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1 George Beadle Center for Genetics, University of Nebraska-Lincoln, Lincoln, NE 68588-0666, USA
2 Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE 68583-0915, USA
Correspondence
Paul Blum
pblum1{at}unlnotes.unl.edu
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ABSTRACT |
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-cyclohexyl fatty acid, consistent with membranes from other Alicyclobacillus species. Very low DNA–DNA hybridization values between CsHg2T and the type strains of Alicyclobacillus indicate that CsHg2T represents a distinct species. On the basis of these results, the name Alicyclobacillus vulcanalis sp. nov. is proposed for this organism. The type strain is CsHg2T (ATCC BAA-915T=DSM 16176T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CsHg2T is AY425985.
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TOPABSTRACTMAIN TEXTREFERENCES |
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-alicylic fatty acid lipid membrane component (Wisotzkey et al., 1992
). Five of the eight described species of Alicyclobacillus, namely Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris (Wisotzkey et al., 1992), Alicyclobacillus hesperidum (Albuquerque et al., 2000), Alicyclobacillus acidiphilus (Matsubara et al., 2002) and Alicyclobacillus sendaiensis (Tsuruoka et al., 2003) contain -cyclohexane fatty acids, whereas Alicyclobacillus cycloheptanicus (Deinhard et al., 1987) and Alicyclobacillus herbarius (Goto et al., 2002) contain -cycloheptane fatty acids. A novel species, Alicyclobacillus pomorum, was described that did not possess -alicyclic fatty acids (Goto et al., 2003). The description of the cellular fatty acid profile of the genus has since been amended to include organisms that possess straight- and branched-chain fatty acids instead of -alicyclic fatty acids (Goto et al., 2003). Three Japanese strains, designated UZ-1, MIH 332 and KHA-31, were also found to contain -cyclohexane fatty acids and, by virtue of 16S rRNA gene sequence analysis and DNA–DNA hybridization assays, were denoted as a genomic species of Alicyclobacillus (Hiraishi et al., 1997).
A. hesperidum and another genomic species, FR-6, were described by Albuquerque et al. (2000). Despite low levels of genomic DNA–DNA hybridization between strain FR-6 and A. acidocaldarius, this strain was designated a genomic species because there were insufficient distinguishing characteristics available to determine whether it constituted a novel species. The latest species that have been described are A. herbarius, which was isolated from herbal tea made from the dried flowers of hibiscus (Goto et al., 2002), A. sendaiensis, isolated from Japanese soil, which has potential biotechnological applications as a source of heat-stable collagenase (Tsuruoka et al., 2003), A. acidiphilus, which can cause ‘off flavour’ in orange juice (Matsubara et al., 2002), and A. pomorum, isolated from spoiled fruit juice (Goto et al., 2003).
The natural habitats for most of these organisms are notable for their unique geochemistries, often rich in metals and metal sulfide minerals. To aid in the characterization of these organisms, metal susceptibility tests were conducted and were found to be useful as a means of distinguishing between Alicyclobacillus species. Metal-based phenotypic analysis provides a new tool for comparing organisms from soil environments.
Strain CsHg2T was isolated from pool water (78 °C, pH 1·7) obtained from an acidic geothermal pool located in Coso Hot Springs in the Mojave Desert, California, USA. The water sample was adjusted to pH 5·5 using calcium carbonate, and cells were recovered by filtration using a 0·22 µm sterile membrane. The filter was immersed in neutralized pool water and transported at ambient temperature to the laboratory. The sample was plated on solid medium using Gelrite (gellan gum; Kelco) plates. The medium employed the basal salts medium of Allen (1959) as modified by Brock et al. (1972) containing 0·2 % (w/v) tryptone and adjusted to a final pH of 2·5 with sulfuric acid. Plates were incubated at 70 °C until growth was observed. CsHg2T was purified by streaking for single colonies on plates three times. Pure cultures of CsHg2T were stored according to the method described by Rolfsmeier & Blum (1995). Other organisms used in the following tests were Alicyclobacillus cycloheptanicus ATCC 49028T, A. acidoterrestris ATCC 49025T and A. acidocaldarius ATCC 27009T, which were obtained from the ATCC. Alicyclobacillus sp. FR-6 (=DSM 11984) and A. hesperidum DSM 12489T were obtained from the DSMZ and three Alicyclobacillus strains, UZ-1, MIH 332 and KHA-31, were kindly provided by Dr Y. Tanimoto of Okayama University, Japan.
