobic cultivation was performed in Horikoshi I medium using the Oxoid AnaeroGen system General physiological, and biochemical tests were performed according to previously described methods [37]. Catalase activity was tested by a bubble production assay with 3 % (v/v) H2O2 [28], and oxidase activity was determined using an oxidase reagent (BioMerieux). The Voges–Proskauer reaction, reduction of nitrate, and urease activity were determined according to the methods described by Pettersson et al. [30]. Hydrolysis of gelatin, casein, and starch, indole production, citrate utilization. M.R. test, and H2S production were tested as described by Dong and Cai [12]. Susceptibility to antibiotics was tested using antibiotic disks method described by Romano (1993) [31]. Utilization of carbon sources was determined by growth in modified Horikoshi I medium containing 1 g yeast extract l-1 and 1 g polypeptone l-1 with a variety of substrates (added at 0.5 %, w/v); controls were grown without addition of any substrate. All tests were carried out in triplicate. Chemotaxonomic Properties The cells of Strain Zby6T were grown in Horikoshi I medium with 3 % (w/v) NaCl at 37 °C for 20 h and harvested by centrifugation, washed with distilled water, and freeze-dried. Determination of the cell wall peptidoglycan composition was performed as described in previous methods [36]. Respiratory quinones were extracted with chloroform/methanol (2:1) from dried cells and purified on TLC [9]. The purified contents were analyzed by reversephase HPLC [17]. For analysis of fatty acids composition, cell lipids were extracted and converted to fatty acid
L. Zhai et al.: Bacillus alkalicola sp. nov.
methyl esters (FAMEs). FAMEs were analyzed with GC (Agilent Technologies 6850) and identified using the TSBA 6.0 database of the Microbial Identification System [35] at the Identification Service of China General Microbiological Culture Collection Center (Beijing, China). Harvested cells of 20 h cultivation were used to measure polar lipid compositions. Polar lipids were extracted, separated by two-dimensional thin-layer chromatography, and identified according to the previous method [32]. Molecular Analyses Chromosomal DNA was extracted using a standard method [23]. The genomic DNA G?C content was determined by the thermal denaturation method [24] and DNA–DNA hybridization (n = 4) was measured using the previous method [10]. The 16S rRNA gene of strain Zby6T was amplified by PCR using the universal primers 27F (50 AGAGTTTGATCATGGCTCAG-30 ) and 1541R (50 AAGGAGGTGATCCAGCC-30 ) [5]. Identification of phylogenetic neighbors and calculation of levels of pairwise 16S rRNA gene sequence similarity were achieved by EzTaxon-e [21]. Multiple alignments with sequences from closely related species were performed using the program CLUSTALW in MEGA5 [38]. Evolutionary distances were calculated using Kimura’s two-parameter model [22]. Phylogenetic trees were constructed with the neighborjoining [34], minimum-evolution [33], and maximumlikelihood [13] algorithms in MEGA5 [38]. Topologies of the resultant trees were evaluated by bootstrap analyses based on 1000 resamplings [14]. Results and Discussions Strain Zby6T showed a range of phenotypic characteristics typical of those of the genus Bacillus. Cells of the strain were Gram-positive, rod-shaped (3.0–4.0 lm in length and 0.6–0.8 lm in width), facultative anaerobic, and motile by polar flagellum. Oval endospores formed at terminal positions in swollen sporangia. (Fig. 1). Colonies that formed after incubation on Horikoshi I agar plate with 3 % (w/v) NaCl at 37 °C for 20 h were circular, smooth, and cream-white with entire margins (3–4 mm in diameter). The temperature range for growth was 10–45 °C with an optimum of around 37 °C. No growth occurred at 4, 45, and 50 °C. Strains grew in medium containing 0–8 % (w/ v) NaCl with optimum NaCl concentration of 3 % indicating that Na? was required but not an essential for the growth. The pH range for growth of strain Zby6T was from 8.0 to 11.0, with an optimum of 10.0. Therefore, strains Zby6T are obligate alkaliphilic and slightly halophilic. Strain Zby6T was sensitive to specinomycin (100 lg), rifampicin (5 lg), penicillin (10 lg), medemycin (15 lg),
