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1 State Key laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
2 Department of Biotechnology, University of the Western Cape, Bellville 7535, South Africa
3 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, UK
4 Genencor International BV, Archimedesweg 30, 2333 CN Leiden, The Netherlands
5 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
Correspondence
Yanhe Ma
mayanhe{at}sun.im.ac.cn
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ABSTRACT |
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; Wiegel & Kevbrin, 2004). Alkaliphilic and thermophilic catalases, for example, have become industrially important enzymes because of their extensive use in industrial processes in the food, dairy, textile and pulp and paper industries (Gudelj et al., 2001; Thompson et al., 2003). Aerobic alkalithermophiles are potentially good sources of alkaliphilic and thermophilic catalases.
Alkalithermophilic bacteria have been isolated from a variety of environments, including some mesophilic ones. The majority of aerobic alkaliphilic and thermophilic species described belong to the genus Bacillus and related genera arising from the splitting of the genus Bacillus (Demharter & Hensel, 1989; Pikuta et al., 2000; Wiegel & Kevbrin, 2004). During the course of our search for aerobic alkaliphilic and thermophilic bacteria, strain HA6T was isolated from an alkaline hot spring. This strain is able to produce relatively high levels of catalase activity, optimally at pH 10 and 60–65 °C. Here we present the characterization and taxonomy of strain HA6T, and its assignment to a new genus.
Strain HA6T was isolated from water samples taken from an alkaline hot spring (80 °C, pH 9.0), Drumbeat Spring in Rehai Park in the Tengchong area of China. Enrichment and isolation were done at 60 °C on medium A containing the following (g l–1): NH4NO3, 1.3; K2HPO4, 0.28; MgSO4.7H2O, 0.25; CaCl2.2H2O, 0.075; NaCl, 5.0; tryptone (Oxoid), 1; yeast extract (Oxoid), 1; Na2CO3, 5.0. The concentrated carbonate stock solution was sterilized separately and added to the medium prior to incubation. The final pH (measured at 25 °C) was 9.0 (pH 8.5 at 60 °C). Solid media contained 2.0 % (w/v) agar. The isolate was routinely grown aerobically at 60 °C on modified medium A (MMA) containing (l–1) 5.0 g yeast extract, 5.0 g tryptone and 15 g NaCl. On MMA at 60 °C, strain HA6T formed yellow–white, circular, translucent colonies.
Cell morphology was observed by using light microscopy and scanning electron microscopy. Gram staining was used to determine the cell-wall structure, in parallel with KOH testing (Gregersen, 1978). General physiological and biochemical tests (including the Voges–Proskauer reaction, the methyl red test, tests for H2S production, indole production, nitrate reduction and biopolymer hydrolysis and tests for catalase, oxidase, urease and phosphatase activity) were performed as described previously (Smibert & Krieg, 1981). Unless otherwise indicated, all tests were performed in triplicate in media containing 1.5 % (w/v) NaCl and 0.5 % (w/v) Na2CO3 and incubated at 60 °C. The NaCl concentrations, pH values and temperatures for growth were determined in liquid MMA. NaCl concentrations in the range 0–10 % (w/v) (0.5 % increments) were tested in medium at 60 °C and pH 8.5. The pH values tested were in the range 7.0–10.5 (at 60 °C, with increments of 0.5 pH units). Temperatures ranging from 35 to 75 °C were tested (at pH 8.5, with 5 °C increments). The pH of the MMA was adjusted prior to inoculation with concentrated Na2CO3 and was measured at 60 °C with a model PB-10 pH meter (Sartorius) equipped with a combination pH electrode and temperature probe. Growth was monitored by assessing the turbidity as OD600. Substrate utilization was tested in MMA containing 1 g tryptone l–1 and 1 g yeast extract l–1. Substrates in sterile stock solutions were each added to the medium at a final concentration of 20 mM, except for organic acids (0.2 %, w/v). Control cultures were grown without any substrate additions. Growth of the third subculture was determined, after two transfers of culture material under the same conditions.