Morphological examination of the cells was via a phase-contrast microscope. Presence of spores was checked by spore staining and culture smears were Gram stained (Hucker & Conn, 1923). Growth of CsHg2T at different temperatures was determined in modified Allen medium with shaking. Optical density of the culture was monitored over a 24 h period at a wavelength of 600 nm. The optimum pH for growth was determined in ATCC 573 medium [1·3 g (NH4)2SO4, 0·37 g KH2PO4, 0·25 g MgSO4.7H2O, 0·07 g CaCl2.2H2O, 0·02 g FeCl3.6H2O, 0·35 % (w/v) tryptone and 0·4 % (w/v) glucose per litre of solution] adjusted to different pH values using sulfuric acid. Cultures were grown at 55 °C with shaking and changes in turbidity of the cultures were monitored over a 24 h period. Acid production from selected carbohydrates was determined using API 50 CH test strips (bioMérieux) in BAM basal salts medium (Albuquerque et al., 2000). Sensitivity to metal salts was determined as the occurrence of growth using initial inocula of 107 cells ml–1 in 10 ml modified Allen medium containing metal salts. Tubes were incubated with shaking for 24 h at 55 °C and a positive test result reflected final turbidities that exceeded initial values.
Cell biomass for lipid analysis was obtained from overnight cultures grown in ATCC 573 medium at 55 °C. Methylated ester-linked fatty acids were prepared as described by Bligh & Dyer (1959), Kates (1986) and White et al. (1979), and separated on a Hewlett Packard 5890 Series II GC containing an Ultra2 HP column (50 m, 0·2 mm i.d., 0·33 µm film thickness) in split mode (44 : 1) with a 0·75 min purge time. Oven temperature was increased from 50 °C to 160 °C at 40 °C min–1 and held for 2 min, then increased at 3 °C min–1 to 300 °C and held for 30 min. Fatty acids were identified using equivalent chain-length standards (Bacterial Acid Methyl Esters CP mix; Supelco) and confirmed by GC-MS. The identity of the cyclo fatty acids was confirmed by GC-MS of their methyl esters (Oshima & Ariga, 1975) and their dimethyloxazoline derivatives (Yu et al., 1989).
Genomic DNA was prepared as described by Partridge et al. (1993). 16S rRNA genes were PCR-amplified (Haseltine et al., 2001) using primers Bac11, 5'-AGAGTTTGATCCTGGCTCAG-3', and Bact1492, 5'-GGTTACCTTGTTACGACTT-3' (De Long, 1992), and purified and sequenced as described by Haseltine et al. (1999). Sequence identities were calculated using BESTFIT included in the Wisconsin sequence analysis package, version 8 (GCG). An alignment of 1412 nucleotides was generated using the CLUSTAL W (Thompson et al., 1994) function included in BIOEDIT (Hall, 1999). A phylogenetic distance tree with bootstrap analysis from 1000 replications was generated using the neighbour-joining method included in MEGA, version 2.1 (Kumar et al., 2001).
Genomic hybridization was performed as described by Urbance et al. (2001) with the following modifications: the hybridization buffer contained 50 % formamide, and hybridization was conducted for 48 h at 45 °C on a shaking water bath, while probe binding was measured by liquid scintillation counting (Liquid Scintillation System LS 1701; Beckman). Hybridization of CsHg2T-labelled genomic DNA was tested against all Alicyclobacillus species and strains mentioned above. The DNA G+C content was determined by a fluorometric method that measures thermal denaturation of DNA (Gonzalez & Saiz-Jimenez, 2002). G+C content of CsHg2T was estimated from a calibration curve prepared by plotting the Tm of three known bacteria, Bacillus subtilis ATCC 6051T, A. acidocaldarius ATCC 27009T and Pseudomonas auruginosa ATCC 10145T, against their G+C contents.