ampicillin (10 lg), josamycin (15 lg), vancomycin (30 lg), oxacillin (1 lg), chloramphenicol (30 lg), erythrocin (15 lg), kanamycin (30 lg), ciprofiloxacin (5 lg), spiramycin (15 lg), azithromycin (15 lg), kitasamycin (15 lg), and nitrofurantoin (300 lg). The specific characteristics of strain Zby6T are summarized in Table 1. Chemotaxonomic analyses indicated that strain Zby6T contained meso-diaminopimelic acid as a diagnostic diamino acid in the cell wall peptidoglycan. Phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine were the major polar lipidand, and MK7 was the major isoprenoid quinone. The cellular fatty acid profiles of strain Zby6T were iso-C15: 0 (27 %), anteiso-C15: 0 (26.7 %), C16: 0 (10.4 %) and iso-C16: 0 (7.6 %), anteiso-C17: 0 (7.0 %), iso-C17: 0 (6.1 %), iso-C14: 0 (4.3 %), C16: 1 x11c (3.2 %), and C16: 1 x7c (1.5 %). These chemotaxonomic features of strain Zby6T are typical of those found in members of the genus Bacillus. The nearly complete nucleotide sequence (1458 bp) was determined by direct sequencing, and the EZTaxon analysis showed that strain Zby6T was most closely related to B. cellulosilyticus DSM 2522T (16S rRNA gene sequence similarity of 97.7 %) and had 96.8 % sequence similarity to B. vedderi, 96.1 % sequence similarity to Bacillus clarkii DSM 8720T, and less than 95.9 % sequence similarity to other recognized species of Bacillus.. The phylogenetic tree based on the maximum-likelihood algorithm (Fig. 1) showed that strain Zby6T formed a phylogenetic group with B. cellulosilyticus DSM 2522T within the genus Bacillus and was distantly related to other members of the genus. Similar tree topologies were obtained by the minimum-evolution method (Supplementary Fig. S3). The genomic DNA G?C content of strain Zby6T was 38.9 mol %. The DNA–DNA relatedness value between strain Zby6T and the most closely relative B. cellulosilyticus DSM 2522T was 59.2 ± 1.8 % (n = 4). Strain Zby6T could be distinguished by several reactions from each of its phylogenetic relatives, including oxidase and catalase activities, hydrolysis of starch, casein and cellulose, nitrate reduction, indole production, and utilization of several carbon sources (Table 1). Furthermore, major cellular fatty acids of strain Zby6T and B. cellulosilyticus DSM 2522T are both iso-C15:0, anteiso-C15:0, and C16:0; however, the amount of the fatty acids differs widely (Supplementary Table 1). Therefore, on the basis of the taxonomic data presented here, it is suggested that the isolate represents a novel species for which the name Bacillus alkalicola sp. nov. is proposed. Description of Bacillus alkalicola sp. nov. Bacillus alkalicola (al.ka.li’co.la. N.L. n. alkali (from Arabic article al, the; Arabic n. qaliy, ashes of saltwort), alkali; L. suff. -cola (from L. n. incola), inhabitant,
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L. Zhai et al.: Bacillus alkalicola sp. nov. Table 1 Differential characteristics of strain Zby6T and phylogenetically related Bacillus species Characteristic
1
2
3
4
5
Range
15–40
20–40
15–45
5–47
ND-50
Optimum
37
37
30
19–31
40
Growth temperature (°C)
Growth pH Range
8.0–11.0
8.0–10.0
7.5–11.0
8.0–12.0
8.9–10.5
Optimum
10.0
10.0
10.0
10.0
10.0
0–8
0–12
0–15
3–14
0–7
3
6
8
5
ND
NaCl concentration for growth (%, w/v) Range Optimum Oxidase
?
–
?
–
?
Catalase Nitrate reduction
– –
? ?
? –
? ?
– ND
Indole production
–
–
?
–
ND –
Hydrolysis of Starch
–
?
?
–
Casein
–
–
?
?
–
Cellulose
–
?
–
–
–
D-Xylose
?
–
?
?
–
D-Lactose
?
?
?