Isoprenoid quinones, extracted and purified from freeze-dried cells using the method of Collins (1985), were analysed by HPLC. The peptidoglycan composition was analysed by one-dimensional chromatography as described by Schleifer (1985), using cellulose thin-layer plates. Cellular fatty acids were determined at the DSMZ (German Collection of Micro-organisms, Braunschweig, Germany). Genomic DNA was extracted by using a method described previously (Pitcher et al., 1989). The G+C content of the genomic DNA was determined by the thermal denaturation method, according to Marmur & Doty (1962). The methods used for PCR amplification of the 16S rRNA gene, sequencing of the PCR products and determination of the phylogenetic position were described by Zhang et al. (2002).
The physiological, biochemical and morphological characteristics of strain HA6T are given in the genus and species descriptions and in Table 1. Strain HA6T was found to be a moderately thermophilic, spore-forming bacillus (Fig. 1). Growth was observed at temperatures of 45–65 °C. The strain grew in liquid MMA at pH 7.5–9.5 at 60 °C, but did not grow without Na2CO3 (pH60 °C 7.2), which means that the novel isolate is obligately alkaliphilic. The strain grew with NaCl at concentrations in the range 0–6 % (w/v); only weak growth was observed on MMA agar with 0.5 % (w/v) K2CO3 in the absence of NaCl or Na2CO3. In the test for glucose oxidation, no gas was visualized in inverted Durham tubes, but weak acidification of glucose was detected. The pH of cultures after growth decreased by 0.1–0.2 pH units relative to the control. This pH change may have resulted from the absorption by the bicarbonate buffer of CO2 produced by the culture. Further studies will be necessary to clarify the products of glucose oxidation by strain HA6T.
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The 16S rRNA gene sequence (1485 bp) of strain HA6T was determined and compared with sequences available in the EMBL database by using the FASTA3 program (Pearson & Lipman, 1988). The 16S rRNA gene sequence similarity, calculated using FASTA3 at the EBI, and the phylogenetic tree, constructed using the neighbour-joining method in the TREECONW software, showed that strain HA6T was phylogenetically related to members of the family Bacillaceae. Pairwise similarity values for strain HA6T with respect to taxa with validly published names were as follows: 93.1 % with thermophilic Bacillus smithii DSM 4216T, 91.8 % with alkaliphilic Bacillus horti K13T, 90.4 % with alkalithermophilic Bacillus thermocloacae DSM 5250T and 91.3–91.7 % with members of the genus Geobacillus. Sequence similarities of less than 91.0 % were found with respect to other species of the Bacillaceae with validly published names. On the phylogenetic tree (Fig. 2), strain HA6T is located close to the species of the genus Bacillus and other related genera, namely Geobacillus and Anoxybacillus. Its position occurs on a separate lineage within the family Bacillaceae. Despite the presence of the short branch of B. smithii giving the higher sequence-similarity value, there is no bootstrap support for this relationship. B. smithii belongs to a different cluster within the family Bacillaceae. However, strain HA6T clustered consistently with B. horti K13T and branched at the periphery of the other related groups. Other thermophilic bacteria of Bacillus rRNA group 5 and alkaliphilic bacteria of Bacillus rRNA group 6 formed separate groups and were remotely related to strain HA6T. These data indicate that strain HA6T is phylogenetically distinct from the known species and genera of the family Bacillaceae and represents a novel genus and species.
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). For example, strain HA6T is an obligate alkaliphile that does not grow at pH 7 and grows optimally at pH 8.5, whereas B. smithii DSM 4216T and Geobacillus stearothermophilus DSM 22T are neutrophilic, showing no growth above pH 9. Strain HA6T is moderately thermophilic, being unable to grow at 37 °C and growing optimally at 60 °C, whereas B. horti K13T, the closest species (on the basis of the 16S rRNA gene sequence), shows good growth at 37 °C and does not grow at 60 °C. Strain HA6T has only one predominant isoprenoid quinone component (MK-7), while B. thermocloacae DSM 5250T possesses two principal components (MK-7 and MK-8). In addition, the genomic DNA G+C content of strain HA6T is higher than those of B. horti K13T, B. smithii DSM 4216T and B. thermocloacae DSM 5250T and lower than that of G. stearothermophilus DSM 22T. Finally, significant differences between the cellular fatty acid composition of strain HA6T and those of type strains of the genera Geobacillus and Bacillus also support the separation of strain HA6T in a novel taxon.
