| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Microbiology, College of Biological Sciences, China Agricultural University, Key Laboratory for Agro-Microbial Resource and Application, Ministry of Agriculture, Beijing, 100094, P. R. China
2 Division of Mineral Resources, Metallurgy and Materials, General Research Institute for Nonferrous Metals, Beijing, 100088, P. R. China
Correspondence
S. S. Yang
yangssh{at}cau.edu.cn
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the almost-complete 16S rRNA gene sequences of strains D-8T and AD-6T are AY351395 and AY351389, respectively.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). The spore-forming moderately halophilic bacteria, an important group found mostly in marine or hypersaline environments, were formerly placed in the genus Bacillus (Claus & Berkeley, 1986). Based on 16S rRNA gene sequence and chemotaxonomic analysis, several phylogenetically distinct lineages in the genus Bacillus have been revealed (Ash et al., 1991; Nielsen et al., 1994; Priest, 1981) and subsequently many spore-forming bacteria have been placed in separate genera. Some moderately halophilic spore-forming bacteria were described as genus Halobacillus (Spring et al., 1996). Initially, the genus Halobacillus was proposed with two novel species, Halobacillus litoralis DSM 10405T and Halobacillus trueperi DSM 10404T. In addition, Halobacillus halophilus DSM 2266T was transferred from Sporosarcina halophila (Claus et al., 1983). Recently, the moderately halophilic bacterial strains HSL-3T, MSS-155T and MA-2T were proposed as representatives of three novel species: Halobacillus salinus JCM 11546T (Yoon et al., 2003), Halobacillus locisalis KCCM 41687T (Yoon et al., 2004) and Halobacillus karajensis DSM 14948T (Amoozegar et al., 2003). In order to investigate the moderately halophilic bacteria of China, we isolated some strains from the hypersaline waters and saline deposits of the Daban and Aiding salt lakes, which are located in the arid region of Xinjiang, China. Isolates D-8T and AD-6T were considered to be Halobacillus-like strains from the results of 16S rRNA gene sequence analysis. The aim of this study was to determine the exact taxonomic position of D-8T and AD-6T by using a polyphasic taxonomic approach, including the investigation of phenotypic characteristics, chemotaxonomic properties, DNA G+C content, DNA–DNA relatedness and a phylogenetic analysis.
Bacterial strains were isolated from the samples according to the method of Spring et al. (1996). The complex medium used for the isolation and maintenance of bacterial strains contained 5 g tryptone, 10 g yeast extract, 5 g Casamino acids, 3 g citrate sodium, 2 g KCl and 20 g MgSO4.7H2O dissolved in 1 l of water (Xu et al., 1995). If required, media were solidified by the addition of agar (15 g l–1). The pH value was adjusted to 7·5. The sea water medium described by Spring et al. (1996) was used for the investigation of spore formation. Unless indicated, all tests were carried out in medium with 10 % NaCl (w/v), at pH 7·5 and incubated at 35 °C. The recommended media and conditions for growth were used to culture the reference strains, including H. halophilus DSM 2266T, H. litoralis DSM 10405T, H. karajensis DSM 14948T, H. salinus JCM 11546T, H. locisalis KCCM 41687T and H. trueperi DSM 10404T (Amoozegar et al., 2003; Spring et al., 1996; Yoon et al., 2003, 2004).
To determine cell morphology, bacterial cultures were grown on complex medium plates for 16 h and then examined by phase-contrast microscopy. Photomicrographs of spores were obtained from cultures grown on sea water medium for 30 h. Transmission electron microscopy was used to observe bacterial flagellation. Motility was analysed by the hanging-drop method with cultures in the early exponential growth phase. The Gram reaction was determined by the KOH lysis method of Gregersen (1978). Colony morphology was examined after 3 days incubation on solid complex medium. The NaCl concentration for growth was determined in liquid media containing 0–30 % (w/v) NaCl. The pH range for growth was determined by adjusting the pH to values between pH 4·0 and pH 11·0. The temperature range for growth was determined by incubation in liquid medium at temperatures between 5 and 60 °C. Physiological and biochemical studies were carried out according to Quesada et al. (1984) and Ventosa et al. (1982).
The interpeptide bridge present in the cell-wall peptidoglycan was analysed using the method described by Schleifer & Kandler (1972). The preparation and hydrolysis of the cell wall were carried out according to Schleifer & Kandler (1972) and Schleifer (1985). Cell-wall hydrolysates were separated by one-dimensional chromatography on micro-cellulose thin layers using H. litoralis as a reference. Menaquinones were analysed as described previously (Komagata & Suzuki, 1987), using reverse-phase HPLC and employing H. locisalis as a reference. For quantitative determination of fatty acid composition, the fatty acid methyl ester mixtures were prepared and identified following the manufacturer's instructions for the Microbial Identification System (MIDI).