–
–
D-Galactose
?
?
?
–
–
Ribose
?
–
?
–
–
D-Mannose
–
?
?
?
?
Inulin
–
–
?
–
–
Glycerol
–
–
?
?
–
Utilization of
Mannitol
–
–
?
?
?
Sorbitol Inositol
? –
– ?
? –
– –
– –
–
?
?
–
–
38.9
40.3
42.8
42.9
38.3
Salicin DNA G?C content (mol%)
Strain 1 Zby6T, 2 Bacillus cellulosilyticus DSM 2522T, 3 Bacillus clarkii DSM 8720T (strain 1–3 from this study), 4 Bacillus polygoni JCM 14604T [2], 5 Bacillus vedderi DSM 9768T [1]. All strains are motile, Gram-positive rods and facultatively anaerobic. All strains are positive for citrate utilization, Voges–Proskauer reaction, and glucose fermentation, utilization of D-glucose, D-fructose, D-sucrose, and D-raffinose as sole carbon sources. Negative for utilization of ethanol, erythritol, rhamnose and dulcose, H2S production, urease activity, methyl red test, hydrolysis of Tween 80, gelatin, and xylan. All strains have phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine as the predominant polar lipids, meso-diaminopimelic acid as diagnostic diamino acid, and MK-7 as the main isoprenoid quinone. Data are from this study. ? Positive, - negative
dweller; N.L. n. alkalicola, an inhabitant of alkaline environments). Cells are Gram-positive rods (3.0–4.0 lm in length and 0.6–0.8 lm in width), facultatively anaerobic and motile by polar flagellum. Oval endospores form at terminal positions in swollen sporangia. Colonies are circular and white-creamy, 3–4 mm in diameter with regular margins after cultivation at 37 °C on Horikoshi agar medium I with 3 % NaCl for 24 h. Growth occur between 15 and 40 °C, with the optimum growth at 37 °C. The pH range for growth is from 8.0 to 11.0, with the optimum growth at pH 10.0. Cells are able to grow at NaCl concentration of
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0–8 % (w/v), with the optimum growth at 3 %. Positive for oxidase, citrate utilization, and VP test, but negative for hydrolysis of starch, gelatin, casein, xylan, cellulose and Tween 80, catalase, urease activity, indole test, nitrate reduction, H2S production, and M.R. test. Strain Zby6T is able to grow using D-glucose, D-lactose, D-fructose, D-mannose, D-xylose, D-ribose, glucosamine, gluconate, D-raffinose, D-cellobiose, D-galactose, D-sucrose, maltose, melitose, melicitose, inulin, mannan, D-trehalose, and esculin as sole carbon sources in modified Horikoshi medium I. Acids are produced fermentatively from D-glucose. The major cellular fatty acids are iso-C15:0, anteiso-
L. Zhai et al.: Bacillus alkalicola sp. nov. Fig. 1 Phylogenetic tree showing the relationship between strain Zby6T and related strains based on 16S rRNA gene sequences. Numbers at nodes represent the levels of bootstrap support (%) based on a maximum-likelihood analysis of 1000 resampled datasets, only values [50 % are given. Nucleotide sequence accession numbers are given in parentheses. Bar 1 % sequence divergence
C15:0, and C16:0. Phosphtidyglycerol, diphosphatidylglycerol, and phosphoethanolamine are the predominant polar lipids. MK-7 is the predominant isoprenoid quinone, and meso-diaminopimelic acid is a diagnostic diamino acid in the cell wall peptidoglycan. The accession number for the 16S rRNA gene sequence of strain Zby6T is GU583650. Strain Zby6T was obtained from sludge of Lake Zhabuye of Tibet, China. The type strain is Zby6T (=CGMCC 1.10368T = JCM 17098T = NBRC 107743T), isolated from a water sample of Zhabuye Lake of Tibet, China. The genomic DNA G?C content of the type strain is 38.9 mol% (Tm). Acknowledgments This work was supported by the Grants from the Chinese Academy of Sciences (Knowledge Innovation Program, KSCX2-EW-G-3) and the Ministry of Science and Technology of China (2013CB733900 and 2012AA022100).
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