An alkalithermophilic genus, Anoxybacillus, has already been described (Pikuta et al., 2000). However, strain HA6T is clearly distinguishable from species of the genus Anoxybacillus, as this genus was defined as being able to grow anaerobically and being able to produce acid from glucose fermentation, unlike strain HA6T. In addition, there are significant differences in the cellular fatty acid compositions of strain HA6T and species of the genus Anoxybacillus. The iso-C17 : 0 content of the novel isolate, as a percentage of the total fatty acid content, is significantly higher than that in Anoxybacillus species.
It is known that the complete oxidation of organic compounds is a feature of complex ecosystems. Strain HA6T was isolated from a hot spring, a habitat in which nutrients are limited and the substrate-mineralization roles of the various members of the microbial community are not well understood. The ability of the novel strain to grow on acetate suggests that this micro-organism may play a significant role in the complete oxidation of organic compounds within the hot-spring microbial community.
On the basis of its phenotypic and genotypic characteristics, strain HA6T cannot be confidently assigned to any of the currently known spore-forming genera of the family Bacillaceae, and therefore represents a novel genus and species. The 16S rRNA gene sequence of strain HA6T, when compared with those available in the EMBL database, showed a very high level of similarity (99.5 %) with respect to strain TA1.A2, a bacterium isolated from a thermal spring in New Zealand and which has not been assigned to any existing species or given a validly published species name. Peddie et al. (1999) studied the bioenergetics of strain TA1.A2: it is also thermophilic and alkaliphilic, showing optimal growth at pH 9.2 and 70 °C. The different optima for pH and temperature for growth and the high sequence similarity may indicate that the two strains belong to different species of the same genus. Therefore, strain HA6T should be placed in a novel genus and species, for which we propose the name Caldalkalibacillus thermarum gen. nov., sp. nov.
Description of Caldalkalibacillus gen. nov.
Caldalkalibacillus (Cal.dal.ka.li.ba.cil'lus. L. adj. caldus hot; N.L. n. alkali alkali; L. n. bacillus small rod; N.L. masc. n. Caldalkalibacillus bacillus living under hot and alkaline conditions).
Cells are Gram-positive, non-motile, rod-shaped and produce terminal spherical endospores. Obligately aerobic, alkaliphilic and thermophilic. Chemo-organotrophic. Catalase-positive. Cell-wall peptidoglycan contains meso-diaminopimelic acid. Major isoprenoid quinone is MK-7. The G+C content of the genomic DNA of the type species is 45.2 mol% (Tm). Predominant cellular fatty acids are iso-C15 : 0 and iso-C17 : 0. Phylogenetically, the genus belongs to the family Bacillaceae. The type species is Caldalkalibacillus thermarum.
Description of Caldalkalibacillus thermarum sp. nov.
Caldalkalibacillus thermarum (ther.ma'rum. L. gen. pl. n. thermarum of warm springs).
Cells are non-motile and rod-shaped (width, 0.5 µm; length, 3.0–6.5 µm). Spherical endospores are formed terminally in swollen sporangia. Gram-positive and KOH-test-negative. Colonies are yellow–white, translucent, circular, smooth, low convex and entire. Strictly aerobic, moderately thermophilic and obligately alkaliphilic. The temperature for growth is 45–65 °C, with an optimum at 60 °C. The pH range for growth is 7.5–10, with an optimum at pH 8.5. Growth occurs in the presence of NaCl at 0–6 % (w/v) and optimally at 1.5 % NaCl. Oxidase- and catalase-positive. Negative for nitrate reduction, nitrite reduction, the Voges–Proskauer test, the methyl red reaction and H2S production. Hydrolysis of starch is weak. Aesculin, cellulose, pectin, chitin, casein, gelatin and Tween 80 are not hydrolysed. Phosphatase- and urease-negative. Positive for the production of indole and ammonia from tryptone. Grows on D-glucose, D-mannose, L-rhamnose, sucrose, D-trehalose, cellobiose, D-melibiose, D-melezitose, inulin, erythritol, D-sorbitol, D-mannitol, glycerol, acetate, lactate, pyruvate, butyrate, citrate, succinate, galacturonic acid and glucuronic acid. Shows weak growth on D-lactose, D-raffinose and D-salicin. Shows no growth on D-fructose, D-galactose, L-sorbose, L-arabinose, D-ribose, D-xylose, maltose, glycogen, adonitol, inositol, gluconic acid, formate, oxalate, propionate, malonate, isocitrate, ketoglutarate or malate. Cells are resistant to novobiocin and bacitracin, but susceptible to ampicillin, erythromycin, norfloxacin, neomycin, rifampicin, tetracycline, streptomycin, chloramphenicol, kanamycin and ciprofloxacin. The major cellular fatty acids are iso-C15 : 0 and iso-C17 : 0. The main menaquinone type is MK-7. Cell-wall peptidoglycan contains meso-diaminopimelic acid. The G+C content of the genomic DNA of the type strain is 45.2 mol% (Tm).