Chromosomal DNA was extracted and purified according to standard methods (Mellado et al., 1996; Sambrook et al., 1989). The 16S rRNA gene sequence was amplified as described by Duckworth et al. (1996). PCR products were sequenced with a DNA Sequencer (373A; Applied Biosystems) using the software provided by the manufacturer. The almost-complete 16S rRNA gene sequences of strains D-8T and AD-6T were compared with sequences from the GenBank, EMBL, DDBJ and PDB databases using the MEGABLAST program (NCBI). Alignment of sequences was carried out using CLUSTAL W software (Thompson et al., 1994). The phylogenetic tree was inferred by using the TREECON application. Evolutionary distance matrices for the neighbour-joining method were calculated with the algorithm of Kimura (1980) and the stability of the relationships was assessed by 1000 bootstrap analyses.
The DNA G+C content was determined by the thermal denaturation method using a BIO-20 UV spectrophotometer according to De Ley et al. (1970) and Huß et al. (1983). For the calculation of DNA G+C content, the equation of De Ley et al. (1970) was used and corrected using Escherichia coli strain K-12 as a reference. Levels of DNA–DNA relatedness were estimated spectrophotometrically (De Ley et al., 1970; Huß et al., 1983). DNA was sheared by sonication to generate fragments of 2–5x105 Da and renaturation was performed in 2x SSC at the optimum temperature.
Strains D-8T and AD-6T were Gram-positive and cells occurred singly, in pairs or in short chains. Under optimal conditions, cells of strain D-8T were rods with round ends and measured 2·2–4·2x0·6–0·9 µm (Fig. 1). Cells of strain AD-6T were club-shaped rods, 1·0–2·8x0·6–1·2 µm in size (Fig. 1). After incubation on sea water medium plates for 30 h, both strains formed endospores located at a central or subterminal position. Both strains were motile with peritrichous flagella. After incubation for 3 days, colonies of strain D-8T were about 3–4 mm in diameter, twice as large as those of strain AD-6T. Colonies of both strains were circular, smooth and slightly raised. Colonies of strain AD-6T were orange. Colonies of strain D-8T were initially cream coloured with orange pigment appearing after a few additional days incubation.
|
Strains D-8T and AD-6T were aerobic and showed catalase, oxidase and DNase activities. For both strains, acid was produced from D-fructose, D-glucose, D-trehalose, D-mannitol, maltose and sucrose, but not from D-galactose. Starch and casein were hydrolysed but no hydrolysis of tyrosine, aesculin or Tween 80 was observed. There were some minor differences between strains D-8T and AD-6T: acid was produced from D-xylose by strain D-8T and gelatin was hydrolysed by strain AD-6T (Table 1). From the results of the cell-wall analysis, the peptidoglycan type of the strains D-8T and AD-6T was determined as L-orn–D-Asp. The predominant menaquinone found in the two strains was MK-7 as found in the type species of the genus, H. locisalis. The major branched fatty acids found in the two isolated strains were anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C16 : 0 (Table 2).
|
|
).
|
In conclusion, strains D-8T and AD-6T showed high levels of 16S rRNA gene sequence similarity to the type strains of Halobacillus species. Phylogenetic analysis revealed that these strains fall within the branch of the genus Halobacillus. Chemotaxonomic analyses indicated that the two strains have the same properties as other Halobacillus species (Amoozegar et al., 2003; Spring et al., 1996; Yoon et al., 2003), although there are a few differences in cellular fatty acid content and some phenotypic properties. Thus, it is evident that the newly isolated strains are members of the genus Halobacillus.
Strains D-8T and AD-6T are similar to Halobacillus species in their morphological characteristics and in most of the physiological characteristics. However, there are some minor differences between the two strains and other Halobacillus species, including the range of temperatures and pH values for growth, tolerance of NaCl, the ability to hydrolyse some substrates and in acid production (Table 1
). The fatty acid profiles of the two strains also differ from those of the reference strains (Table 2). DNA–DNA hybridization is deemed to be a key marker for the identification of a novel species (Wayne et al., 1987). In the present study, levels of DNA–DNA hybridization were low enough to classify strains D-8T and AD-6T as separate species in the genus Halobacillus. On the basis of phenotypic, chemotaxonomic and genetic characteristics, it is proposed that strains D-8T and AD-6T should be placed in the genus Halobacillus as novel species, with the names Halobacillus dabanensis sp. nov. and Halobacillus aidingensis sp. nov., respectively.