Cells of strain CsHg2T were Gram-positive rods, 1·5–2·5 µm long and 0·4–0·7 µm wide, possessing terminal spores. CsHg2T grew aerobically at 35–65 °C with an optimum of 55 °C and over a range of pH values from 2·0 to 6·0 with an optimum of 4·0. Phenotypic characteristics of CsHg2T and related species of Alicyclobacillus (those sharing more than 94 % sequence similarity) are shown in Table 1. CsHg2T hydrolysed starch on starch plates, was oxidase and catalase negative and grew on solid media with up to 2 % (w/v) sodium chloride. Bacterial colonies were semi-transparent to white, convex and about 1 mm in diameter after growth for 28 h at 55 °C. CsHg2T produced acid from a variety of pentoses and hexoses, some disaccharides and sugar alcohols (Table 1). Some results for acid production from various carbohydrates did not match the results given by Albuquerque et al. (2000) and Tsuruoka et al. (2003), the reasons for which were discussed by Goto et al. (2002). The total fatty acids of CsHg2T included 46 % -cyclohexyl 17 : 0 and 22·6 % -cyclohexyl 19 : 0 (Table 2), placing CsHg2T in the group of Alicyclobacillus that possess -cyclohexyl fatty acids.
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). To clarify the extent of relatedness between CsHg2T and these species, genomic DNA–DNA hybridization was conducted. Although the 16S rRNA gene sequence of CsHg2T shared the highest relatedness to A. acidocaldarius ATCC 27009T, DNA–DNA relatedness between these two strains was only 9 %. DNA–DNA hybridization values of CsHg2T did not exceed 24 % for any of the Alicyclobacillus species tested. Strain FR-6 exhibited the highest binding to CsHg2T, at 24 %. These results indicate that CsHg2T is distinct from other alicyclobacilli and can be classified as a separate species (Wayne et al., 1987). The G+C content of CsHg2T was 62 mol%. This falls within the range of values of the different Alicyclobacillus species (51·6–62·3 mol%).
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Aerobic, Gram-positive, spore-forming rods measuring 1·5–2·5x0·4–0·7 µm. Oxidase and catalase negative, grows in 2 % NaCl and utilizes starch. Colonies appear semi-transparent to white and grow to 1 mm diameter in 28 h at 55 °C in modified Allen medium. -Cyclohexyl fatty acid is the major fatty acid component of the cell. Grows at a temperature range of 35–65 °C (optimum 55 °C) and over a pH range of 2·0–6·0 (optimum 4·0). Resistant to 0·2 mM molybdic acid 0·002 mM mercuric chloride, 10 mM sodium selenate and 40 mM zinc chloride and sensitive to 8 mM cupric chloride, 1 mM cobalt chloride, 10 mM sodium iodide and 0·8 mM cadmium chloride. Produces acid from glycerol, L-arabinose, ribose, D-xylose, galactose, D-glucose, D-fructose, D-mannose, inositol, mannitol, cellobiose, maltose, melibiose, sucrose, trehalose, D-raffinose, glycogen and D-turanose, but not from D-arabinose, L-xylose, adonitol, methyl 
-xyloside, L-sorbose, rhamnose, sorbitol, dulcitol, methyl 
-D-mannoside, N-acetylglucosamine, amygdalin, aesculin, salicin, lactose, inulin, melezitose, starch, xylitol, -gentiobiose, D-lyxose, D-tagalose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate and 2-ketogluconate. The DNA G+C content is 62 mol%.
The type strain, CsHg2T (=ATCC BAA-915T=DSM 16176T), was isolated from a hot spring in Coso Hot Springs in the Mojave Desert, California, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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