The type strain, HA6T (=CGMCC 1.4242T=JCM 13486T), was isolated from an alkaline hot spring in Tengchong in China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Demharter, W. & Hensel, R. (1989). Bacillus thermocloacae sp. nov., a new thermophilic species from sewage sludge. Syst Appl Microbiol 11, 272–276.
Gregersen, T. (1978). Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J Appl Microbiol Biotechnol 5, 123–127.[CrossRef]
Gudelj, M., Fruhwirth, G. O., Paar, A., Lottspeich, F., Robra, K. H., Cavaco-Paulo, A. & Gubitz, G. M. (2001). A catalase-peroxidase from a newly isolated thermoalkaliphilic Bacillus sp. with potential for the treatment of textile bleaching effluents. Extremophiles 5, 423–429.[CrossRef][Medline]
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 4, 109–118.
Nakamura, L. K., Blumenstock, I. & Claus, D. (1988). Taxonomic study of Bacillus coagulans Hammer 1915 with a proposal for Bacillus smithii sp. nov. Int J Syst Bacteriol 38, 63–73.
Nazina, T. N., Tourova, T. P., Poltaraus, A. B. & 8 other authors (2001). Taxonomic study of aerobic thermophilic bacilli and descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51, 433–466.[Abstract]
Pearson, W. R. & Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 2444–2448.
Peddie, C. J., Cook, G. M. & Morgan, H. W. (1999). Sodium-dependent glutamate uptake by an alkaliphilic, thermophilic Bacillus strain, TA2.A1. J Bacteriol 181, 3172–3177.
Pikuta, E., Lysenko, A., Chuvilskaya, N., Mendrock, U., Hippe, H., Suzina, N., Nikitin, D., Osipov, G. & Laurinavichius, K. (2000). Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavithermus comb. nov. Int J Syst Evol Microbiol 50, 2109–2117.[Abstract]
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure of murein. Methods Microbiol 18, 123–156.
Smibert, R. M. & Krieg, N. R. (1981). General characterization. In Manual of Methods for General Bacteriology, pp. 409–443. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology.
Thompson, V. S., Schaller, K. D. & Apel, W. A. (2003). Purification and characterization of a novel thermo-alkali-stable catalase from Thermus brockianus. Biotechnol Prog 19, 1292–1299.[CrossRef][Medline]
Wiegel, J. (1998). Anaerobic alkalithermophiles, a novel group of extremophiles. Extremophiles 2, 257–267.[CrossRef][Medline]
Wiegel, J. & Kevbrin, V. (2004). Alkalithermophiles. Biochem Soc Trans 32, 193–198.[CrossRef][Medline]
Yumoto, I., Yamazaki, K., Sawabe, T., Nakano, K., Kawasaki, K., Ezura, Y. & Shinano, H. (1998). Bacillus horti sp. nov., a new Gram-negative alkaliphilic bacillus. Int J Syst Bacteriol 48, 565–571.
Zhang, W., Xue, Y., Ma, Y., Zhou, P., Ventosa, A. & Grant, W. D. (2002). Salinicoccus alkaliphilus sp. nov., a novel alkaliphile and moderate halophile from Baer Soda Lake in Inner Mongolia Autonomous Region, China. Int J Syst Evol Microbiol 52, 789–793.[Abstract]
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