Description of Halobacillus dabanensis sp. nov.
Halobacillus dabanensis (da.ban.en'sis. N.L. masc. adj. dabanensis from Daban salt lake, where the type strain was isolated).
Cells are Gram-positive rods with round ends, 2·2–4·2x0·6–0·9 µm, occurring singly, in pairs or in short chains (Fig. 1). Cells are motile by means of peritrichous flagella. Ellipsoidal endospores are located at a central or subterminal position. Colonies are circular, smooth, entire, slightly raised and 3–4 mm in diameter after incubation for 3 days. Colonies are initially cream coloured but orange pigment is produced after a few additional days incubation. Growth occurs in 0·5–25 % (w/v) NaCl with 10 % (w/v) optimal for growth. The temperature range for growth is 15–50 °C; optimum is 35 °C. The pH range for growth is pH 5·0–11·0; optimum pH value is 7·5. Catalase, oxidase and DNase are produced, but urease is negative. Acid is produced from D-fructose, D-glucose, D-xylose, D-trehalose, D-mannitol, maltose and sucrose but not from D-galactose. Starch and casein are hydrolysed; tyrosine, aesculin, gelatin and Tween 80 are not hydrolysed. Voges–Proskauer test is negative. Nitrate is not reduced to nitrite. The cell-wall peptidoglycan is of the L-orn–D-Asp type. The predominant menaquinone is MK-7. The major cellular fatty acids are anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C16 : 0. The DNA G+C content is 41·4 mol%.
The type strain, D-8T (=JCM 12772T=CGMCC 1.3704T), was isolated from the Daban salt lake in Xinjiang region, China.
Description of Halobacillus aidingensis sp. nov.
Halobacillus aidingensis (ai.ding.en'sis. N.L. masc. adj. aidingensis from Aiding salt lake, where the type strain was isolated).
Cells are club-shaped rods, 1·0–2·8x0·6–1·2 µm. Gram-positive, occurring singly or in pairs (Fig. 1) and motile by means of peritrichous flagella. Ellipsoidal endospores are located at a central or subterminal position. Colonies are round, smooth, orange in colour, entire and 1–2 mm in diameter after incubation for 3 days. Growth occurs in 0·5–20 % (w/v) NaCl with an optimum concentration of 10 % (w/v) NaCl. The temperature range for growth is 15–40 °C; optimum temperature is 32 °C. Growth occurs at pH 6–10; optimum pH is 7·5. Acid is produced from D-fructose, D-glucose, D-trehalose, D-mannitol, maltose and sucrose but not from D-galactose and D-xylose. Catalase, oxidase and DNase are produced, but urease is negative. Starch, gelatin and casein are hydrolysed but tyrosine, aesculin and Tween 80 are not hydrolysed. Phosphatase, lecithinase, phenylalanine deaminase and arginine dihydrolase are not produced. The Voges–Proskauer test is negative. Nitrate is not reduced to nitrite. The cell-wall peptidoglycan type is L-orn–D-Asp. The predominant menaquinone is MK-7. The major cellular fatty acids are anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C16 : 0. The DNA G+C content is 42·2 mol%.
The type strain, AD-6T (=JCM 12771T=CGMCC 1.3703T), was isolated from Aiding salt lake in Xinjiang region, China.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). Phylogenetic heterogeneity of genus Bacillus as revealed by comparative analysis of small-subunit-ribosomal RNA sequence. Lett Appl Microbiol 13, 202–206.
Claus, D. & Berkeley, R. C. W. (1986). Genus Bacillus Cohn 1872. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1105–1139. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Claus, D., Fahmy, F., Rolf, H. J. & Tosunoglu, N. (1983). Sporosarcina halophila sp. nov., an obligate, slightly halophilic bacterium from salt marsh soils. Syst Appl Bacteriol 4, 496–506.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Duckworth, A. W., Grant, W. D., Jones, B. E. & van Steenbergen, R. (1996). Phylogenetic diversity of soda lake alkaliphiles. FEMS Microbiol Ecol 19, 181–191.
Gregersen, T. (1978). Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J Appl Microbiol Biotechnol 5, 123–127.[CrossRef]
Huß, V. A. R., Festal, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Komagata, K. & Suzuki, K. (1987). Lipids and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–203.
Mellado, E., Moore, E. R. B., Nieto, J. J. & Ventosa, A. (1996). Analysis of 16S rRNA gene sequences of Vibrio costicola strains: description of Salinivibrio costicola gen. nov., comb. nov. Int J Syst Bacteriol 46, 817–821.
Nielsen, P., Rainey, F. A., Outtrup, H., Priest, F. G. & Fritze, D. (1994). Comparative 16S rDNA sequence analysis of some alkaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus. FEMS Microbiol Lett 117, 61–66.[CrossRef]
Priest, F. G. (1981). DNA homology in genus Bacillus. In The Aerobic Endospore-Forming Bacteria, pp. 33–57. Edited by R. C. Berkeley & M. Goodfellow. London: Academic Press.
Quesada, E., Ventosa, A., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1984). Deleya halophila, a new species of moderately halophilic bacteria. Int J Syst Bacteriol 34, 287–292.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure of murein. Methods Microbiol 18, 123–156.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Spring, S., Ludwig, W., Marquez, M. C., Ventosa, A. & Schleifer, K. H. (1996). Halobacillus gen. nov., with descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov. Int J Syst Bacteriol 46, 492–496.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Ventosa, A., Quesada, E., Rodriguez-Valera, F., Ruiz-Beraquero, F. & Ramos-Cormenzana, A. (1982). Numerical taxonomy of moderately halophilic Gram-negative rods. J Gen Microbiol 128, 1959–1968.
Ventosa, A., Nieto, J. J. & Oren, A. (1998). Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62, 504–544.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Xu, D. Q., Huang, J. J., Zhang, J. Z., Fan, Q. & Liu, D. L. (1995). Halomonas huanghaiensis sp. nov., a new species of genus Halomonas. Acta Microbiol Sin 35, 315–321 (in Chinese).
Yoon, J.-H., Kang, K.-H. & Park, Y.-H. (2003). Halobacillus salinus sp. nov., isolated from a salt lake on the coast of the East Sea in Korea. Int J Syst Evol Microbiol 53, 687–693.
Yoon, J.-H., Kang, K.-H., Oh, T. K. & Park, Y.-H. (2004). Halobacillus locisalis sp. nov., a halophilic bacterium isolated from a marine solar saltern of the Yellow Sea in Korea. Extremophiles 8, 23–28.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  I. J. Carrasco, M. C. Marquez, Y. Xue, Y. Ma, D. A. Cowan, B. E. Jones, W. D. Grant, and A. Ventosa Sediminibacillus halophilus gen. nov., sp. nov., a moderately halophilic, Gram-positive bacterium from a hypersaline lake Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1961 - 1967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Romano, I. Finore, G. Nicolaus, F. J. Huertas, L. Lama, B. Nicolaus, and A. Poli Halobacillus alkaliphilus sp. nov., a halophilic bacterium isolated from a salt lake in Fuente de Piedra, southern Spain Int J Syst Evol Microbiol, April 1, 2008; 58(4): 886 - 890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Yoon, S.-J. Kang, and T.-K. Oh Halobacillus seohaensis sp. nov., isolated from a marine solar saltern in Korea Int J Syst Evol Microbiol, March 1, 2008; 58(3): 622 - 627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Soto-Ramirez, C. Sanchez-Porro, S. Rosas-Padilla, K. Almodovar, G. Jimenez, M. Machado-Rodriguez, M. Zapata, A. Ventosa, and R. Montalvo-Rodriguez Halobacillus mangrovi sp. nov., a moderately halophilic bacterium isolated from the black mangrove Avicennia germinans Int J Syst Evol Microbiol, January 1, 2008; 58(1): 125 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-Y. An, K. Kanoh, H. Kasai, K. Goto, and A. Yokota Halobacillus faecis sp. nov., a spore-forming bacterium isolated from a mangrove area on Ishigaki Island, Japan Int J Syst Evol Microbiol, November 1, 2007; 57(11): 2476 - 2479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Carrasco, M. C. Marquez, Y. Xue, Y. Ma, D. A. Cowan, B. E. Jones, W. D. Grant, and A. Ventosa Salsuginibacillus kocurii gen. nov., sp. nov., a moderately halophilic bacterium from soda-lake sediment Int J Syst Evol Microbiol, October 1, 2007; 57(10): 2381 - 2386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Yoon, S.-J. Kang, Y.-T. Jung, and T.-K. Oh Halobacillus campisalis sp. nov., containing meso-diaminopimelic acid in the cell-wall peptidoglycan, and emended description of the genus Halobacillus Int J Syst Evol Microbiol, September 1, 2007; 57(9): 2021 - 2025. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N.-P. Hua, A. Kanekiyo, K. Fujikura, H. Yasuda, and T. Naganuma Halobacillus profundi sp. nov. and Halobacillus kuroshimensis sp. nov., moderately halophilic bacteria isolated from a deep-sea methane cold seep Int J Syst Evol Microbiol, June 1, 2007; 57(6): 1243 - 1249. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
